Neolynx Research

NeoLynx Research Application Manager
NeoLynx Research 3.5

Application Manager Users Guide
Version 3.5
th
15 April 2001
CAUTION: For Research Use Only. Not for use in diagnostic procedures.
No use of this product as a diagnostic tool is implied or should be inferred.
Page i
NeoLynx Research Application Manager Users Guide

Important Notice
Micromass Limited (Manchester, UK) and its subsidiary Micromass Inc. (Beverley,
MA) do not offer for sale or support the NeoLynx Research Application Manager for
non-research applications within the USA.
No use of the NeoLynx Research Application Manager as a diagnostic device is

implied or should be inferred.
Copyright Notice
Micromass Ltd believes that the information in this publication is accurate. However
the information is subject to change without notice and should not be construed as a
contractual undertaking by Micromass Ltd. Despite the care which has been given to
the preparation of this publication, Micromass Ltd accepts no responsibility for any
loss or any other matter which may arise from any error or inaccuracy which may
inadvertently have been included.
Copyright (C) 1993-1999 Micromass Ltd. All Rights Reserved.
No part of this publication may be copied without the express written permission of
Micromass Ltd.
Trademarks
Micromass ® is a registered trade mark of Micromass Limited

(Reg. U.S. Pat. & Tm. Off.).
MassLynx and NeoLynx are registered trademarks of Micromass Ltd.
Windows is a trademark of Microsoft Corporation. Other product names mentioned

in this manual may be trademarks or registered trademarks of their respective
companies and are hereby acknowledged.
Page ii
Table of Contents
Introduction 1
Overview 1
Introduction 2
Electrospray Ionisation 3
Analytical Procedures 4
Analysis Of Acylcarnitines 4
Analysis Of Amino Acids 7
Modes Of Data Acquisition 14
Sample Preparation and Data Acquisition 15

Sample Preparation 15
Sample Introduction 20
Mass Calibration 20
Mass Spectrometer Tuning 21
Data Acquisition 24
Overview 24
Projects 24
Sample Lists 26
Scan Function Editor 30
Data Processing with NeoLynx 39

Overview 39
Introduction 40
NeoLynx Test File Editor 41
The Test Editor Toolbar 42
Getting Started 43
Processing Options 46
Function Names Table 49
Mandatory Mass Table 50
Rule Definition Table 52
Specifying Parameters 55
Processing 59
Print Control 64
NeoLynx Browser 65
Overview 65
Accessing the NeoLynx Browser 65
The NeoLynx Browser Screen 66
The NeoLynx Browser Toolbar 67
Getting Started 68
View Options 68
The Spectrum Page 68
The Chromatogram Page 69
The Default Plate Page 70
The Colors Page 73
The List Columns Page 74
Other Display Options 76
Other Menu Options 77
File Menu 77
View Menu 77
Window Menu 77
The Plate Pane 78
The Results Table Pane 79
The Function Pane 80
Page iii
The Results Summary Pane 80

The Sample Description Pane 81
The Spectrum Pane 82
The Chromatogram Pane 83
Printing and Reporting 83
Report Schemes 86
Report Scheme Settings 86
The Report Control Page 87
The Batch Summary Page 89
The Sample Report Page 91
The Electronic Report Pages 94
Selected References 99
Appendix A - Data Compendium 105

Selected acylcarnitine profiles 105
Selected neutral / acidic amino acid profiles 109
Selected basic amino acid profiles 114
Alternative scan function for Glycine and Alanine 115
Appendix B - Quantification 117
Appendix C - Examples Of Data Processing Using NeoLynx121

Data Files Supplied 121
Manual Examination of Spectra for Example 3 140
Appendix D - Importing Worksheets 155

Overview 155
Import Worksheet 155
Import Data 158
Appendix E - NRS Configurations 161

Sample_Conc1.rep 162
Sample_Anon.rep 175
Sample_Summary.rep 177
Index 183
Page iv
Conventions
The MassLynx Users Guide follows these typographic conventions
This Represents
bold Anything you must type exactly as it appears
italic Place holders for information you must provide. For example
if you are asked to type filename, you would type the actual
name for a file instead of the word shown in italic type.
ALL Directory names, filenames and acronyms.

CAPITALS
Keyboard Formats
Key combinations and key sequences appear in the following formats.
KEY1+KEY2 A plus sign (+) between key names means to press and hold
down the first key while you press the second key. For
example, "press ALT+ESC" means to press and hold down the
ALT key and press the ESC key. Then release both keys.
KEY1,KEY2 A comma sign (,) between key names means to press and
release the keys one after the other. For example, "press
ALT,F" means to press and release the ALT key and then press
the F key.
Page v
Page vi
Introduction
Introduction
Chapter 1
Overview
The objective of this document is to explain the processes required to return a
quantitative analysis of metabolites extracted from blood spots. The chemical and
instrumental background on the analyses is covered in some depth, with the aim of
allowing the users to develop their own analytical strategies. The principles and
operation of the NeoLynx suite of processing software are discussed, and a
comparison of the quantification results with classical integration methods is
presented. The ultimate goal of this type of analysis is to provide a result giving the
endogenous concentration of the targeted metabolites.
There are seven basic stages involved in automated analysis which are:
1. Creation of a Project.
2. Definition of Tune page, Inlet and Scanning Method files.
3. Definition of NeoLynx Test Files.
4. Creation of a list of samples using the Sample List Editor.
5. Preparation of Samples.
6. Acquisition of sample data.
7. Processing of acquired data and production of reports using the NeoLynx

Browser.
A data compendium of samples from a number of patients with inherited metabolic

diseases is included Appendix A. The samples were provided by Dr Mohamed
Rashed of the King Faisal Specialist Hospital and Research Centre.
Page 1
Introduction
Introduction
In the study of metabolic diseases in new-born babies, blood spot samples are taken
on filter papers soon after birth. The adsorption of blood onto filter papers has been
well characterised, and it is possible to utilise a reasonably well-defined amount of
blood by punching a disc out of the dried filter paper.
In the analyses described in this document, the metabolites are typically extracted
from the dried blood sample on the filter paper disc, chemically derivatised where
necessary and subjected to analysis by electrospray tandem mass spectrometry.
The function of a tandem mass spectrometer is to:
1. Produce ions from the compounds in the sample under analysis.
2. Select PRECURSOR ions according to their mass in the first analyser of the
instrument.
3. Fragment the mass selected PRECURSOR ions, by colliding them with argon
gas in the collision cell, to give PRODUCT ions - a process called
COLLISION-INDUCED DISSOCIATION (CID).
4. Analyse the PRODUCT ions according to their mass in the second analyser of
the tandem instrument.
The overall process is often called MS/MS (mass spectrometry - mass spectrometry
or tandem mass spectrometry) and is a highly specific way for detecting specific
substances in complex mixtures
When data from samples has been acquired NeoLynx processes the results to:-
• determine the metabolite concentrations of interest
• compare them to a standard set of rules
• produce reports detailing both normal and abnormal results.
Page 2
Introduction
Electrospray Ionisation
Electrospray ionisation is a soft ionisation technique that allows for the mass
spectrometric analysis of labile, polar compounds that were previously extremely
difficult to analyse. A soft ionisation technique is one in which the majority of the
ionic species observed are attributable to molecular related species or facile
fragmentation (e.g. dehydration) from the parent molecule. A solution of the analyte
of interest is nebulised from a fine nozzle through a high electric field, forming the
ions to be analysed.
Probe
Exhaust
Sample Cleanable Exhaust
Baffle Liner
Purge Gas
(Megaflow only)
Sample
Cone
RF
Isolation Lens
Valve
Nebuliser Desolvation
Gas Gas
Extraction Analyser
Cone
Source
Enclosure
Rotary Turbomolecular
Pump Pumps
Figure 1.1 The z-spray ionisation source
In a typical experiment the mobile phase pumped from an LC system, or an infusion

pump, enters through the probe and is pneumatically converted to an electrostatically
charged aerosol spray. The solvent is evaporated from the spray by means of a
drying gas passed through the desolvation heater. The resulting analyte and solvent
ions are drawn through the sample cone aperture into the ion block, from where they
are extracted into the analyser.
The electrospray ionisation technique allows rapid, accurate and sensitive analysis of
a wide range of analytes from low molecular weight polar compounds (less than 200
Da) to biopolymers larger than 100 kDa. Generally, compounds of less than 1000
Da produce singly charged protonated molecules ([M+H]+) in positive ion mode.
Likewise, these low molecular weight analytes yield deprotonated molecules ([M-H]-
) ions in negative ion mode, although this is dependent upon compound structure.
High mass biopolymers, for example peptides, proteins and oligonucleotides,

produce a series of multiply charged ions. The acquired data can be transformed by
the software to give a molecular weight profile of the biopolymer.
The Z-spray source can be tuned to fragment ions within the 'ion block', although the
'cone voltage' is usually tuned for optimum sensitivity. The optimum cone voltage
setting is normally compound-class dependent. Cone voltage fragmentation can
provide valuable structural information of low molecular weight analytes, although it
is considerable less specific than the experiments detailed below.
Electrospray is a concentration dependant technique, and it is this characteristic that

is taken advantage of for the quantification process. Analytes and the isotopically
labeled analogs both have a similar sensitivity under ionisation, and the relative peak
intensities detected will be proportional to their concentration in the solution under
analysis.
Page 3
Introduction
Analytical Procedures
The following analyses have been developed and reported in the literature, and are in
use in one form or another in a number of laboratories around the world. They are
explained in more detail in the references cited at the end of this document.
Analysis Of Acylcarnitines
L-carnitine (3-hydroxy-4-aminobutyrobetaine) plays a vital role in the mitochondrial

β-oxidation of fatty acids by acting as a transporter of acyl groups across
mitochondrial membranes. Free fatty acids are activated by the cytosol to the
coenzyme A thioesters (acyl CoA) and conjugated with carnitine by long-chain
carnitine acyltransferase. This is also known as carnitine palmitoyl transferase and is
located on the inner and outer mitochondrial membranes. When the carnitines cross
the membrane, the carnitine is regenerated with an equivalent release of the acyl-
CoA. Successive cycles of β-oxidation are carried out by a closely coupled system
of enzyme complexes, of which the acyl-CoA dehydrogenases are essential
components. Three specific systems exist with overlapping chain-length
specificities, very long-chain (VLCAD), medium-chain (MCAD) and short-chain
(SCAD), whose preferred substrates are palmitoyl (C16) CoA, octanoyl (C8) CoA and
butyryl (C4) CoA, respectively. One of the major products of the β-oxidation, acetyl
(C2) CoA, is transported out of the mitochondrion as a carnitine conjugate.
Defects have been identified and characterised in many of these enzymes and, in
disorder of fatty acid and branched chain amino acid catabolism affecting the
mitochondrial enzymes, the potential exists for the accumulation of abnormal acyl
CoA intermediates. These compounds may exhibit toxicity by inhibiting other
essential enzymes and may also sequester the limited pool of coenzyme A in the
mitochondrion. Carnitine plays an important role in the transport of these
intermediates out of the mitochondrion using the carnitine acyltransferases. The
ratio of bound carnitine (acylcarnitine) to free carnitine in plasma reflects the acyl
CoA to free CoA thioester ratio, and the ratio is usually elevated in patients with
fatty acid oxidation disorders. The secondary role of carnitine is therefore one of
detoxification when there is an abnormal accumulation of abnormal acyl CoA.
The generation of a profile of the acylcarnitines present in the blood should yield a
significant amount of information, relating to the fatty acid catabolism of the patient.
Acylcarnitines can be analysed in the native state but exist in solution as zwitter ions.
If a positive ion analysis is to be performed considerable increases in sensitivity are
achieved by blocking the carboxylate group through esterification. If free acyl-
carnitines are to be analysed, the addition of a small amount of acid will serve to
promote the fraction of the positive ions in solution. The discussions in this
document relate primarily to the anaysis of butyl esters.
The acylcarnitines may be studied by an analysis where all the molecular species that
decompose to yield a common fragment ion are determined. In mass spectrometric
terms, this is called a PRECURSOR or PARENT ion scan. In the analysis of
acylcarnitines, a common fragment of mass 85 Da can be used to provide
information on all the butylated acylcarnitines present in the sample. The
derivatisation and fragmentation is shown in Figure 1.2.
Page 4
Introduction
Figure 1.2 Butylation and fragmentation of acylcarnitines to yield a common

fragment at m/z 85
The acylcarnitine profile is derived by scanning the first mass analyser (MS1), and
only allowing fragment ions of m/z 85 to pass through the second mass analyser
(MS2) to be detected. The PARENT or PRECURSOR ion scan is depicted in
Figure 1.3.
MS1 (Scanning) Collision cell MS2 (Static)
Figure 1.3 A schematic representation of the parent or precursor ion scan in which
the first mass analyser (MS1) is scanned and the second mass analyser (MS2) is set
to pass only one specific fragment
A typical acylcarnitine profile from a healthy patient is shown in Figure 1.4. The
spectrum shows the presence of free carnitine (m/z 218), acetyl carnitine (m/z 260),
propionyl carnitine (m/z 274) and palmitoyl carnitine (m/z 456). Four internal
standards are used for quantification purposes (tri-deuterated free carnitine,
propionyl carnitine, octanoyl carnitine and palmitoyl carnitine).
normal01 1 (2.902) 1: Parents of 85ES+

100 2.08e6
221.2
%
260.2
460.6
277.3 456.5
347.4
484.5
289.3 302.4 380.3
0 m/z
225 250 275 300 325 350 375 400 425 450 475 500
Figure 1.4 Acylcarnitine profile from a healthy patient
Page 5
Introduction
Below is a table showing a list of the masses that may be observed for a number of
acylcarnitine butyl esters.
Notation Common Name Mass of butyl ester /Da

Free Free carnitine 218.18
d3-Free d3-free carnitine (internal standard) 221.20
C2 Acetyl 260.19
d3-C2 d3-acetyl (internal standard) 263.21
C3 Propionyl 274.20
d3-C3 d3-propionyl (internal standard) 277.22
C4 Butyryl 288.22
d3-C4 d3-butyryl (internal standard) 291.24
C5:1 Tiglyl 300.22
C5 Isovaleryl 302.23
C6 Hexanoyl 316.25
C5-OH 3-hydroxy-isovaleryl 318.23
C8:1 Octenoyl 342.27
C8 Octanoyl 344.28
d3-C8 d3-octanoyl (internal standard) 347.30
C10:2 Decadienoyl 368.28
C10:1 Decenoyl 370.30
C10 Decanoyl 372.31
C4-DC Methylmalonoyl 374.29
C5-DC Glutaryl 388.31
C12:1 Dodecenoyl 398.33
C12 Dodecanoyl 400.34
C6-DC Adipyl 402.33
C14:2 424.34
C14:1 426.36
C14 Myristoyl 428.37
C8-DC Subaryl 430.41
C14-OH 3-hydroxy-myristoyl 444.37
C16:1 Palmitoleyl 454.39
C16 Palmitoyl 456.40
C10-DC Sebacyl 458.45
d3-C16 d3-palmitoyl (internal standard) 459.42
C16:1-OH 3-hydroxy palmitoleyl 470.40
C16-OH 3-hydroxy palmitoyl 472.40
C18:2 Linoleyl 480.40
C18:1 Oleyl 482.42
C18 Stearoyl 484.44
C18:2-OH 3-hydroxy linoleyl 496.40
C18:1-OH 3-hydroxy oleyl 498.42
Table 1.1 Masses of butyl esters
Page 6
Introduction
Analysis Of Amino Acids
A number of inherited diseases result in the disruption of the metabolism and

transformation of amino acids. This results in an accumulation of certain amino
acids, depending on the reduction in enzyme efficiency that has occurred.
In phenylketonuria a deficiency in phenylalanine hydroxylase inhibits the conversion

of phenylalanine to tyrosine, with an accumulation of phenylalanine and a decrease
in tyrosine.
An elevation of methionine in blood may be indicative of two inherited amino acid

metabolic disorders: isolated hypermethioninemia and homocystinuria due to
cystathionine β-synthase deficiency.
Maple syrup urine disease is an inherited disorder characterised by the accumulation

of the branched-chain amino acids and their corresponding α-keto acids in blood and
urine. It arises from defects in the oxidative decarboxylation by branched-chain α-
keto-acid dehydrogenase (a multienzyme complex).
Early detection and dietary treatment of these and other disorders can lead to a
significantly improved outcome for a patient.
A large number of amino acids (though not all) may be analysed using tandem mass
spectrometry. The metabolites are extracted from the blood spots using methanol
and derivatised using the same procedure as for the acylcarnitines. The
fragmentation of the esterified amino acids is somewhat different to the
acylcarnitine, and amino acids fall into two distinct classes for their detection by
tandem mass spectrometry, based on structural and chemical differences. In
addition, both glycine and arginine show significantly improved sensitivity if
different scanning methods are used.
Neutral and Acidic Amino Acids
An analysis where the molecular species of interest lose an uncharged common

fragment from within their chemical structures. In the case of the derivatised amino
acids it is possible for all the amino acids to lose a neutral molecule of mass 102 Da.
In mass spectrometric terms, detection of all molecular species that undergo a
CONSTANT NEUTRAL LOSS of 102 Da will provide information on the different
butylated amino acids present in the sample.
The fragmentation for this transition is shown in Figure 1.5.
Page 7
Introduction
Figure 1.5 Butylation and fragmentation of amino acids to show the common loss of
butyl formate (102 Da)
The neutral and acidic amino acid profile is derived by scanning the first and second
mass analysers together with the difference between the first mass analyser (MS1)
and the second mass analyser (MS2) set to the neutral loss mass, 102 Da. The only
time an ion will be detected is when the fragmentation of the species in the collision
cell results in the appropriate loss from the precursor ion. The CONSTANT
NEUTRAL LOSS scan is depicted in Figure 1.6.
MS1 (Scanning) Collision cell MS2 (Scanning)
Figure 1.6 A schematic representation of the constant neutral loss scan in which
both mass analysers (MS1 and MS2) are scanned at a pre-defined offset of 102 Da
for the detection of neutral amino acids
A neutral amino acid spectrum from a healthy patient is shown in Figure 1.7.
normal01 1 (2.902) 3: Neutral Loss 102ES+

100 3.91e6
191.3
188.2
% 227.3
150.2 244.2
146.2 222.2
174.3
162.2
182.3 238.3
209.2 260.3
192.3
0 m/z
140 160 180 200 220 240 260
Figure 1.7 Neutral amino acid profile from a healthy patient
Page 8
Introduction
The amino acids detected in this spectrum are detailed in Table 1.2. In addition to
the endogenous species detected, a number of deuterated internal standard analogues
of the amino acids have also been included for quantification purposes. The internal
standards used in the experiment shown in Figure 1.7 include: d4-alanine (m/z 150),
d8-valine (m/z 182), d3-leucine (m/z 191), d3-methionine (m/z 209), d5-phenylalanine
(m/z 227) and d6-tyrosine (m/z 244).
Basic Amino Acids
In instances where the side chain of the amino acid contains a basic group, it is
energetically more favourable for the derivatised compounds to lose an ammonia
molecule (an additional 17 Da) as well as the butyl formate molecule. Again, the
loss from the precursor molecule will be neutral (losing two species instead of one),
but in such cases all ions undergoing a CONSTANT NEUTRAL LOSS of 119 Da
will need to be reported.
Figure 1.8 Butylation and fragmentation of amino acids to show the common loss of
butyl formate and ammonia (119 Da)
The basic amino acid profile is derived by scanning the first and second mass
analysers together with the first mass analyser (MS1) set to the neutral loss mass,
119 Da. The only time an ion will be detected is when the fragmentation of the
species in the collision cell results in the appropriate loss from the precursor ion. The
CONSTANT NEUTRAL LOSS scan is depicted in Figure 1.9.
MS1 (Scanning) Collision cell MS2 (Scanning)
Figure 1.9 A schematic representation of the constant neutral loss scan in which
both mass analysers (MS1 and MS2) are scanned at a pre-defined offset of 119 Da
for the detection of basic amino acids
A basic amino acid spectrum from a healthy patient is shown in Figure 1.10.
Page 9
Introduction
normal01 1 (2.902) 4: Neutral Loss 119ES+

100 7.86e5
203.2
221.6
244.2
%
234.1
174.2 238.1
176.2 189.1 204.1 212.2 231.1 245.2

0 m/z
170 180 190 200 210 220 230 240 250
Figure 1.10 Basic amino acid profile from a healthy patient
The amino acids detected in this spectrum are detailed in Table 1.2. In addition to
the endogenous species detected, a single deuterated internal standard, d6-ornithine
(m/z 195) has been included for quantification purposes.
Note: some amino acids appear in both the 102Da and 119Da Neutral loss spectra.
Glycine and Arginine
Two exceptions to the general classifications for amino acids described above relate
to glycine and arginine.
Although glycine can be detected by using a constant neutral loss of 102 Da,
examination of the fragmentation of the glycine butyl ester using a daughter ion scan
shows that the most abundant fragment results from the neutral loss of C4H8 (56 Da).
A DAUGHTER or PRODUCT ion scan may be generated by passing the analyte ion
of interest through the first mass analyser (MS1), fragmenting the selected ion by
collision with a target gas in the collision cell, and scanning the second mass
analyser (MS2) in order to detect all the fragments generated from the selected
precursor. This is illustrated in Figure 1.11.
MS1 (Static) Collision cell MS2 (Scanning)
Figure 1.11 A schematic representation of the daughter or product ion scan in

which the first mass analyser (MS1) is fixed to pass the ion of interest and the
second mass analyser (MS2) is scanned to detect all the fragment ions generated in
the collision cell
The product ion spectrum from the glycine butyl ester is shown in Figure 1.12.
Page 10
Introduction
daughters of 132.2 15/15, 15/15, collision 15, cone 20

gly02 1 (0.839) Daughters of 132ES+
100 1.80e6
57.2
30.4 41.3 48.3

29.4 86.2
0 m/z
20 30 40 50 60 70 80 90 100 110 120 130 140
Figure 1.12 Product ion spectrum of the butyl ester of glycine. Cone voltage = 20V,
Collision energy = 15eV
The product ion spectrum of the glycine butyl ester shows the protonated molecule
(M+H)+ at m/z 132. As the fragment ion at m/z 76 (loss of 56 Da) is approximately
five times more abundant than the normal neutral loss of 102Da, it is more
appropriate to analyse for glycine using a neutral loss of 56Da.
A neutral loss of 56Da is also observed for the other butyl esters of the amino acids,
but is not the most intense transition in the product ion spectra.
Arginine is another anomally from the normal fragmentations described above.

Although arginine is observed in the basic amino acid profile (neutral loss of
119 Da), the most abundant transition is the loss of 161Da from the m/z 231
precursor to give a dominant product ion at m/z 70.
Page 11
Introduction
A list of the common amino acids, their masses, and preferred neutral loss, is given
below.
Amino Acid Sigma # Mass of ester +H Preferred loss

Alanine A7503 146.12 102.07
Arginine A4881 231.18 161.11
Asparagine A8256 189.12 119.09
Aspartic acid A9006 246.18 (2 Bu) 102.07
Citrulline C6131 232.17 119.09
Glutamic acid G1126 260.19 (2 Bu) 102.07
Glycine G7126 132.10 56.06
Histidine H7875 212.14 102.07
Isoleucine I5393 188.16 102.07
Leucine L7875 188.16 102.07
Lysine L6001 203.18 119.09
Methionine M9500 206.12 102.07
1-Me-Histidine M9005 226.16 102.07
3-Me-Histidine M3879 226.16 102.07
Norleucine N1398 188.16 102.07
Ornithine O2250 189.16 119.09
Phenylalanine P1876 222.15 102.07
Proline P0255 172.13 102.07
Serine S4375 162.11 102.07
Threonine T8375 176.13 102.07
Tryptophan T0129 261.16 102.07
Tyrosine T3379 238.14 102.07
Valine V0375 174.15 102.07
Table 1.2 List of common amino acids and preferred neutral loss
Bile Acids
Bile acid concentrations are elevated in the blood of some neonates with cholestatic
hepatobiliary disorders, providing a means of screening for treatable conditions
including biliary atresia. Bile acids can be eluted from the Guthrie blood spots using
methanol containing deuterated internal standards, the glycine and taurine conjugates
of d4-chenodeoxycholic acid and d4-cholic acid. The samples are evaporated to
dryness and reconstituted in propan-2-ol/water (60/40 v/v) and analysed directly by
negative ion electrospray tandem mass spectrometry.
R4
R3
CH3
O
CH3
HO R2
R1
Page 12
Introduction
Name R1 R2 R3 R4 (M-H)-
Lithocholic H H H OH 375.29
Chenodeoxycholic H OH H OH 391.28
Deoxycholic H H OH OH 391.28
Hyodeoxycholic OH H H OH 391.28
Cholic H OH OH OH 407.28
Hyocholic OH OH H OH 407.28
Glycolithocholic H H H NH-CH2-COOH 431.31
Glycodeoxycholic H H OH NH-CH2-COOH 448.31
Glycocholic H OH OH NH-CH2-COOH 464.30
Glycochenodeoxycholic H OH H NH-CH2-COOH 448.31
Taurochenodeoxycholic H OH H NH-(CH2)2-SO3H 498.29
Taurodeoxycholic H H OH NH-(CH2)2-SO3H 498.29
Taurolithocholic H H H NH-(CH2)2-SO3H 482.29
Taurocholic H OH OH NH-(CH2)2-SO3H 514.28
Table 1.3 Structure of generic bile acids
Bile acids form strong deprotonated molecules, giving rise to a related molecular
species at the m/z value shown in Table 1.3. Predominant losses from the
deprotonated molecules are the consecutive losses of water (H2O), depending on the
number of substituent hydroxyl groups. Neutral losses of 36 Da and 54 Da may be
used for the determination of di- and trihydroxy bile acids in, for example an LC-
MS separation experiment.
The majority of work analysing bile acids from neonatal blood spots has been
performed using the common fragmentation of the glycine and taurine conjugated
bile acids. Taurine conjugated bile acids show a common fragment (relating to the
taurine sub-unit) of m/z 80, formed from the deprotonated molecule. The glycine
conjugated bile acids show a common fragment (relating to the glycine sub-unit) of
m/z 74, formed from the deprotonated molecule.
Owing to the small number of species to be analysed (it is not possible to

differentiate the different bile acid isomers in a simple MS/MS experiment), it is
usual to use multiple reaction monitoring to monitor specific species (and the
internal standards) for quantification purposes. The limit of detection of the
conjugated bile acids extracted from 5mm blood spots is less than 0.5µM for each
isobaric species.
Inclusion of the bile acid screening into a common experiment with amino acids and
acylcarnitines has been undertaken on a limited basis. One problem that occurs
during the derivatisation (butylation) process is the hydrolysis of the glycine groups
from the conjugates. The taurine conjugates are not affected.
Other Analyses
A considerable amount of research has been devoted to the analysis of haemoglobin

by mass spectrometry and tandem mass spectrometry. It is possible to use single
stage mass spectrometry to measure the level of total haemoglobin glycation by
using a simple dilution of whole blood. The same sample may also be used in the
preliminary stages of the detection and characterisation of haemoglobin variants.
The experimental procedures involved in such characterisations fall outside the
scope of this document, but are available on request.
Page 13
Introduction
Modes Of Data Acquisition

If a number of different classes of metabolites are to be screened for, the mass
spectral data may be acquired in one of two ways:
1. A method where a mass spectrum is acquired in order to detect all the species
that undergo a specific decomposition (e.g. PARENTS of m/z 85 will yield a
spectrum to show all masses of all the butylated acylcarnitines present in the
sample). This method will give rise to a spectrum that may indicate a known
disorder, or may give rise to a signal that has not previously been observed.
This method will be referred to as the full-scan MCA mode of operation.
2. A method where only specific fragmentations are detected by the mass

spectrometer using a multiple reaction monitoring technique (MRM). In this
mode of operation only specific user-defined metabolites will be detected and
all other metabolites will be ignored. This method will be referred to as the
MRM mode of operation.
The choice of scanning method depends on a number of factors, including legislative

and ethical considerations. It might be considered that the MRM mode of operation
would be preferred for a neonatal screening application as it is usual to just test for a
specific disorder. MRM data files are considerably smaller than MCA data files
acquired for similar compound classes. However, in cases where samples from acute
patients are to be studied it will be necessary to examine the spectra for all possible
disorders, and the full-scan MCA method will be required. These different modes of
scanning the mass spectrometer to derive information are termed FUNCTIONS.
Despite the complex physiology involved, the levels of the acylcarnitine and amino
acid metabolites present in blood are reasonably constant from person to person. If a
metabolic disorder is apparent then the levels of the appropriate metabolite are
altered severely, usually increased, by a measurable difference from what is
considered to be 'normal'.
In order to develop a consistency in the measurements, known amounts of

isotopically labelled acylcarnitines and amino acids are added to the blood sample
while it is being extracted from the filter paper. The internal standards, which are
identical in every way to the endogenous metabolites apart from the isotopic
labelling, behave chemically in exactly the same way as the metabolites they are
mimicking. This means that they will also be detected by the different scan functions
of the mass spectrometer. The relative levels of the endogenous metabolites to the
internal standards can easily be determined by measuring the relative intensities of
the peaks in the acquired mass spectra. It is not economically viable to provide an
internal standard for every analyte that may appear in a physiological sample, so only
a limited number of internal standards are used. The range of internal standard
materials available is discussed in more detail in the next section.
Page 14
Sample Preparation and Data Acquisition
Sample Preparation and Data

Acquisition
Chapter 2
Sample Preparation
Blood samples are typically collected on Guthrie filter cards (Schleicher and Schuell
903 filter paper), dried and submitted to central laboratories for analysis. These
cards have been in use since the mid 1960s and remain in routine use around the
world. Blood spots are punched from these filter cards and used in a variety of ways.
A number of different sizes of blood spots are typically used, corresponding to
different amounts of whole blood equivalent, and these are summarised in the table
below.
Spot diameter /mm Area / mm2 Whole blood content / µL

3 7.1 3.1
4.7 17.3 7.6
6 28.3 12.4
2 x 4.7 34.7 15.2
Table 2.4 Blood spot diameter showing whole blood equivalent
Preparation of samples for introduction into the mass spectrometer is perhaps the
most time-consuming part of the whole analysis. The sample preparation procedure
described here is that derived by the group at Duke University Medical Centre (D S
Millington, D L Norwood, N Kodo, C R Roe and F Inoue, Anal. Biochem., 180
(1989) 331-339 and D H Chace, D S Millington, N Terada, S G Kahler, C R Roe
and L F Hoffman, Clin. Chem., 39 (1993) 66-71) and is based on the analysis of
blood extracted from Guthrie cards
Reagents
The reagents used for the derivatisation are;
Reagent Grade Use
Methanol HPLC grade for extraction
Butanolic HCl see below for derivatisation
Acetonitrile HPLC grade for reconstituting the derivatised samples
Water HPLC grade for reconstituting the derivatised samples
Butanolic Hydrochloric acid

The derivatising reagent is probably the most critical of the reagents used. The
derivatisation requires the presence of acid in order to catalyse the reaction. The
concentration of the hydrochloric acid is normally stated as 3M. However, the
reagent is required to be anhydrous, as the presence of water during the
derivatisation stage will result in hydrolysis of the acylcarnitines, and to some extent,
the amino acids, thus reducing the sensitivity of the overall analysis. Butanolic HCl
Page 15
is commercially available in some countries, but there are limitations on shipping,

making access to such sources troublesome.
Butanolic HCl may be prepared in one of the following ways:
Adding acetyl chloride to dry n-butanol in the ratio 1:9. This is an exothermic
process, and due care must be exercised during the mixing. It is usual to
prepare the reagent with the mixing vessel in an ice bath.
Passing hydrochloric acid gas through dry n-butanol. The gas may be sourced from
either a cylinder or from the reaction of sodium chloride with sulphuric acid.
Passing the gas for 45 minutes should result in an appropriate acid
concentration in the solution. The concentration of the acid may be verified
by back titration.
Extraction and Derivatisation Procedure
Extraction and derivatisation of the metabolites should be performed in appropriate

vessels. Glass vials have been used successfully, but the requirement for analysing
large numbers of samples makes the use of single vials for each sample somewhat
unwieldy. Preference is currently being given to the use of 96-well microtitre plates.
The plate material and well-depth should be chosen with care as some materials have
been shown to degrade with the reagents used. Polypropylene plates have been
shown to be particularly resistant to degradation. The profile of the wells is not
critical, but round-bottomed vials will result in a concentration of the analytes of
interest in the bottom of the wells during the drying procedures.
The blood spot samples are punched into the chosen vessels and a volume of
methanol is added to effect the selective extraction of the metabolites from the filter
paper. In this process the blood proteins remain attached to the filter paper. The
samples are then gently agitated for approximately 30 minutes, although there have
been reports of efficient extraction by simply allowing the samples to stand.
The methanol also contains the isotopically labelled internal standards that will
subsequently be used for quantification purposes. Isotopically labelled standards are
used as they will have similar chemical and ionisation characteristics to the analytes
of interest, but will differ in their molecular masses, and will be differentiated during
the analysis by the tandem mass spectrometer. Adding the internal standards at the
point of sample extraction also serves as a check that the derivatisation has been
effective. If Internal Standard peaks are not at the expected intensity then the
derivatisation may not have worked.
The choice of which internal standards to add will depend on the particular analytes
of interest. In an ideal situation every analyte to be measured would require the
addition of an appropriately labelled chemical analogue, although this may not be
cost effective. The concentration of the internal standards in the methanol will
depend on:
(i) The volume of blood (number and size of blood spot punches) used.
(ii) The volume of methanol used for the extraction.
(iii) The clinical decision point for the concentration of a particular analyte.
This final point is regiospecific, and it is recommended that the internal standards are
added at a concentration equivalent to the clinical concentration that prompts follow
up (e.g. approximately 200µM/L equivalent for d5-phenylalanine for measuring
phenylalanine in whole blood). The highest accuracy of measurment will be
Page 16
achieved when the intensity of the analyte and internal standard peaks are
approximately the same intensity.
Once the metabolites have been extracted, it is necessary to either transfer the
methanolic solution to another vessel, or remove the blood spot. The methanol is
then evaporated under a stream of air or nitrogen (possibly with gentle heating to aid
the evaporation), to leave a residue of the extracted metabolites in the bottom of the
well. The evaporation time will depend on a number of factors and may take up to
20 minutes.
An amount of butanolic HCl (60-100µL) is then added to each dried sample. The
sample vessels then need to be sealed to prevent evaporation of the reagents before
the derivatisation is complete. A number of commercial, inert rubber plates are
available for this purpose. The sealed plate is then heated at 60°C for approximately
15 minutes. The optimum conditions for butylation should be carefully established.
Some evidence exists for the hydrolysis of acylcarnitines if a prolonged butylation
period is used.
Following the derivatisation, it is necessary to remove the butanolic HCl by

evaporation, similar to the process described above. Hydrochloric acid vapours are
both toxic and corrosive, and care must be exercised. As the viscosity of n-butanol
is somewhat higher than methanol, this stage is usually the most time-consuming of
the whole procedure.
Once the derivatised samples have been dried down, they are reconstituted in an
appropriate solvent (e.g. 80% acetonitrile) in preparation for injection into the mass
spectrometer.
The derivatisation procedure described above is shown schematically in Figure 2.1 .
A
B C D
F G H
A. Punch out selected spot from Guthrie card.

B. Add MeOH (e.g 200µL, including internal standard) and agitate for 20 minutes.
C. Decant off supernatant or remove bloodspot.
D. Evaporate to dryness under nitrogen or air with mild (40oC) heating.
E. Add 60-100µL butanolic HCl. Cap or seal. Heat at 60oC for 15 minutes.
F. Evaporate to dryness under nitrogen or air with mild (40oC) heating.
G. Add suitable solvent (approx. 50-100µL) e.g. 80% aqueous MeCN
H. Inject appropriate amount (typically 10-20µL) into mobile phase flowing to the
mass spectrometer.
Figure 2.1 Schematic diagram of sample preparation
Page 17
It is possible that the operations in the derivatisation process may be automated such
that batches of samples are prepared in a single session.
Isotopically Labelled Internal Standards
The primary source of isotopically-labelled internal standards is:
Cambridge Isotope Laboratories

50 Frontage Road
Andover, MA 01810-5413
USA
Phone: 508-749-8000
FAX: 508-749-2768
Please check for a local supplier. The UK catalogue numbers and prices (Sterling)
are shown below (Correct at Spring 2001).
Compound CIL Catalogue # Amount (g) Price (£)

2
H4- alanine DLM-1276 1.0 187
2
H3-aspartic acid DLM-832 1.0 279
2
H3-glutamic acid DLM-335 1.0 192
2
H5-glycine DLM-280 5.0 128
13
C15N-glycine CNLM-507 1.0 892
2
H3-leucine DLM-1259 1.0 267
2
H3-methionine DLM-431 1.0 109
2
H5-phenylalanine DLM-2986 1.0 225
2
H7-proline DLM-487 0.25 725
2
H2-serine DLM-1073 1.0 275
2
H2-tyrosine DLM-449 1.0 185
2
H4-tyrosine DLM-451 1.0 489
2
H8-valine DLM-311 1.0 345
============= ============= ============= =============
2
H3-carnitine DLM-1871 P.O.A.
2
2
2
H3-C2-carnitine DLM-754 P.O.A
2
H6-C2-carnitine DLM-3821 P.O.A.
2
2
2
2
2
2
Page 18
A primary source of kits of isotopically labelled internal standards is:
NeoGen Screening Inc.

110 Roesslar Road
Pittsburgh, PA 15220, USA
Phone: 412-341-8658
FAX: 412-341-8926
NeoGen Screening supply two kits that contain certified solutions of L-amino acids
(Kit A) and L-carnitine and acylcarnitines (Kit B). Each kit should provide
sufficient internal standards to allow for the preparation of up to 20,000 3mm blood
spots.
Kit A Kit B
15
N13C-glycine 2
H9-L-carnitine
2 2
H4-alanine H3-acetylcarnitine
2 2
H8-valine H3-propionylcarnitine
2 2
H3-leucine H3-butyrylcarnitine
2 2
H3-methionine H9-isovalerylcarnitine
2 2
H5-phenylalanine H3-octanoylcarnitine
2 2
H4-tyrosine H9-myristoylcarnitine
2 2
H3-aspartic acid H3-palmitoylcarnitine
2
H3-glutamic acid
2
H2-ornithine
2
H2-citrulline
2
H413C-arginine
It is advisable to maintain close links with the suppliers, as it is understood that new
labelled compounds that might be suitable for this type of work are constantly under
development.
Some isotopically labelled acylcarnitines are also available from:
Dr Herman ten Brink

Department of Pediatrics
Academic Hospital V U Metabolic Unit
P O Box 7057
1007 Amsterdam
Netherlands
Phone: + 31-20-444-2883
FAX: + 31-20-444-0305
Page 19
Sample Introduction
Once the sample has been prepared and is in solution, it needs to be passed into the
ion source of the mass spectrometer. This is normally performed by injecting the
sample into an appropriate mobile phase that is flowing continuously into the
electrospray ion source. There are no special restrictions on the type of LC pump
and autosampler that may be used for the NeoLynx application.
The mobile phase is usually delivered to the mass spectrometer by an LC pump at

flow rates from 10µL/min to 100µL/min, depending on the application. A typical
mobile phase for the majority of applications is 50-80% aqueous acetonitrile or
aqueous methanol containing no additives. The lack of additives allows for simple
switching between positive and negative ion analyses with minimal loss in
sensitivity. A number of LC pumps may be directly controlled from MassLynx,
allowing user-defined flow rates to be programmed.
In order to introduce the sample into the mobile phase some form of sample injection
is necessary. This may be performed using a manual LC injector (e.g. Rheodyne
7125), but is more commonly performed using an LC autosampler. Autosamplers
allow large batches of samples to be injected without user intervention. A range of
LC autosamplers may be controlled via MassLynx, and used for synchronising the
sample injection with a 'start' signal to the mass spectrometer
The facility is also available for the inclusion of an ultra-violet (UV) or diode array
(DAD) detector to be controlled from the data system, if required (this will only be
required for adding an extra dimension of information when performing
chromatographic separations).
For details of how to configure the pump, autosampler and detector see the
Controlling Inlet Systems and Autosamplers chapter in the MassLynx Guide to Data
Acquisition.
Mass Calibration
Periodic verification of the mass scales of both analysers is recommended for
optimum performance in both full-scan and MRM acquisitions.
It is advised that the mass scales should be calibrated to either m/z 23 (sodium) using
a mixture of sodium iodide and rubidium iodide (nairb.ref), or to m/z 45 using a
mixture of PEG 400 and 2mM ammonium acetate (PEG_NEO.ref). The upper limit
of the mass calibration should be greater than m/z 600.
For full details of calibration procedures and recipes for calibration solutions see the
Mass Calibration section in the Quattro LC User's Guide.
Page 20
Mass Spectrometer Tuning

In order to get the best analysis from the mass spectrometer, it is important that the
instrument is tuned effectively for the analysis. It is recommended that a 'standard'
normal blood spot is prepared with each batch, and that this sample is used for
tuning the mass spectrometer. Ideally, a copy of the tune page should be printed and
stored for future reference and quality control.
When tuning the instrument, it is possible to monitor up to four peaks on the tune
page. A typical tune page setting for neonatal blood spot analysis on amino acids
and acylcarnitines is shown in Figure 2.2.
To access the tune page press the button on the MS panel of the MassLynx top
level screen.
Figure 2.2 Typical tune page for Quattro LC Z-spray
Page 21
Figure 2.3 Typical tune page for Quattro LC Z-spray
The collision gas pressure for argon (recommended) is approximately 2.0 x 10-3
mbar (indicated on the readbacks).
The italicised parameters are intended as a guide only and must be set according to
the resolution required.
Source
Capillary 3.50 kV
Cone set in scan function editor
Extractor 3-5V
RF Lens 0.0V
Block temperature 140ºC
Desolvation Temperature 300°C
Analyser
LM resolution1 15.0
HM resolution1 15.0
Ion energy 1 0.0 - 1.0 eV
Collision entrance ±2 V
Collision Energy set in scan function editor
Collision exit 0-3V
LM resolution2 15.0
HM resolution2 15.0
Multiplier 650V
Page 22
Data Acquisition
Overview
NeoLynx operates in an automated MassLynx environment, and data acquisition is
usually achieved via the Sample List using some form of automated sample
introduction. It is possible to acquire data manually for post-processing in a batch
fashion. Multiple Sample Lists can be generated and queued up on the mass
spectrometer. This manual has been written with a view to all the data acquisition
and processing being performed from the Sample List.
Projects
MassLynx utilises a project structure that has been designed to assist laboratories in
GLP compliance. All acquisition settings files, raw data files, Sample Lists etc. are
stored within a project. The Project structure is particularly useful for the NeoLynx
application, as it is conceivable that a large number of samples will be analysed on a
daily basis.
It is strongly recommended that a Master project is created for each laboratory

containing the basic information for the control of the instrumentation and data
acquisition. The master project can then be used as a template for the creation of
individual projects. It may be appropriate to create a project on a daily basis, as this
will make data archiving and retrieval simpler.
MassLynx comes with some predefined projects NeoLynx.pro and Default.pro are
the two relevant to metabolite investigation. NeoLynx.pro is an example metabolite
investigation project, only available when NeoLynx is installed. Default.pro is the
default project where all data are stored until a new project has been selected or
created.
For more information on projects see the Getting Started section of the
MassLynx NT User Guide.
When creating a new project, the Project name must not contain
spaces.
Calibration Note
If the instrument is recalibrated, the new calibration experiment should be performed

in the <MASTER.PRO> project, used as the project template. In this way the
calibration is automatically included in any new project created from the template.
ROOT DIRECTORY
The project root directory (<PROJECT_NAME>.PRO) contains seven

sub-directories of which Acqudb, Data and Sampledb are the most important. The
root directory also contains the NeoLynx Results files generated after NeoLynx
processing of a Sample List. These files are discussed in more detail later.
Page 23
ACQUDB
This directory contains all the instrument control files used in the data acquisition.
For some files, different extensions will be used depending on the type of interface
card selected when the software was installed (TDA or Ethernet)
mass spectrometer tuning *.dbf (TDAT) or *.ipr (Ethernet)
mass spectrometer acquisition *.mdb (TDAT) or *.exp (Ethernet)
HPLC pump control *.h11 for HP1100
*.gil for Gilson
*.w60 for Waters 600
Calibration files *.cal
DATA
This directory contains all the raw data directories associated with a particular
project. Each raw directory contains the raw mass spectral information and any
saved mass spectral processes. The folders are given the name of the acquisition
with the format: <datafilename>.RAW.
SAMPLEDB
This directory contains the sample list files (*.SPL) that contain the run summary
information. It is recommended that an individual SPL file is created for each batch
(approximately one plate or 100 samples) as the SPL filename is used as a prefix for
the NeoLynx Results files generated by the NeoLynx processing.
Page 24
Sample Lists
The Sample List is part of the top level MassLynx screen. NeoLynx uses it to
prepare a list of samples for analysis. The list of samples is set up using a
spreadsheet style editor, which can be tailored to suit different requirements.
A NeoLynx Sample List format is supplied with the software. A Sample List format
is a definition of the columns to be used in a Sample List. To use the NeoLynx
format select Load Format from the MassLynx Samples menu, select NeoLynx
from the dialog displayed and press the OK button. See the MassLynx NT User
Guide for details.
Figure 2.4 The Load Sample List Format dialog.
Propagation of the sample list is available to input large numbers of samples, with
incremental descriptors, into the Sample List. A copy of a typical, propagated
sample list for this application is shown in Figure 2.5 .
Figure 2.5 Sample List Editor
Page 25
Full details on editing the sample list can be found in the Sample List section of the
MassLynx NT User Guide. The parameters used in the Sample List in this example
are:
File Name The raw data filename under which the data will be stored.
File Text The text that will be appended to each file. This may be used to
contain patient details.
MS File The Scan Function Editor file that will be used to control the
mass spectrometer during the data acquisition.
Inlet File The Inlet Control File that will be used to control the LC pump
and autosampler.
Bottle The location of the sample in the autosampler tray.
Inject Volume The volume of sample to be injected in microlitres. It is

recommended that the injection loop is over-filled for the best
reproducibility. E.g. for a 20µL loop, inject 25-30µL.
Sample Type The type of sample. This is used to differentiate the different
sample types when processing data. Available Sample Types are
Standard, Blank, QC and Analyte. If no sample type is entered
or Blank is selected then the sample will be processed as an
analyte. The Sample Type definition is assigned when data is
processed not acquired.
Process The type of processing that will be performed on the data. In this
case it should be NeoLynx.
Parameter File The name of the NeoLynx Test File (*.ntf) to use.
Process In NeoLynx 3.2 and 3.3 the parameters '4 4' had to be entered in
Parameters this column. In NeoLynx 3.5, this column must be empty.
For the MS File, Inlet File, Sample Type, Process and Parameter File fields files
created and saved in the appropriate directories of the current project can be
included in the sample list by double clicking on a cell, with the left mouse button,
and selecting the file from the drop down list displayed. See the MassLynx NT User
Guide for further details.
If the file required is in another project then click on a cell, with the left mouse
button, and enter the full path name.
If any of the required columns do not appear in the sample list press the toolbar
button (or from the Samples menu select Fields, Customize Display) and check the
boxes for the required fields.
The columns labelled Spare1 to Spare5 in the Sample List can be used to enter extra
information. This information can be used for variables in rule calculations or for
printing on reports.
Once the Sample List has been prepared, it must be saved (File, Save or Save As)
before continuing.
Page 26
Important Note: The name of the Browser Report file generated is based on the
Sample List Name. E.g. Sample List Neo1.SPL will create a Browser Report file
called Neo1.nrf. It is therefore essential that Sample Lists are saved with different
names to ensure that Browser Report files are not overwritten. The Sample List
name must not contain spaces.
To Start a Sample List Run
Select Start from the Run menu or press the toolbar button. This will display
the dialog box shown in Figure 2.6 .
Figure 2.6 The Start Sample List Run dialog for processing only (Acquire Data box
not checked)
The Project box shows where the acquired data will be stored, or from which
directory the previously acquired data will be retrieved for processing. If this is
incorrect you must press the Cancel button and select the correct project before
starting the Sample List run again (see the Getting Started section of the MassLynx
NT User Guide).
The Acquire Sample Data box allows the user to specify whether or not data is to
be acquired. Do not check this box if only post-processing of previously acquired
data is to be performed.
The Auto Process Samples box allows the user to specify whether or not data is to
be processed. Checking both this box and the Acquire Sample Data box will
acquire and process the data automatically. Do not check this box if only data
acquisition is to be performed.
The Auto Quantify Samples box is used to automatically Quantify the results and is
not applicable to the NeoLynx process.
Page 27
The Run from Sample n To Sample m boxes automatically default to the first and
last samples in the current Sample List. It is possible to alter these values, to select a
smaller range, by typing new values into the relevant boxes, or to highlight a range
of samples in the Sample List with the mouse before displaying the dialog.
Priority Checking this box will flag the Sample List as a priority process and put it
above all non-priority lists in the queue.
Night Time Process Check this box if the sample list is to be run at night.
Process allows for routines to be executed before, during and after the Sample List
has been run. For the NeoLynx application there are no pre- or post-run processes.
Pressing OK will add the Sample List to the Sample List queue on the mass
spectrometer. The queue appears at the bottom of the MassLynx screen and allows
the user to delete, prioritise and pause Sample Lists. See the Getting Started
section of the MassLynx NT User Guide for more details.
Page 28
Scan Function Editor
MassLynx allows for up to eight separate scan functions to be acquired. It is

recommended that data acquired from the mass spectrometer is separated into
discrete functions, that relate to a specific class of metabolites and their related
internal standards. It is possible to include full-scan MCA functions with MRM
functions. For a full explanation of the Function List Editor, see the MassLynx NT
Guide to Data Acquisition.
Scan Functions for Butyl Ester Derivatives
The following experiments have been described in the literature, and are included as
a guide only. It is recommended that tuning and collision conditions are optimised
for each compound on each instrument.
Compound Class Mass range Cone Collision Polarity Scan

Function Voltage Energy Time
Acylcarnitines Parents of m/z m/z 200 - 500 35 25 +ve 3.0s
85.0
Neutral and acidic Neutral Loss of m/z 140 - 270 25 12 +ve 1.5s
amino acids 102.1 Da
Basic amino acids neutral Loss of m/z 160 - 270 28 25 +ve 1.0s
119.1 Da
Glycine and Alanine Neutral Loss of m/z 120 - 160 22 8 +ve 0.5s
56.0 Da
Arginine Neutral Loss of m/z 220 - 240 40 35 +ve 0.2s
161.1 Da
Argininosuccinic Daughters of m/z m/z 60 - 150 35 25 +ve 1.0s
acid 459.4
Full-Scan Acquisitions
A typical acquisition sequence from the MassLynx scan editor is shown in Figure
2.7 .
There are two functions to be acquired in full scan MCA mode.
1. Scans for the Parent ion of m/z 85.2 (acylcarnitines) for 30 seconds, followed
by:
2. A scan for a constant Neutral Loss of 102.1 Da (amino acids) for 30 seconds.
In this analysis, acylcarnitines and amino acids are detected sequentially. The exact
timing sequence will be determined by
(i) the size of the injection.
(ii) the flow rate of the mobile phase.
(iii) the length of the transfer tubing from the injector to the ion source.
Page 29
Figure 2.7 The scan function editor for sequential full-scan MCA acquisition
The acylcarnitines and amino acids can also be detected simultaneously in a looped
experiment, as in Figure 2.8 .
Figure 2.8 The scan function editor for simultaneous full-scan MCA acquisition
Parameters currently used for these scan functions are shown in Figure 2.9 ,
Figure 2.10 and Figure 2.11 , along with Collision gas pressure (Ar) = 2.0x10-3
mbar.
Figure 2.9 Scan function details for MRM scan (Function 1)
This will allow for the injection profile to be monitored for each sample.
Page 30
Figure 2.10 Scan function details for constant neutral loss scan (Function 2)
This will give a mass spectrum of all the butylated amino acids present in the sample.
Page 31
Figure 2.11 Scan function details for precursor ion scan (Function 2).
This will give a mass spectrum of the butylatedacylcarnitines present in the sample.
Note that, in order to maintain mass spectral resolution when scanning in Parent or
Constant Neutral Loss modes, a scan speed of between 100 and 150 m/z per sec is
recommended.
Page 32
MRM Acquisitions
If the analytical method only requires specific analytes to be monitored then a

multiple reaction monitoring sequence should be used. Each function within the
MassLynx scan editor is capable of monitoring up to 32 transitions. Again, it is a
requirement of NeoLynx that each function is dedicated to one class of analytes and
the associated internal standards.
Two functions used for the specific detection of acylcarnitines and amino acids are
shown below. The number of channels actually used in a particular analysis will
depend on the analytes of interest and the availability of internal standards.
Function 1 - For butylated acylcarnitines

Function type: MRM of 28 channels
Chan Reaction Dwell Cone Volt Col.Energy Analyte

1 : 218.20 > 85.00 0.05 35 25 Free carnitine
2 : 221.20 > 85.00 0.05 35 25 d3-carnitine (i.s.)
3 : 260.20 > 85.00 0.05 35 25 C2-carnitine
4 : 263.20 > 85.00 0.05 35 25 d3-C2-carnitine (i.s.)
5 : 274.20 > 85.00 0.05 35 25 C3-carnitine
6 : 288.20 > 85.00 0.05 35 25 C4-carnitine
7 : 300.20 > 85.00 0.05 35 25 C5:1-carnitine
8 : 302.20 > 85.00 0.05 35 25 C5-carnitine
9 : 316.20 > 85.00 0.05 35 25 C6-carnitine
10 : 318.20 > 85.00 0.05 35 25 C5-OH-carnitine
11 : 342.30 > 85.00 0.05 35 25 C8:1-carnitine
12 : 344.30 > 85.00 0.05 35 25 C8-carnitine
13 : 347.30 > 85.00 0.05 35 25 d3-C8-carnitine (i.s.)
14 : 370.30 > 85.00 0.05 35 25 C10:1-carnitine
15 : 372.30 > 85.00 0.05 35 25 C10-carnitine
16 : 400.30 > 85.00 0.05 35 25 C12-carnitine
17 : 426.40 > 85.00 0.05 35 25 C14:1-carnitine
18 : 428.40 > 85.00 0.05 35 25 C14-carnitine
19 : 444.40 > 85.00 0.05 35 25 C14-OH carnitine
20 : 454.40 > 85.00 0.05 35 25 C16:1-carnitine
21 : 456.40 > 85.00 0.05 35 25 C16-carnitine
22 : 459.40 > 85.00 0.05 35 25 d3-C16-carnitine (i.s.)
23 : 470.40 > 85.00 0.05 35 25 C16:1-OH-carnitine
24 : 472.40 > 85.00 0.05 35 25 C16-OH-carnitine
25 : 480.40 > 85.00 0.05 35 25 C18:2-carnitine
26 : 482.40 > 85.00 0.05 35 25 C18:1-carnitine
27 : 496.40 > 85.00 0.05 35 25 C18-carnitine
28 : 498.40 > 85.00 0.05 35 25 C18:1-OH-carnitine
Page 33
Function 2 - For butylated amino acids

Function type: MRM of 23 channels
Chan Reaction Dwell(secs) Cone Volt. Col.Energy Analyte

1 : 132.10 > 30.00 0.05 25 12 Gly
13 15
2 : 134.10 > 32.00 0.05 25 12 C N-Gly (i.s.)
3 : 146.10 > 44.00 0.05 25 12 Ala
4 : 150.10 > 48.00 0.05 25 12 d 4-Ala (i.s.)
5 : 162.10 > 60.00 0.05 25 12 Ser
6 : 172.10 > 70.00 0.05 25 12 Pro
7 : 174.10 > 72.00 0.05 25 12 Val
8 : 176.10 > 74.00 0.05 25 12 Thr
9 : 182.10 > 80.00 0.05 25 12 d 8-Val (i.s.)
10 : 188.10 > 86.00 0.05 25 12 Leu/Ile
11 : 191.10 > 89.00 0.05 25 12 d3-Leu (i.s.)
12 : 206.20 > 104.10 0.05 25 12 Met
13 : 209.20 > 107.10 0.05 25 12 d 3-Met (i.s.)
14 : 212.20 > 110.10 0.05 25 12 His
15 : 215.20 > 113.10 0.05 25 12 Cit
16 : 222.20 > 120.10 0.05 25 12 Phe
17 : 227.20 > 125.10 0.05 25 12 d 5-Phe (i.s.)
18 : 238.20 > 136.10 0.05 25 12 Tyr
19 : 242.20 > 140.10 0.05 25 12 d 4-Tyr (i.s.)
20 : 246.20 > 144.10 0.05 25 12 Asp
21 : 260.20 > 158.10 0.05 25 12 Glu
22 : 261.20 > 159.10 0.05 25 12 Trp
23 : 263.20 > 161.10 0.05 25 12 d 3-Glu (i.s.)
This acquisition would again be prepared in the Scan Function Editor and would be
similar to that seen in Figure 2.11.
Figure 2.12 Scan function editor for MRM acquisition
In the MRM analysis shown, the two functions are acquired in parallel, this means
that all the channels for the first function are acquired, immediately followed by the
channels for the second function, and so on. On completion of the acquisition of all
the functions, the acquisition returns to the start of the first function. The details of
the first function are shown in Figure 2.13 .
Page 34
Figure 2.13 MRM function details
When using an MRM acquisition, the data acquisition is initiated as soon as the
sample has been injected into the mobile phase passing into the electrospray source
of the mass spectrometer. The reaction chromatograms for each function are stored
in a single data directory, each in a separate data file. The total ion chromatogram
for the first function (acylcarnitines in this instance) is shown in Figure 2.14 .
24-Oct-1995, 03:01:24
TEST01 1: MRM of 28 Channels ES+
0.55 TIC
100
8.81e4
Area
0 Time
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20
Figure 2.14 Total ion chromatogram for acylcarnitine MRM function (time
abscissa)
Page 35
As can be seen from Figure 2.14 , the analyte passes from the injector to the ion
source in less than 20 seconds, lasting for approximately 30 seconds. In this time,
each reaction channel is tested approximately 10 times, and is sufficient to provide
good chromatographic definition. (Note a chromatogram is acquired even though no
separation takes place). The benefit of this type of acquisition is that the mobile
phase in the mass spectrometer may flow somewhat quicker (up to 40 µL/min) than
in the full-scan MCA acquisition (up to 10 µL/min), and the inter-sample time may
be reduced.
In both full scan and MRM acquisitions, it is recommended that the sample bolus is
sampled at least 7 times as it passes into the ion source. This is to average out any
dispersion (and 'chromatography') that may have occurred in the tubing between the
injection loop and the mass spectrometer.
Page 36
NeoLynx Data Processing
Data Processing with NeoLynx

Chapter 3
Overview
The intensity of a mass spectral peak in electrospray ionisation is proportional to the
concentration of the compound in the analysis. NeoLynx operates by calculating the
ratio of the intensity of the mass spectral peak for the analyte to the intensity of the
mass spectral peak for a pre-defined internal standard (e.g. d5-phenylalanine). If the
concentration of the internal standard is known, then the concentration of the analyte
may be calculated by simply multiplying the peak intensity ratio by the concentration
of the internal standard.
In addition to the calculation of simple peak intensity ratios, and thus analyte
concentrations, it is also possible to monitor individual peak intensities so as to
monitor the performance of the complete assay (extraction, derivatisation and mass
spectral analysis).
In recent publications, there has been considerable discussion relating to the utility of
complex ratios of analytes e.g. [Phe]/[Tyr]. It is possible to use NeoLynx to perform
such calculations.
All of the values reported by NeoLynx are compared to a set of maxima and minima
that are specific for each test performed. If the calculated peak intensity or peak
intensity ratio falls outside the acceptable limits, a user-defined message is returned.
NeoLynx uses a number of specific files for processing and viewing results.
NeoLynx Test Files (*.ntf) These files contain the ratio test information, spectral
(and chromatogram) processing information and test file modification history for
audit purposes.
NeoLynx Result Files (*.nrf) These files contain the results for the specified
Sample List, including the mass spectral and chromatographic data, the test results
and information about the NeoLynx Test File used to generate it.
NeoLynx Report Scheme (*.nrs) These files define the reporting options used by
the NeoLynx Browser for generating printed and electronic summary reports.
The NeoLynx Test Files and the NeoLynx Report Schemes are controlled by the
MassLynx Security system and may not be edited or saved without appropriate
permissions. The NeoLynx Test Files and NeoLynx Results Files are also encrypted
to prevent access by unauthorised personnel from a non-MassLynx application.
Page 37
Introduction
NeoLynx is an integral part of MassLynx that has been designed for the
post-processing of data acquired in a neonatal screening-type experiment.
NeoLynx requires that the analytes and their associated internal standards have both
been acquired in the same mass spectrometric function. Regardless of the type of
data (full-scan MCA, continuum or MRM), NeoLynx processes the data to give a
mass measured or combined centroided spectrum. NeoLynx automatically
recognises the type of data to be processed.
Processing information is stored with each sample in the NeoLynx Results file.
Once the centroided data has been prepared, NeoLynx calculates ratios of certain
peaks within the processed spectrum and tests the derived ratios against limits
defined in the NeoLynx Test file. A report is generated that contains a summary of
all the spectral information, processing data, tests performed and the results of the
ratio comparisons.
The NeoLynx Results file is saved to the root directory of the current project
(*.PRO) and can be viewed using the NeoLynx Browser (see the NeoLynx Browser
chapter).
Note: The name of the NeoLynx Results file generated is based on the Sample List
Name. E.g. Sample List Neo1.SPL will create a Browser Report file called
Neo1.nrf. It is therefore essential that Sample Lists are saved with different names to
ensure that Browser Report files are not overwritten. The Sample List name must
not contain spaces.
All the information required to calculate the peak intensity ratios and to generate the
report is defined in the NeoLynx Test File.
Page 38
NeoLynx Test File Editor

The NeoLynx Test File Editor can be accessed from the top level MassLynx screen
by selecting NeoLynx Test Editor from the NeoLynx menu or by pressing the
toolbar button.
Figure 3.1 NeoLynx Test File Editor
Warning
NeoLynx uses rule editor files in which the fields are separated by commas. In
certain countries it is common to use a comma instead of a decimal point, and a
period as a thousand separator (e.g. 1.0 → 1,0 and 1,000.23 → 1.000,23).
MassLynx is configured with the computers regional settings appropriately set in the
Control Panel. This must not be changed under any circumstances.
Security
If the User is not authorised to modify the NeoLynx Test Files, or if the NeoLynx
Test File is opened without MassLynx Security running, it will not be possible to
modify and save the file, and all relevant buttons will be greyed out.
Page 39
The Test Editor Toolbar

The Toolbar is displayed at the top of the test editor window and allows you to
perform some common operations with a single click of the appropriate Toolbar
button.
Press to create a new NeoLynx Test file.
Press to open an existing NeoLynx Test file.
Press to save the current NeoLynx Test file.
Press to copy the selected part of the table.
Press to paste the previously copied data onto the table. Note: The Paste area
must be the same size as the copied area.
Press to add a test.
Press to delete a test.
Press to view the Mandatory Mass table.
Press to view the Function Name table.
Press to view the Rule Definition table.
Press to view the NeoLynx Combine parameters.
Press to view the NeoLynx Mass Measure parameters.
Press to view the NeoLynx Peak Detect parameters.
Press to view the Processing Options.
Press to print the current NeoLynx Test file.
Press to display the About box.
Page 40
Getting Started
To Create a NeoLynx Test Editor File
Press the Toolbar button, or select New from the Test Editor File menu. A new
blank table is created.
Enter the required test data as described later in this chapter.
Press the toolbar button, or select Save or Save As from the Test Editor File
menu. Enter a name for the new Test Editor file and press OK.
The Test Modification History dialog will be displayed.
Figure 3.2 Test Modification History dialog
This dialog is displayed when a file is saved and is used to keep a record of changes
made to a test file. Comments added here can be viewed by selecting Modification
History from the NeoLynx Test Editor View menu.
Enter comments as required (up to 50 characters) and press OK.
To Open an Existing Test Editor File
Press the Toolbar button, or select Open from the Test Editor File menu.
Select the required NeoLynx Test Editor file (*.ntf) and press Open.
To Convert a Rule Editor File
NeoLynx V3.2 and V3.3 Rule files (*.rle) created in previous version of NeoLynx
can be converted into NeoLynx Test Files (*.ntf).
Press the Toolbar button, or select Open from the Test Editor File menu.
NeoLynx Test Files(*.ntf) are the default file types. Click on the arrow, at the
end of the Files of type box and select NeoLynx Rules Files (*.rle).
Select the required file and press Open to display the Convert RLE File dialog.
Page 41
Figure 3.3 Convert RLE file dialog
Before the table can be saved the Combine, Mass Measure and Peak Detect
parameters must be defined. Press the relevant buttons and enter values in the
dialogs displayed or press the Accept Defaults button to use the default values
listed below. Files cannot be saved until all the required fields have been
entered.
Combine Mass Measure
Average = 5:10 Background subtract selected

Peak separation = 1.000 Polynomial order = 30
Subtract = 2:3 Below curve (%) = 10.00
X = 1.000 Smooth selected
Peak width (Da) = 1.00
Peak Detection
Number of smooths = 2
Peak Selection = Savitzky Golay selected
Largest Peak in Window Min peak width at half height
(channels) = 2
Mass Window (a.m.u.) = 1.00
Top selected
The following parameters on the Options dialog are also defined.
Process Options tab

Pass Message = All Tests Within Limits
Output File = Up Issue
Create MassLynx Process selected
Variables tab
Mass 1 = 111.1 X=3
Mass 2 = 222.2 s1 = 10
Mass 3 = 333.3 s2 = 20
Mass 4 = 444.4 s3 = 30
IS Conc = 1 s4 = 40
RRF = 2 s5 = 50
Page 42
The Function Table, Mass Table, Rule Table and Options parameters can also be
defined by pressing the relevant button and entering values in the dialogs displayed.
All dialogs are described in detail later in this chapter. Values can be modified after
the table has been saved.
When the required values have been defined press the Save As button, enter the
name of the files and press Save.
The Test Modification History dialog is displayed, enter comments as required (up
to 50 characters) and press OK.
Test Modification History
The Test History provides an audit trail for NeoLynx Test File modifications and is
displayed by selecting Modification History from the Test Editor View menu.
Figure 3.4 NeoLynx Test History dialog
The Modification History contains:
• the date and time of modification
• the action performed
• the name of the operator logged on to MassLynx
• a comment entered in the Test Modification History dialog.
The modification history is stored as part of the NeoLynx Test file.
Page 43
Processing Options
The Process Options dialog may be accessed by pressing the toolbar button or
selecting Options from the Test Editor Processing menu. This dialog contains
information relating to the automatic generation of printed and electronic reports.
Print Options Page
Figure 3.5 The Processing Options: Print Options page
Run Scheme after batch Check this box to generate printed reports and electronic
reports on completion of each Sample List (if NeoLynx processing is specified).
Results Browser This defines the location of the NeoLynx Browser program. Press
the Browse button, locate the NeoBro.exe program, highlight it and press Open to
define the path. By default this is the C:\MassLynx directory.
Report Scheme This defines the location of the NeoLynx Report Scheme used to
generate the printed and electronic reports. Press the Browse button, locate the
required *.nrs file, highlight it and press Open to define the path. By default this is
the C:\MassLynx directory. See the NeoLynx Browser chapter for details on Report
Schemes.
Page 44
Process Options Page
Figure 3.6 The Processing Options: Process Options page
Pass Message Enter the text to be displayed in the Browser Report if the results for
all tests lie within normal limits.
Output File Select Overwrite or Up Issue. NeoLynx processes raw data files and
creates a NeoLynx Results file whose name is based on the Sample List name.
If Overwrite is selected then any existing Results File with the same name will be
overwritten.
If Up Issue is selected then the file name will have an issue number added to it (e.g.
filename_001.nrf. Each time a report with the same name is generated this number is
increased by 1. The maximum number of issues is 999, after which a warning
message is generated.
Save Options In NeoLynx V3.5 the processed data and the processing parameters
are stored in the NeoLynx Results File. To save this information as a process in the
data file check the Create MassLynx Process box. This option is provided to
provide back-compatibility with previous versions of NeoLynx, and is not required
for NeoLynx 3.5 unless specified in the laboratory protocol.
Data Threshold %BPI Use the arrows to enter a data threshold value. Any
peak that is less than this percent of the base peak intensity will not be stored in the
NeoLynx Results file. Range 0.0 to 3.0%. Setting a threshold value of 1.0%
(depending on the noise of the system) can reduce the size of the NeoLynx Results
File significantly. The threshold value does not affect the calculated data in the
NeoLynx Results file, only the spectral display data.
Page 45
Variables Page
Figure 3.7 The Processing Options: Variables page
This dialog is used to define values for evaluating the results of a rule, and testing
the mathematical validity of a rule expression. See Test on page 54 for more details.
Page 46
Function Names Table
NeoLynx provides a feature where the names of the mass spectrometer's functions
can be defined. This is for Test Editor display only purpose and does not relate
interactively with the MS Method editor definitions. Once completed, the functions
can be selected from a drop down list box for insertion into a NeoLynx Test File. To
access the Function Names dialog press the toolbar button or select Function
Name Table from the View menu.
It is possible to set the scaling for the spectra to be viewed in the NeoLynx Browser.
For each function enter a mass and a nominated intensity (Percent). E.g. for
consistency, it may be required to set the intensity of an internal standard peak to be
50% of the mass spectral scale. If these parameters are set to zero, default spectral
scaling will be used.
The Function Names Table must be defined before the Mandatory Mass Table as
Mandatory Masses cannot be defined without a Function.
Figure 3.8 Function Names dialog
To Add a Function Name
1. Enter a function Number and Function name.
2. Press the Add button, the details will be added to the table.
To Modify a Function Name
1. Position the cursor on the entry to be modified. This can be done by clicking
on the field, with the left mouse button, or by using the arrow keys.
Page 47
2. The current details are displayed in the Number and Function fields. Change
the function name as required.
3. Press the Add button and the entry will be updated in the table.
Note: If the function Number is changed then a new entry will be added to the table.
To Delete a Function Name
1. Position the cursor on the entry to be deleted. This can be done by clicking on
any field in the row, with the left mouse button, or by using the arrow keys.
2. Press the Delete button and the entry will be deleted from the table.
Page 48
Mandatory Mass Table
To access the Mandatory Masses dialog press the toolbar button or select
Mandatory Mass Table from the View menu.
Figure 3.9 Mandatory Masses dialog
The Mandatory Mass Table is used to define peaks that must be found by the
NeoLynx processing algorithms. For example, peaks corresponding to internal
standard compounds should be present in each sample and labelled as mandatory.
Masses defined as mandatory are displayed in bold in the Test Editor table.
Page 49
Peak Detect
A mandatory mass will only be defined as being missing if it falls below a threshold
of the base peak intensity. This can be defined by selecting the Peak Detect button.
Figure 3.10 - Peak detection dialog for Mandatory Mass
Peak Selection Select Largest Peak in Window or Nearest Peak to Centre from
the drop down list box.
Mass Window Enter the window, around the mandatory mass, to look for the peak
in.
Intensity Threshold Enter the percentage intensity of the base peak, below which
the mandatory mass will be flagged as being absent.
If the processing parameters are correct there should only be one peak in the window
of interest. The software will inspect the Mass Window looking for the largest peak
or the peak nearest to the centre. If the intensity of the peak is above the Intensity
Threshold it will be marked as found, if not it will be marked as missing and the
Mandatory Mass Message will be displayed.
Mandatory Mass Message
The Mandatory Mass Message is generated if a mandatory mass peak is not found.
This message can be one of the following:
• A plain text message (e.g. Mandatory Mass Missing).
• A message containing the name defined in the compound field. Enter

%Compound% as in Figure 3.9 .
• A message containing the mass defined in the mass field. Enter %Mass% as in
Figure 3.9 .
To Add a Mandatory Mass
1. Enter a Compound name, the Mass of the compound and the Function to be
associated with this mass.
Note: A Mass and Function combination must be unique.
A Mandatory Mass can also be added by clicking, with the right mouse button, on a
mass in the Test Editor table and selecting Mandatory Mass Table from the pop up
Page 50
menu displayed. The Mandatory Mass Table dialog is displayed with the mass and
function copied from the Test Editor table. Enter the compound name and press the
Add button.
To Modify a Mandatory Compound Name
Position the cursor on the entry to be modified. This can be done by clicking on the
field, with the left mouse button, or by using the arrow keys.
The current details are displayed in the Compound, Mass and Function fields.
Change the compound name as required.
Press the Add button and the entry will be updated in the table.
Note: If the Mass or Function is changed then a new entry will be added to the table.
To Delete a Mandatory Mass
Position the cursor on the entry to be deleted. This can be done by clicking on any
field in the row, with the left mouse button, or by using the arrow keys.
Press the Delete button and the entry will be deleted from the table.
Page 51
Rule Definition Table
To access the Rule Table dialog press the toolbar button or select Rule Table
from the View menu.
Figure 3.11 Rule Table dialog
The Rule Definition Table is used to define the calculations performed for a rule.
# The number of the Rule. Note: Rules 0, 1 and 2 are pre-defined and correspond to
the Rules defined in NeoLynx V3.3. For these three rules only the rule name,
multiplier and units fields can be modified.
Rule A user-definable name that makes selection of the different rules in the
NeoLynx Test Table possible from a drop-down menu with easy reference.
Concentration formula The equation used as the test on the mass spectral data.
The result of this equation is tested against the Min Conc and Max Conc fields in
the NeoLynx Test Editor table. Available variables: m1, m2, m3 and m4 for the
masses of interest. Three variables (IS, RRF, X) are defined in the NeoLynx Test
Editor table, and five variables (s1, s2, s3, s4 and s5) are available from the Sample
List fields Spare1 - Spare5. All mathematically valid equations may be used.
Units - User-defined units used on reports.
Page 52
To Add a Rule
1. Enter a rule Number, Rule description, Concentration Formula and Units

value in the appropriate fields.
To Modify a Function Name
1. Position the cursor on the entry to be modified. This can be done by clicking
2. The current details are displayed in the Number and Function fields. Change
the function name as required.
3. Press the Add button and the entry will be updated in the table.
Note: If the function Number is changed then a new entry will be added to the table.
To Delete a Function Name
1. Position the cursor on the entry to be deleted. This can be done by clicking on
2. Press the Delete button and the entry will be deleted from the table.
Variables
Figure 3.12 Variable Settings dialog
This dialog is used to define values for evaluating the results of a rule, and testing
the mathematical validity of a rule expression. Enter values similar to the ones
expected and press OK. Press the Test button on the Rule Table dialog to see the
results of the equation.
The values used to derive the results saved in the NeoLynx Report file are those
defined in the NeoLynx Test File table (IS, RRF and X columns) and/or the
Sample List (Spare1-5).
Page 53
Test
Figure 3.13 Evaluated Expression dialog
Press the Test button to check the results of a Rule. This operation tests the
mathematical validity of the Ion Ratio and the Multiplier and checks that the syntax
of the variables is correct. The Evaluated Expression dialog is displayed showing
the result of the rule using the values defined on the Variable Settings dialog.
Page 54
Specifying Parameters
The columns in the NeoLynx Test Editor must be completed by the user, as they are
dependent on the metabolites being analysed and the internal standards used.
When a new Test Editor file is created the Mass fields and the IS, RRF and X fields
are greyed out. The Mass fields default to 0.00 and the others to 1.00. When a Rule
is selected for a test the fields required to perform the calculation for the rule will no
longer be greyed out (See Figure 3.14 below). Values must be entered in these
fields.
Figure 3.14 Required fields
To Add a Test
Press the toolbar button, select Add Test from the Test Editor Edit menu or
click, with the right mouse button, anywhere in the table and select Add Test
from the pop up menu displayed.
Figure 3.15 Pop up menu
This will add a new blank line at the bottom of the table. Until all values for
the test are correctly defined the number of the test is colored red and has a
line through it (strikethrough) e.g. .
Enter data as required. See below for an explanation of the fields. When all relevant
fields have been defined the test number turns grey and the strikethrough is
removed.
Note: The table should then be saved by selecting Save or Save As from the Test
Editor File menu.
Note: A maximum of 255 tests can be defined in one grid.
To Modify a Test
1. Position the cursor on the field to be modified. This can be done by clicking
Enter data as required. See below for an explanation of the fields.
Note: The table should then be saved by selecting Save or Save As from the Test
Editor File menu.
Page 55
To Delete a Test
2. Position the cursor on the test to be deleted. This can be done by clicking on
3. Press the toolbar button, select Delete Test from the Test Editor Edit
menu or click, with the right mouse button, anywhere in the table and select
Delete Test from the pop up menu displayed. This will delete the selected
row.
Editing Data in a Cell
For the Rule and Function cells double click, with the left mouse button, on the cell
and select a value from the drop down list box displayed. These values are defined
in the Rule Definition and Function Name tables.
For all other fields to overwrite data in a cell click on the cell, with the left mouse
button, and type in a new value. If data for the same field type has been copied, by
pressing the toolbar button or Copy from the Edit menu, then the value can be
pasted into the current cell by pressing the toolbar button or selecting Paste
from the Edit menu.
To edit data within a cell (e.g. to change C4-carnitine a to C4-carnitine b) double

click, with the left mouse button, on the cell. All data in the cell is highlighted. Use
the left mouse button or the keyboard arrow keys to position the cursor within the
cell and change values as required.
Data from a single cell or multiple cells can be copied and pasted to other parts of
the table. Areas may be selected with the mouse or the keyboard.
With the Mouse
To select Click with the left mouse button on
A single cell The required cell
A block of cells The first cell in the block, hold down the left mouse
button and drag until the required cells are highlighted.
A row The row number
With the keyboard
Use the arrow keys to position the cursor at the top left corner of the area to be
selected, hold down the shift key and use the arrow keys to select an area.
When the required area has been selected press the toolbar button or select
Copy from the Edit menu. Move to the first cell of the paste area (using the arrow
keys or by clicking on it with the left mouse button) and press the toolbar button
or select Paste from the Edit menu. Note: The paste area must be of the same type
as the copy area. I.e. if a Min Conc is copied it can only be pasted to another Min
Conc cell.
Page 56
• Column ordering priorities
The tests within a test table may be ordered in ascending or descending order. The
Function column MUST be ordered first, in ascending or descending function
number order. Up to five more columns can be ordered within the function number
ordering.
To select the ordering for a column, click with the left mouse button, on the row
below the column header. This will set the priority in ascending alphabetical or
numerical order and 1° Asc, 2° Asc etc will be displayed. Click again to change the
ordering to a descending alphabetical or numerical order (1° Desc, 2° Desc etc will
be displayed).
When a new Test file is created the default ordering parameters are:
Name The name of the test to be performed. Usually the name of a

specific analyte, e.g. C3 carnitine. Test names must be unique, e.g.
C4-carnitine a, C4-carnitine b, etc.
Rule The type of measurement to be performed. Three rules are already

defined when a new table is created:
0:Absolute Intensity = use absolute peak intensities from the

spectral data
1:2 Peak Ratio = determine the peak intensity ratio

(Mass 1 / Mass 2)
2:4 Peak Ratio = determine the peak intensity ratio

((Mass 1 / Mass 2)/ (Mass 3 / Mass 4))
New ones can be added as required.
Function The number and description of the mass spectral function within the
datafile to be processed. E.g. Parents of m/z 85 (acylcarnitines) and
Neutral Loss of 102 Da (amino acids) may be functions
1:Acylcarnitines and 2:Neutral AA, respectively.
Min Conc The minimum concentration below which a message will be

displayed.
Max Conc The maximum ratio or absolute peak intensity (Ion Ratio) above
which a message will be displayed. Remember that the ratios will
probably be small (2, 1, 0.2, etc.) and that absolute intensities will
be quite high (e.g. >104).
Note: Max Conc must exceed Min Conc.
Mass1 The first mass of interest. This value must be greater than 20.
Mass2, 3, 4 When a new Test Editor file is created the Mass fields are greyed
out. When a Rule is selected for a test the fields required to
perform the calculation for the rule will no longer be greyed out.
Values must be entered in these fields. This value must be greater
than 20.
MsgLow Message written to the result column of reports if the absolute
Page 57
intensity or ratio falls below the Min Conc value.
MsgHigh Message written to the result column of reports if the absolute

intensity or ratio lies above Max Conc value.
MsgOK Message written to the result column of reports if the absolute

intensity or ratio lies between Min and Max Conc values.
IS The concentration of the Internal Standard. A value must be

entered, the default is 1.
RRF The relative response factor, to be used if an internal standard is

used that is not an analogue of the analyte of interest.
X A user definable variable that may be used in individual test

calculations.
NeoLynx has inbuilt checking routines which verify certain criteria within a rule. If
erroneous values are entered the number of the test turns red and has a line through it
(strikethrough). An error message is also displayed as a tool-tip when the cursor is
placed on the highlighted test number.
Page 58
Processing
The Processing menu on the top line of the NeoLynx Test Editor allows access to
the Combine and Mass Measure and Peak Detection parameters. The parameters
defined here are stored with the individual NeoLynx Test Files and are unique to a
test file.
Mass Measure
Full-scan MCA data is prepared by performing a single Mass Measure operation on

the raw data. Mass measure allows the operator to perform mass measurement of a
continuum (or MCA) spectrum in one command. Mass measure performs peak
centering with optional background subtraction and/or smoothing.
The type of data acquired in a full-scan MCA experiment is shown in Figure 3.16
for constant neutral loss of 102 Da.
08-Aug-1994, 15:48:
MAG1 1 (2.222) Sm (SG, 2x1.00); Sb (30,10.00 ) 2: Neutral Loss 102ES
172.0 2.48e5
100
191.0
134.0 150.0 227.1

% 146.0
188.0 242.0
209.0 260.1
132.0 182.1 238.0 263.1
161.9 192.1
212.0 243.1
0 m/z
120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
Figure 3.16 Constant Neutral Loss (102 Da) for an amino acid mixture after
background subtraction and smoothing
The data shown in Figure 3.16 has been partially processed. In order for NeoLynx
to be able to compare peak intensities, it is necessary to further process the data in
order to generate a centroided spectrum as shown in Figure 3.17 .
08-Aug-1994, 15:48:
MAG1 1 (2.222) Cn (Top,4, Ht); Sm (SG, 2x1.00); Sb (30,10.00 ) 2: Neutral Loss 102ES
172.0 2.48e5
100
191.0
134.0 150.0 227.1

% 146.0
188.0 242.0
209.0 260.1
132.0 182.1 222.0 238.0 263.1
162.0 192.1 243.0
212.9 264.1
0 m/z
120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
Figure 3.17 The same spectrum as shown in Figure 3.16 after centroiding
In Mass Measure, Background subtraction takes place if the Background subtract

control is checked. The Mass Measure dialog gives access to the Polynomial order
Page 59
and Below curve (%) parameters which are described in the Background Subtract
section of the MassLynx NT User Guide.
Mean smoothing takes place if the Mean smooth control is checked. The Mass
Measure dialog gives access to the Peak width, Number of smooths, Mean, and
Savitzky Golay parameters which are described in the Smoothing section of the
MassLynx NT User Guide.
Peak Centering always takes place when the Mass Measure process is performed.
The Mass Measure dialog gives access to the Peak width at half height, Top, and
Centroid top parameters which are described in the Center section of the MassLynx
NT User Guide.
The Mass Measure dialog box always retains the last set of parameters used.
When preparing data for analysis by NeoLynx, the Mass Measure parameters shown
in Figure 3.18 are recommended. To access the dialog press the toolbar
button or select Mass Measure Parameters from the Processing menu.
Figure 3.18 Mass measure dialog
Page 60
Combine
For MRM and Continuum acquisitions, in order to prepare a spectrum that can be
analysed by the NeoLynx routine it is necessary to combine the spectra from across
the chromatogram peak and to subtract any background noise that may appear on
either side of the chromatographic peak.
24-Oct-1995, 03:01:24
TEST01 1: MRM of 28 Channels ES+
0.55 TIC
100
8.81e4
Area
0 Scan
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Figure 3.19 Total ion chromatogram for acylcarnitine MRM function (scan number
abscissa)
Note: The figure above displays the same data as Figure 2.14 but on a Scan rather
than Time axis. To change the axis select View from the Chromatogram Display
menu and select the required Axis Label.
The Combine Spectrum dialog is shown in Figure 3.20 . To access it press the
toolbar button or select Combine Parameters from the Processing menu.
Three Scan ranges are specified, and a Background factor (X). The Average
range contains the scans of interest across the chromatographic peak top. The
Subtract ranges contain scans from the background of the chromatogram, on either
side of the peak of interest. Typical values for the chromatogram shown in Figure
3.19 are shown in the dialog box in Figure 3.20 .
Figure 3.20 Combine spectrum dialog
Page 61
Values can be entered by:-
• Typing values into the Average and Subtract boxes.
• Clicking with the right mouse button and dragging across a chromatogram peak
at half height to enter the Average value, and then clicking and dragging either
side of the peak to enter the Subtract ranges. The software will automatically
enter these values in the correct boxes. Note: Ensure that the Multiple
Average box is not checked as the first six ranges are automatically entered in
the Average box and the next two in the Subtract box.
The scans across the peak top (scans 6 to 13) are averaged together, and from the
result is subtracted the average of all the specified background scans (scans 2 to 4
and scans 17 to 19) multiplied by the Background factor (typically 1.000).
The Peak separation parameter is the spectral peak width in amu. For the MRM
data acquired in this application, the peak separation should be set to 1.0.
A combined spectrum is shown in Figure 3.21 and contains the ions of interest, as
specified in the original scan function. Note: The appearance of the data is similar
to Figure 3.17 .
24-Oct-1995, 03:01:
TEST01 9 (0.553) Cm (6:13-(2:4+17:19)) 1: MRM of 28 Channels ES
260.3 1.06e4
100
263.3 459.5 482.5
347.4
%
374.4 456.5
484.5
274.3 480.5
342.4 372.4
288.3302.3316.3 388.4 400.4424.5428.5 454.5 472.5 498.5
0 m/z
260 280 300 320 340 360 380 400 420 440 460 480 500 520
Figure 3.21 The combined spectrum derived from the acylcarnitine MRM function
Page 62
Peak Detection
Figure 3.22 Peak Detection dialog
To access this dialog press the toolbar button or select Peak Detection
Parameters from the Processing menu.
Peak Selection is the method by which NeoLynx selects the centroided peak of
interest. The choices are:
Largest peak in window: select the largest peak within the Mass window
around the mass specified in the test editor.
Nearest to center: select the nearest peak within the Mass Window to the
mass specified in the test editor.
This option was included in the event that the smoothing and peak centering
parameters were incorrectly set. The peak of interest will most likely be the largest
(and only) peak in the window. The default setting is for the largest peak in the
window.
Mass window (a.m.u.) is the mass window that is used to look for centered peaks
around the specified mass of interest. In this application, a window of 1.0 Da will
give ±0.5 Da around the target mass of interest.
Page 63
Print Control
Printing of sample results reports is now controlled from the NeoLynx Browser.
The Print Control dialog allows access to the printing options available with the
NeoLynx Test Editor. To access it press the toolbar button or select Print from
the File menu.
Figure 3.23 Print Control dialog
The report options dialog is used to control the type of reports sent to the printer.
Check the relevant boxes to print the required information.
In order to fit the Test Editor Table on one page it is recommended that the default
paper orientation is set to Landscape in the Print Setup dialog. Some of the
columns may also need to be made narrower for all the fields to be printed. To do
this position the cursor on the line between the column headings until ↔appears,
hold down the left mouse button and drag until the column is the required width.
The appearance of the report may be viewed by selecting Print Preview from the
File menu.
Page 64
NeoLynx Browser
NeoLynx Browser
Chapter 4
Overview
Processing of samples using the NeoLynx algorithm results in a NeoLynx Results
file (*.nrf) being generated. This file is created in the root directory of the project in
which the data are stored. The NeoLynx Results File is viewed using the NeoLynx
Browser.
The NeoLynx Results files contain:
• All sample-related results
• All reduced mass spectral data used to generate the results
• All chromatographic data used to generate the results (if available)
• Complete NeoLynx Test File(s) used during the processing
• Sample-related information
The title bar of the NeoLynx Browser displays the name of current NeoLynx Results
File (*.nrf) and the name of the currently active NeoLynx Report Scheme (*.nrs).
The Report Scheme controls the type of printed and electronic reports generated.
Accessing the NeoLynx Browser
To access the NeoLynx Browser press the toolbar button or select NeoLynx
Results Browser from the NeoLynx menu on the top level MassLynx Screen.
Page 65
NeoLynx Browser
The NeoLynx Browser Screen
Figure 4.1 The NeoLynx Browser
The screen is split into 7 panes. The Chromatogram Pane may not be displayed,
depending on the type of data acquired.
• The Plate pane showing the plate layout and tested wells.
• The Results Table pane showing a list of detected compounds.
• The Function pane showing a list of the functions used to acquire data.
• The Results Summary pane showing the Abnormal messages or Pass Message.
• The Sample Description pane showing sample information for the currently
selected vial.
• The Spectrum pane showing the spectrum for the vial and test selected.
• The Chromatogram pane showing the chromatogram for the vial and test
selected. Note this is not shown if chromatogram data are not available.
Page 66
NeoLynx Browser
The NeoLynx Browser Toolbar

Toolbar Menu equivalent Purpose
button
File… Open Open an existing NeoLynx Browser report
File… Print Print the results from a NeoLynx Browser

file using the specified NeoLynx Report
Scheme
File… Save Electronic Generate electronic reports using the

Reports specified NeoLynx Report Scheme
NeoLynx Browser go to first Vial
NeoLynx Browser go to previous Vial
NeoLynx Browser go to next Vial
NeoLynx Browser go to last Vial
Display previous plate
Display next plate
Display default range
Display Fails Only
Display Samples sequentially
File… Report Scheme Invoke Edit Report Scheme Settings dialog

Settings
Help… About NeoLynx Display program information, version

Results Browser number and copyright
Getting Started
To Open an Existing NeoLynx Browser File
1. Press the Toolbar button, or select Open from the File menu.
2. Select the required NeoLynx Browser results file (*.nrf) and press Open.
Page 67
NeoLynx Browser
View Options
A number of options are available to change the appearance of the Browser screen,
to access them select Options from the View menu.
The Spectrum Page
Figure 4.2 The Spectrum Page
Peak Annotation Decimal Places Change this value, by pressing the arrows, to
change the number of decimal places displayed on peak annotation. Range 0 to 4.
Horizontal Axis Decimal Places Change this value, by pressing the arrows, to
change the number of decimal places displayed on the horizontal axis. Range 0 to 4.
Daltons Label Type in the text to appear as the label for the horizontal axis, for
mass spectral data. Maximum length is 8 characters.
Default range to data When checked the default mass display range of a spectrum
will be based upon the peaks the spectrum contains, only the mass range which
actually contains data will be displayed.
If unchecked the default display range will be the actual mass range the spectrum
was originally acquired over.
Process description Check this box to display the Process Description in the top
left corner of the Spectrum. This describes the processing used to derive the
displayed spectrum from the raw data.
Select all Spectra If this box is checked then when a new vial is selected all
functions are selected in the Functions pane and all spectra for the vial are displayed.
Individual spectra can be displayed by clicking on the test in the Function or Test
Results panes.
Page 68
NeoLynx Browser
Lock spectra If this box is checked, the spectra will be locked to the mass and
intensity defined for the individual functions in the NeoLynx Test File. In such a
situation, the nominated peak(s) will be set to the pre-defined intensity on the
display, and all other peaks will be scale accordingly - be aware that some peaks may
be sturated in this display mode.
Detected peak colour Allows for selection of the colour of the peaks in the
spectrum display that have been used in the NeoLynx calculations.
The Chromatogram Page
Figure 4.3 The Chromatogram Page
Peak Annotation Decimal Places Change this value, by pressing the arrows, to
change the number of decimal places displayed on peak annotation.
Horizontal Axis Decimal Places Change this value, by pressing the arrows, to
change the number of decimal places displayed on the horizontal axis.
Label Type in the text to appear as the label for the horizontal axis.
Show Combine Parameters Check this box to display the combine parameters
(Spectrum Average and Background Subtract) used to generate the Spectrum in the
Spectrum pane. The Combine parameters are also displayed on the chromatogram
(green = average, red = background subtract). The colors used to display the
combine and background subtract regions are user-selectable.
Page 69
NeoLynx Browser
The Default Plate Page
Figure 4.4 The Default Plate Page
Rows Enter the number of rows on the plate to be used. Range 1 to 32.
Columns Enter the number of columns on the plate to be used. Range 1 to 32.
Origin This is the corner of the rack that the vial grid referencing starts from.
Select one of Top Right, Top Left, Bottom Right or Bottom Left from the drop down
list box.
Method This is the method of numbering the vials on a plate. There are three
options
• XY which references the vials A1, B1 etc.
• Sequential Discontinuous which numbers the vials 1, 2, 3 across a row, left to

right if origin is top left, and then starts the next row from the left again.
• Sequential Continuous which numbers the vials 1, 2, 3 across a row, left to

right if origin is top left, then continues number the next row, right to left etc.
If a Gilson autosampler or a Waters 2700 autosampler is used with NeoLynx then the
vial referencing must be set to either sequential continuous or sequential
discontinuous.
Horizontal If the method chosen is X,Y then this box becomes enabled. It allows
horizontal referencing of the plate to be a number or a letter.
Vertical If the method chosen is X,Y then this box becomes enabled. It allows
vertical referencing of the plate to be a number or a letter.
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NeoLynx Browser
Horizontal Priority Check this box if samples are to be acquired horizontally

across the plate.
If Referencing = X,Y, Horizontal = Letter, Vertical = Number and

Horizontal Priority is checked, this will result in samples being acquired in the
order A1, A2, A3. If the Horizontal Priority box is not checked samples will be
acquired in the order 1A, 1B, 1C etc.
If Referencing = sequential continuous or discontinuous and Horizontal Priority

is checked, this will result in samples being acquired from row 1 then row 2. If
the Horizontal Priority box is not checked samples will be acquired from column
1 then column 2 etc.
Plate Display
For autosamplers controlled by the MassLynx software a plate layout will have been
defined (see the MassLynx Guide to Data Acquisition). This information is stored in
the raw data header file and cannot be changed. I.e. when the browser report is
opened the plate will be displayed in the format defined for the acquisition and not
the one defined on the Browser Default Plate page.
For autosamplers not controlled by the MassLynx software, this page is used to
organise the data acquired so that it can be displayed in plate format.
Sample information can be displayed in the format it was acquired or sequentially.

I.e. if data for 16 samples has been acquired on three different plates as shown
below, it can be viewed on the three separate plates by pressing the and
(previous and next plate) toolbar buttons.
Page 71
NeoLynx Browser
Pressing the (sequential samples) button will display the data sequentially in the
format defined on the Default Plate page. This type of display is useful for
examining incomplete runs (Sample(s) not tested) to determine the sequence number
of the missed sample.
Note: Sequential Format is displayed above the plate to indicate that the plate is
displayed in the sequential sample format.
Pressing the button a second time will return to the previous format.
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NeoLynx Browser
The Colors Page
Figure 4.5 The Colors Page
This page allows the user to define the colors used on the Plate pane. Select a color
from the drop down list box.
Standard Pass The sample was defined as Standard in the Sample List and passed
all tests defined in the NeoLynx Test File.
Standard Fail The sample was defined as Standard in the Sample List and failed
one or more tests defined in the NeoLynx Test File.
Analyte Pass The sample was defined as Analyte or Blank (or left blank) in the
Sample List and passed all tests defined in the NeoLynx Test File.
Analyte Fail The sample was defined as Analyte or Blank (or left blank) in the
Sample List and failed one or more tests defined in the NeoLynx Test File.
QC Pass The sample was defined as QC in the Sample List and passed all tests
defined in the NeoLynx Test File.
QC Fail The sample was defined as QC in the Sample List and failed one or more
tests defined in the NeoLynx Test File.
Unused – There was no sample defined for this position on the plate.
Page 73
NeoLynx Browser
The List Columns Page
Figure 4.6 The List Columns Page
This page allows the user to define which information is displayed in the Results
Table pane.
Check the boxes next to the fields required.
Columns can be removed from the display by clicking, with the right mouse button,
on a column and selecting the Remove Column pop-up menu which appears.
Columns can only be restored from the List Columns page.
Show failures only Check this box to show only failed tests. Pressing the
toolbar button will also perform this action, pressing the button a second time will
display all results again.
Function The number and name of the function defined in the Test.
Rule The number and name of the Rule that was applied in the Test. E.g. 1:2 Peak
Ratio = determine the peak intensity ratio (Mass 1 / Mass 2).
Target Mass 1-4 The Target Mass(es) defined in the Test. These will be 0.00 if not
used for the rule.
Result The message defined in the Msg Low, Msg High or Msg OK field for the
Test, depending on the result of the test.
Calculated Conc The Calculated Concentration defined in the Test.
IS Conc The concentration of the internal standard defined in the Test.
RRF The Relative Response Factor defined in the Test.
Page 74
NeoLynx Browser
Formula The complete formula (Ion Ratio * Multiplier) defined in the Test.
X The value of the X variable defined in the Test.
Detected Mass 1-4 The masses detected and used in the calculations.
High Conc The Max Conc defined in the Test.
Low Conc - The Min Conc defined in the Test.
Units The units defined in the Test.
Spare 1-5 The values entered in the Spare 1-5 columns in the Sample List.
Page 75
NeoLynx Browser
Other Display Options
To display two documents simultaneously
1. Select Open from the File menu, this will display a new window over the top
of the previous one.
2. Select Tile or Cascade from the window menu. This will display the two
child windows in the style chosen.
3. Repeat the above steps as often as required.
To display two different views of the same document
Select New Window from the Window menu. A copy of the current window
created, named “filename:2”. Select Cascade, Tile Horizontally or Tile Vertically
from the Window menu to display this new window along side the existing
document to compare results of similar searches.
To change the size of a pane
To change the size of the panes on display position the mouse pointer on the line
between the two panes until a or symbol appears. Hold down the left
mouse button and drag until the pane is the required size.
To change the size of the display
On a standard screen set to 800 by 600 pixels there may not be enough room to
display all the information in all of the panes. Changing the desktop settings will
display more information.
1. Press the Start button, select Settings and then Control Panel.
2. From the Control Panel select Display.
3. On the Settings page change the Desktop Area to 1024 by 768 pixels or
above.
4. Press the Test button and if the test page displays correctly press the OK
button.
Page 76
NeoLynx Browser
Other Menu Options

File Menu
Load on startup If this option is selected, from the File menu, the next time the
Browser is opened then the last Browser file viewed will be automatically loaded. A
tick mark appears next to the item when selected, selecting the option again will turn
it off.
Send To If this option is selected then an e-mail message is created with the
currently selected report attached to it. Enter the address to send it to and press the
send button.
Note to view the report the person receiving the e-mail must have MassLynx (with
the NeoLynx option) or a stand-alone copy of the NeoLynx Browser installed. The
attachment can then be saved as normal and then opened in the NeoLynx Browser.
It can also be opened by double clicking on the report in the e-mail message.
View Menu
Refresh This option rereads the current Browser Report and updates the display
information. This command should be used if the content of the Browser Report has
changed as a result of processing further samples. Pressing the F5 function also
performs this action.
Toolbar If this option is selected, from the View menu, then the Toolbar will be
visible. A tick mark appears next to the item when selected, selecting the option
again will turn it off.
Status Bar If this option is selected, from the View menu, then the Status Bar will
be visible. A tick mark appears next to the item when selected, selecting the option
again will turn it off.
Window Menu
New Window Selecting this item will create a copy of the current window. This is
useful for displaying different views of the same document. The second document
will have a ':2' after the name. To change between documents select the document
required from the documents listed at the bottom of the Window menu. A tick will
appear next to the document which is currently active.
Cascade Arranges document windows so that the title bar of each window is visible.
Tile Arranges open windows side by side on the screen, dividing the available space
equally between the open windows so that all of them are visible.
Arrange icons Arranges all iconised windows into rows.
Page 77
NeoLynx Browser
The Plate Pane
Figure 4.7 The Plate pane
The Plate Pane displays information about all the samples from the currently selected
plate. The vials are color coded to indicate whether they contained a sample which
passed all the tests or failed one or more tests defined in the NeoLynx Test File.
Different colours can be assigned to the different Sample Types defined in the
Sample List.
All Samples Tested is displayed if all the samples submitted for analysis have been
processed. If samples have not been tested, the message Sample(s) not tested is
displayed.
These colors can be changed see "The Colors Page" on page 73.
The currently selected vial is indicated by being shown recessed. Change the current
vial by clicking on a new vial, using the vial selection toolbar buttons ( , ,
and ) or arrow keys.
Clicking on a vial, with the left mouse button, will display detailed information about
that vial in the other panes of the screen.
The current plate number can be changed by using the previous plate and next
plate toolbar buttons.
The appearance of a plate can be modified using the View Options command. This
enables the number of rows and columns, reference labels and vial colors to be set.
See View Options on page 68 for more details.
Pressing the (sequential samples) button will display the data sequentially in the
format defined on the Default Plate page. See Plate Display on page 71 for more
details.
Page 78
NeoLynx Browser
The Results Table Pane
Figure 4.8 The Results Table pane
This pane is used to display information about the Tests performed. How to display
a field and the field descriptions are described fully in The List Columns Page on
page 74.
By default when a well is selected all results are displayed. To display only the
failed tests press the toolbar button, press the button a second time to return to
displaying all results. Failed tests are highlighted as in the example above.
Columns can be removed from the display by clicking, with the right mouse button,
on a column and selecting the Remove Column pop-up menu which appears.
Columns can only be restored from the List Columns page
The width of the columns can be changed, by positioning the mouse pointer on the
heading between two columns until the symbol appears, and then clicking the
left mouse button and dragging until the column is the required width.
The order in which the columns is displayed can be changed. Click on the column
heading, with the left mouse button, hold the button down and drag the column
header to the required position.
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NeoLynx Browser
The Function Pane
Figure 4.9 The Function pane
This pane shows a list of the functions used to acquire data. The description of the
function displayed in this pane is the MassLynx Scan Functions Editor definition, not
the description used in the NeoLynx Test File.
If all the Tests for this function, defined in the NeoLynx Test File, have been passed
a green tick appears next to the function, otherwise a red cross is displayed.
Click, with the left mouse button, on a function to display the results for this function
in the Results Table pane, the Spectrum Pane and the Chromatogram Pane, if active.
Multiple entries can be selected by holding Ctrl key and clicking on the required
entries or holding down the Shift key and clicking on the last entry in a block.
Selection of multiple functions will update the Spectrum, Chromatogram and Results
Pane with the selected functions.
The Results Summary Pane
Figure 4.10 The Results Summary pane
If any test has failed this pane shows the message defined in the MSG HIGH or
MSG LOW fields in the NeoLynx Test file. The Result highlighted corresponds to
the entry selected in the Results Table pane.
If no tests have failed the Pass Message defined in the NeoLynx Test File is
displayed (NeoLynx Test File Editor, Processing, Options, Process Options).
Page 80
NeoLynx Browser
The Sample Description Pane
Figure 4.11 The Sample Description pane
This pane shows the description of the sample for the well selected in the plate pane.
Sample The name of the Sample defined in the Sample List.
Type The Sample Type defined in the Sample List.
Acquired The name of the person logged on to MassLynx at the time of data
acquisition and the time of acquisition.
Processed The name of the person logged on to MassLynx at the time of data
processing and the time of processing.
The name of the NeoLynx Test File used to process the data is shown on the button
at the bottom of the pane. Pressing this button will display the relevant NeoLynx
Test File in read-only mode.
Note: When a NeoLynx Results File is created the NeoLynx Test File used to
process the acquired data is stored as an integral part of it. The file displayed by
pressing the button on this pane is the saved file and so any changes made to the
NeoLynx Test File after the Results File was created will not be displayed.
Page 81
NeoLynx Browser
The Spectrum Pane
Figure 4.12 The Spectrum pane
This pane displays the mass Spectra of the entry (or entries) highlighted in the
Function pane, the Results Table Pane or the Results Summary pane.
Peaks which match the detected peaks in the Results Table pane are displayed in
green.
The Spectrum Pane will appear blank if an unused vial is selected. If the pane is not
large enough to display all the selected spectra, a scroll bar automatically appears on
the right of the pane.
The text in red on the left of the pane shows the mass spectrum retention time. The
process description will also be displayed if the relevant box is checked on the View,
Options, Spectrum page.
The text on the right of the pane shows the acquisition function type and ionisation
mode followed by the absolute intensity of the largest peak in the spectrum, on the
next line.
Altering the range of the horizontal axis (zoom) with the mouse
Press the left mouse button at one end of the region of interest, and without releasing
the button, drag the mouse horizontally to the other end. As you drag the mouse you
will see a "rubber band" stretched out to indicate the range you have selected; don't
go beyond the bounds of the axis. When the mouse button is released the selected
range will be re-displayed to fill the current window.
This operation can be repeated as often as required.
Pressing the toolbar button will restore both the Spectrum and Chromatogram
display to the default range.
Viewing other spectra
Each entry can have one or more spectra associated with it. To view another
spectrum page down using either the scroll bar or the arrow keys.
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NeoLynx Browser
The Chromatogram Pane
Figure 4.13 The Chromatogram pane
This pane displays the processed chromatogram of the entry highlighted in the
Results Table pane. If chromatogram data is available the chromatogram pane is
always displayed beneath the spectrum pane. If a chromatogram is expected, but not
displayed, the NeoLynx Browser may have minimised the chromatogram window.
Position the cursor at the bottom of the spectrum pane until it changes to .
Click with the left mouse button and drag upwards until the chromatogram pane is
the required size.
The part of the chromatogram peak used for the combine process is displayed in
colour if Show Combine Parameters is checked on the View, Options,
Chromatogram page.
Each peak is annotated with the retention time. The text in red on the left of the pane
is the chromatogram description.
The text on the right of the pane is an indication of the maximum intensity of the
chromatogram.
Altering the range of the horizontal axis (zoom) with the mouse
Press the left mouse button at one end of the region of interest, and without releasing
the button, drag the mouse horizontally to the other end. As you drag the mouse you
will see a "rubber band" stretched out to indicate the range you have selected; don't
go beyond the bounds of the axis. When the mouse button is released the selected
range will be re-displayed to fill the current window.
This operation can be repeated as often as required.
Pressing the toolbar button will restore both the Spectrum and Chromatogram
display to the default range.
Viewing other chromatograms
Each entry can have one or more chromatograms associated with it. To view another
chromatogram page down using either the scroll bar or the arrow keys.
Page 83
NeoLynx Browser
Printing and Reporting

A variety of printed and electronic reports can be produced using the NeoLynx
Browser. Formatting of the outputs is described in this section, and examples of the
supplied electronic reports are shown in Appendix E.
To Print NeoLynx Results
1. Press the Toolbar button, or select Print from the File menu. This will
display the Print control dialog.
Figure 4.14 The Print Control dialog
Select Print Current Sample to print information for the currently selected vial.
Select Print All to print information for all vials on all plates.
Select Print Selection and select a From and To vial number from the drop down
list boxes.
Press OK to print the report.
The format of the reports is defined by the selected NeoLynx Report Scheme, which
is covered in detail later in this section.
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NeoLynx Browser
To Generate NeoLynx Electronic Reports
2. Press the toolbar button or select Save Electronic Reports from the File
menu. This will display the advice dialog.
Figure 4.15 Generate Electronic Reports: advice dialog
The Report Settings displayed will depend on the options selected in the NeoLynx
Reoprt Scheme. Pressing OK will result in the electronic reports being
generated.
This message is not shown when electronic reports are generated automatically
from a Sample List – Run Scheme after batch is checked in the NeoLynx Test
File, Print Options.
Note: If the current version of a report is open (e.g. being viewed in Notepad or
Excel) then the file will not be updated or overwritten. All closed files will be
updated.
Page 85
NeoLynx Browser
Report Schemes
Access to NeoLynx Report Schemes is controlled by the MassLynx Security
program. Schemes may not be edited and saved unless accessed by an authorised
operator. If unauthorised access is attempted, the following message will be
generated:
Figure 4.16 Warning dialog
To Open a Report Scheme
1. Press the toolbar button or select Report Scheme Open from the file
menu.
2. Select the required *.nrs file from the dialog displayed.
3. Press Open.
To Save a Report Scheme
1. Select Report Scheme Save As from the file menu.
2. Select the required location and enter a *.nrs filename in the dialog displayed.
3. Press Save.
Report Scheme Settings

The information on printed reports can be defined via the Report Scheme Settings
dialog. To access the dialog select Report Scheme Settings from the NeoLynx
Browser File menu.
The examples shown below use the NeoLynx.nrs report scheme supplied with the
NeoLynx software.
Page 86
NeoLynx Browser
The Report Control Page
Figure 4.17 The Edit Report Settings dialog, Report Control page
Print Reports
Check the Plate Summary box to produce a Plate report. This is a picture of the
plate with the symbols defined in the Plate Summary Symbols representing Passed,
Failed and Unused wells. Different symbols can be used for Standard, QC and
Analyte samples. See below for details, and Figure 4.19 for an example.
Check the Batch Summary box to produce a summary report for a Batch of
Samples. Note the number of samples printed is controlled from the Print Control
dialog (See To Print NeoLynx Results on page 84). For details on the content of this
report see
Page 87
NeoLynx Browser
The Batch Summary Page on page 89.
Check the Sample Report box to produce a more detailed report for a sample.
Electronic Reports
Reports can be saved in an electronic format and/or printed. Check the Report Title
boxes for the required reports and check the corresponding Print box to print a
copy.
Report Headings
Enter text to appear at the top of a report in the Report Header Text field. This text
will be displayed between the Report name and Submitter name if selected, see
below.
Check the Display report name box to display "NEOLYNX <report type> REPORT"
at the top of the report. If this text is not required make sure the box is unchecked
and enter your own text in the Report Header Text field described above.
Check the Display Submitter name box to display the name of the submitter. the
submitter is the name of the person logged on to MassLynx at the time of data
acquisition.
Check the Display Batch Status box to display either "All Samples Tested" or
"Samples Not Tested" in the upper right field to advise of the processing status.
Plate Summary Symbols
Enter a character into each field to be displayed on the Plate Summary of the printed
report.
Check the Report Symbol Key box to print a list of symbol definitions on the
report.
Sample Report Format
To print the spectra associated with a sample check the Spectra height (mm) box
and enter the height required.
To print the chromatograms associated with a sample check the Chromatogram

height (mm) box and enter the height required.
Page 88
NeoLynx Browser
The Batch Summary Page
A Batch Summary report is a tabulated list of all samples that have been processed.
Samples are grouped in sets of 96 (to reflect microtitre plate formats) and represent a
simple summary of the samples analysed. Information may be configured at the top
of the Batch Report (Batch Summary header), and the columns in the main body of
the report (Summary Data Fields). An example is shown in Figure 4.20
In a Batch Summary 'failed' samples are highlighted in bold type.
Figure 4.18 The Batch Summary Page
Batch Summary Header
This section defines the text printed in a Batch Summary Header. Twelve field areas
are available in the Batch Summary Header. The available fields are all batch
dependent, and may be selected from:
Acquire Date/Time The date and time of data acquisition.
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NeoLynx Browser
Instrument The name or serial number of the instrument as defined in the

MassLynx Control Panel.
Job Code The name of the Sample List from which the data were processed.
Lab Name The name of the Laboratory as defined in the MassLynx Control Panel.
Process Date/Time The date and time on which the data were processed.
Results Date/Time The date and time at which the NRF file was created.
Results File The path and name of the NRF file.
Submitter The name of the person logged on to MassLynx at the time of data
acquisition.
Total Samples The total number of samples processed in the reported batch.
User Name The name of the person logged on to MassLynx at the time of data
processing.
Free Text 1 - 12 Twelve fields in which user-defined comments may be added.
Batch Summary Data Fields
The data reported in the columns of the Batch Summary are sample-dependent, the
data fields available are:
Acquire Date The date on which the file was acquired.
Acquire Time The time at which the data file was acquired.
Description The text string defined in the File Text column of the Sample List.
Filename The name of the data file.
ID The ID number defined in the ID column of the Sample List.
Index The sequence number of the sample in the Sample List.
Pass/Fail Whether the sample has Passed or Failed the tests in the NeoLynx Test
File.
Process Date The date on which the data file was processed.
Process Time The time at which the data file was processed.
Sample Type The Sample Type, as defined in the Sample List.
Spare 1-5 Information from the Spare columns in the Sample List.
Test File The name of the NeoLynx Test File used to process the data.
Well The Well location of the processed sample
Page 90
NeoLynx Browser
To Add a Field
Click, with the left mouse button, on a field in the Available Fields box and press the
button. The new field is added to the bottom of the list.
To Remove a Field
Click, with the left mouse button, on the field to remove, in the Available Fields box
and press the button. The field will be removed from the list.
To Change the Order of Fields
Click, with the left mouse button, on the field to move, in the Available Fields box
and press the or button until the field is in the required position.
Field Alias
If the field name in the Available Fields list does not correspond to the description
the user will recognise, another name can be used by entering it in the Field Alias
box. E.g. Pass/Fail could be displayed on the report as State. This field may also be
used to display field names in another language.
To Change the Field Width
Enter a new value in the Width box. Note: this option is not available for the Header
fields.
NeoLynx Plate Report Generated by: Administrator All Samples Tested Page 1
Results File: PKU_MCA_001.nrf Total Samples: 34

Results File Created: 11-Sep-2000 09:49:01
Printed: Tue 10 Apr 2001 09:58:34
Plate Summary:
Symbols
Standard Pass 0
Standard Fail X
QC Pass 0
QC Fail X
Analyte Pass 0
Analyte Fail X
Not Used -
Plate 1
1 2 3 4 5 6 7 8 9 10 11 12
A 0 X X X X X X X 0 0 X X
B X 0 0 X X - - - - - - -
C 0 X X 0 X X X X 0 0 0 X
D X 0 0 X X - - - - - - -
F - - - - - - - - - - - -
G - - - - - - - - - - - -
H - - - - - - - - - - - -
Figure 4.19 A Plate Summary
Page 91
NeoLynx Browser
NeoLynx Summary Report Generated by: Administrator All Samples Tested Page 1
Total Samples: 34 Acquire Date/Time: 19-May-2000 15:36:18 Instrument: QLC 9298

Results File: PKU_MCA_001.nrf Process Date/Time: 11-Sep-2000 09:49:01
Printed: Tue 10 Apr 2001 09:58:34
Sample Summary:
FileName Pass/Fail Well

PKU_MCA_001 Pass 1:A,1
PKU_MCA_002 Fail 1:A,2
PKU_MCA_013 Fail 1:B,1
PKU_MCA_014 Pass 1:B,2
PKU_MCA_015 Pass 1:B,3
PKU_MCA_018 Pass 1:C,1
PKU_MCA_019 Fail 1:C,2
PKU_MCA_030 Fail 1:D,1
PKU_MCA_031 Pass 1:D,2
PKU_MCA_032 Pass 1:D,3
Figure 4.20 A Batch Summary printout
Page 92
NeoLynx Browser
The Sample Report Page
A Sample Report is a report relating to a single sample data file. Two distinct
regions need to be configured - the Sample Report Header, and the Sample Report
Data Area. The fields displayed on the report and the report header are selected in
the same manner as for the Batch Summary (see above). It is also necessary to
select the Sample Type (Standard, Analyte, QC) data to report.
An example of a printout is shown in
Figure 4.22
Figure 4.21 The Sample Report Page
Sample Summary Header This area defines the text printed on a Sample Report
Header and what information is printed on the Individual Sample Report. Twelve
field areas are available in the Sample Summary Header. The available fields are all
sample-dependent, and may be selected from:
Acquire Date/Time The date and time on which the data were acquired.
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NeoLynx Browser
Description The text string defined in the File Text column of the Sample List.
Filename The name of the data file.
ID The ID number defined in the ID column of the Sample List.
Instrument The name or serial number of the instrument as defined in the

MassLynx Control Panel.
Pass/Fail Whether the sample has Passed or Failed the tests in the NeoLynx Test
File.
Process Date/Time The date and time on which the data were processed.
Sample Type The Sample Type, as defined in the Sample List.
Spare1-5 Information from the Spare 1-5 columns in the Sample List.
Submitter The name of the person logged on to MassLynx at the time of data
acquisition.
Test File The name of the NeoLynx Test File used to process the data.
User Name The name of the person logged on to MassLynx at the time of data
processing.
Well The Well location of the processed sample
Free Text 1 - 11 Eleven fields in which user-defined comments may be added.
Sample Report Data Fields
The data reported in the columns of the Sample Summary are test-dependent, the
data fields available are:
Calculated Conc The Calculated Concentration from the Rule defined in the Test.
Calculated Concentration = Ion Ratio * Multiplier.
Detected Mass 1-4 The masses detected and used for the calculations, according to
Target Mass 1-4 and the defined Peak Detection Parameters.
Formula The complete formula (Ion Ratio * Multiplier) defined in the Rule used in
the Test.
High Conc The value defined as the High Concentration in the Test.
IS Conc The name of the Sample List from which the data were processed.
Low Conc The value defined as the Low Concentration in the Test.
RRF The Relative Response Factor defined in the Test.
Rule The name of the Rule used in the Test.
Spare 1-5 Information from the Spare columns in the Sample List.
Target Mass 1-4 The Target Mass(es) sought by the Test. These will be 0.00 if not
required for the rule.
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NeoLynx Browser
Test Name The name of the Test as defined in the NeoLynx Test File.
Test Result The result of the test - from Message Low, Message High or Message
OK fields.
Units The units as defined in the designated Rule.
X The value of the X variable defined in the Test.
The lower region of the window shows three areas relating to different Sample
Types. It is possible to differentiate between Sample Types, and only generate a
Sample Report if the specified criteria are met. Available options are No Results,
Fails Only, Passes Only or All Results.
NeoLynx Processing Report Generated by: Administrator All Samples Tested Page 1
FileName: Sample01 Pass/Fail: Pass Well: 1:A,1

Acquire Date/Time: 08-Aug-1994 15:36:18 Process Date/Time: 11-Sep-2000 09:49:01 Type: Sample
Results File: NeoLynx_Sample_001.nrf Total Samples: 16

Printed: Tue 10 Apr 2001 09:58:56
Sample Report:
1: Parents of 85 (200:500) ES+ 25eV
344.2
100
218.0 260.1 459.5

% 263.1 347.4
456.6
316.1
0 m/z
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
TestName Test Result Calculated Conc Units

C3 Carnitine OK 1.68 µmol/L
C4 Carnitine High C4 2.73 µmol/L
C5:1 Carnitine OK 0.08 µmol/L
C5-OH Carnitine OK 1.18 µmol/L
C10:1 Carnitine High C10:1 3.39 µmol/L
C14:1 Carnitine OK 0.23 µmol/L
2: Neutral Loss 102 (120:270) ES+ 12eV
172.0
100
% 146.0 191.1
134.0 150.0 188.0 227.1
182.2 222.0 238.0 242.0 260.1 263.0
0 m/z
130 140 150 160 170 180 190 200 210 220 230 240 250 260 270
TestName Test Result Calculated Conc Units

Leu/Ile OK 174 umol/L
Met OK 14.3 umol/L
Phe OK 61.8 umol/L
Tyr OK 177 umol/L
Phe/Tyr OK 0.35 -
Figure 4.22 An example of a Sample Report
Page 95
NeoLynx Browser
The Electronic Report Pages
In the NeoLynx Report Scheme supplied with NeoLynx 3.4 (NeoLynx.nrs), the
electronic reports are configured to emulate the electronic reports supplied by default
with NeoLynx 3.3. A summary of a report page is given here. Six different
Electronic Report configurations are available. The default configurations, and the
types of files generated are shown in Appendix E.
Figure 4.23 The Sample_Conc1 Page
The fields displayed on the report and the report header are selected in the same
manner as for the Batch Summary. See
Page 96
NeoLynx Browser
The Batch Summary Page on page 89 for details.
The drop-down Header field allows for pre-defined column headers to be allocated
if the Column Headers box is checked. User-defined column headers are also
allowed.
Test Results Check one of All Tests, Passes Only or Fails Only to display the type
of test result required.
Sample Type Check Standard, Analyte and/or QC to display the results for the
required sample type(s).
Report Format Check Column Headers to show the column header on the first line
of the report. Check the Row Format to show the results for a sample on one row. If
Row Format is not selected each individual test result is printed in column format.
Check Index Column to display the index field.
Save Options Check Append to add data to the end of the current report or
Overwrite to create a new file. A report will be created if it does not already exist.
Blank Line After Check New Sample to print a blank line after each sample.
Check New Table to print a blank line after a change of NeoLynx Test File in the
Sample List..
Delimiter Select the delimiter, to use to separate fields, from the drop down list
box. Available types are tab, comma, space, semi-colon, none. If a different
delimiter is required type the single character in the Delimiter box.
Report Path To save the report to the C:\MASSLYNX directory check the Default
to current project folder box. To write the report to a different location uncheck
this box, press the Folder button and select a folder from the browser displayed.
Note that by using this option carefully, identical reports with identical names may
be written to separate locations on a single computer, or across a network.
Report Name The name of the saved report is defined in this field. To incorporate
the name of the Sample List from which the data was derived, enter %Sample% in
the Report Name field as shown in Figure 4.23 . The file extension may be
user-defined. *.rep is used to reproduce the NeoLynx 3.3 Reports. It is possible to
use other extensions e.g. *.xls, *.txt.
Page 97
NeoLynx Browser
Page 98
NeoLynx Browser
Notes
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Page 100
References
Selected References
This is a list of selected references that may be relevant in the use of mass
spectrometry for neonatal screening, with particular focus on the amino acid and
acylcarnitine metabolites. This list is not exhaustive for the period covered and is
intended as a guide only.
1. Recent Developments in Neonatal Screening - E W Naylor, Seminars in

Perinatology, 9 (1985) 232-249.
2. Newborn Screening: Past, Present and Future - R Guthrie, in Genetic Disease:

Screening and Management, T P Carter and A M Wiley, Eds, Liss (1986)
319-339.
3. Hepatic Bile Acid Metabolism during Early Development Revealed from the
Analysis of Human Fetal Gallbladder Bile - K D R Setchell, R Dumaswala, C
Colombo and M Ronchi, J. Biol. Chem., 263 (1988) 16637-16644.
4. ∆4-3-Oxosteroid 5β-Reductase Deficiency Described in Identical Twins with

Neonatal Hepatitis - K D R Setchell, F J Suchy, M B Welsh, L Zimmer-
Nechemias, J Heubi and W F Balistreri, J. Clin. Invest., 82 (1988) 2148-2157.
5. New-born Screening Fact Sheets - Committee on Genetics of the American

Academy of Pediatrics, Pediatrics, 83 (1989) 449-464.
6. Identification of 3α,4β,7α trihydroxy-5β-cholanoic acid in Human Bile:

Reflection of a New Pathway in Bile Acid Metabolism in Humans - R
Dumaswala, K D R Setchell, L Zimmer-Nechemias, T Iida, J Goto and T
Nambara, J. Lipid Res., 30 (1989) 847-856.
7. Acyl-CoA Dehydrogenase Deficiencies - C R Roe and P M Coates, Metabolic

Basis of Inherited Diseases, McGraw Hill (1989) 889-914.
8. Dienoyl-Coenzyme A Reductase Deficiency : A Possible New Disorder of

Fatty Acid Oxidation - C R Roe, D S Millington, D L Norwood, N Kodo, H
Sprecher, B S Mohammed, M Nada, H Schulz and R McVie, J. Clin. Invest.,
85 (1990) 1703-1707.
9. Comprehensive Study of the Biliary Bile Acid Composition of Patients with

Cystic Fibrosis and Associated Liver Disease Before and After UDCA
Administration - M Nakagawa, C Colombo and K D R Setchell, Hepatology,
12 (1990) 322-334.
10. Tandem Mass Spectrometry: A New Method for Acylcarnitine Profiling with
Potential for Neonatal Screening for Inborn Errors of Metabolism - D S
Millington, N Kodo, D L Norwood and C R Roe, J. Inher. Metab. Dis., 13
(1990) 321-324.
11. Bile Acid Metabolism in Early Life: Studies of Amniotic Fluid - M Nakagawa
and K D R Setchell, J. Lipid Res., 31 (1990) 1089-1098.
12. The Analysis of Diagnostic Markers of genetic Disorders in Human Blood and
Urine using Tandem Mass Spectrometry with Liquid Secondary Ion Mass
Spectrometry - D S Millington, N Kodo, N Terada, D Roe and D H Chace,
Int. J. Mass Spectrom. Ion Processes, 111 (1991) 211-228.
Page 101
References
13. New Technologies in New-born Screening - E W Naylor, Yale J. Biol. Med.

64 (1991) 21-24.
14. Failure of Ursodeoxycholic Acid to Prevent a Cholestatic Episode in a Patient

with Benign Recurrent Intrahepatic Cholestasis: A Study in Bile Acid
Metabolism - A Crosignani, M Podda, E Bertolini, P M Battezzati, M Zuin
and K D R Setchell, Hepatology, 13 (1991) 1076-1083.
15. The Role of Tandem Mass Spectrometry in the Diagnosis of Fatty Acid
Oxidation Disorders - D S Millington, N Terada, D H Chace, Y-T Chen, J-H
Ding, N Kodo and C R Roe, New Developments in Fatty Acid Oxidation,
Wiley-Liss (1992) 339-354.
16. Carnitine and Acylcarnitines in Metabolic Disease Diagnosis and Management

- D S Millington and D H Chace, in Mass Spectrometry: Clinical and
Biomedical Applications Vol. 1, D M Desiderio, Ed., Plenum (1992) 299-318.
17. Carnitine and Acylcarnitine Analysis in the Diagnosis of Metabolic Diseases:

Advantages of Tandem Mass Spectrometry - D S Millington, N Terada, N
Kodo and D H Chace, in Advances in Chemical Diagnosis and Treatment of
Metabolic Disorders, Volume 1, I Matsumoto, Ed., Wiley (1992) 59-71.
18. Neonatal Screening for Duchenne/Becker Muscular Dystrophy;

Reconsideration Based on Molecular Diagnosis and Potential Therapeutics - E
W Naylor, E P Hoffman, J Paulus-Thomas, H B Wessel, K S Reid, B Mitchell
and B J Schmidt, Screening, 1 (1992) 99-113.
19. Oral Bile Acid Treatment and the Patient with Zellweger Syndrome - K D R
Setchell, P Bragetti, L Zimmer-Nechemias, C Daugherty, M A Pelli, R Vaccaro,
G Gentili, E Distrutti, G Dozzini, A Morelli and C Clerici, Hepatology, 15
(1992) 198-207.
20. Rapid Diagnosis of Phenylketonuria by Quantitative Analysis for

Phenylalanine and Tyrosine in Neonatal Blood Spots by Tandem Mass
Spectrometry - D H Chace, D S Millington, N Terada, S G Kahler, C R Roe
and L F Hofman, Clin. Chem., 39 (1993) 66-71.
21. Medium-Chain Acyl-CoA Dehydrogenase (MCAD) Deficiency: Diagnosis by

Acylcarnitine Analysis in Blood - J L K vanHove W Zhang, S G Kahler, C R
Roe, Y-T Chen, N Terada, D H Chace, A K Iafolla, J-H Ding and D S
Millington, Am. J. Hum. Genet., 52 (1993) 958-966.
22. Neonatal Screening for Cystic Fibrosis: Addition of Molecular Diagnostics to

Increase Specificity - W C Spence, J Paulus-Thomas, D M Orenstein and E W
Naylor, Biochem. Med. Metab. Biol., 49 (1993) 200-211.
23. The Analysis of Acylcarnitines - B M Kelly, M E Rose and D S Millington, in

Advances in Methodology - Two, W W Christie, Ed., The Oily Press, (1993)
247-289.
24. Ion spray liquid chromatography / mass spectrometric characterization of bile

acids - B M Warrack and G C DiDonato, Biol. Mass Spectrom., 22 (1993)
101-111.
25. Screening techniques for the detection of inborn errors of bile acid metabolism by
direct injection and micro-high performance liquid chromatography-continuous flow
/ fast atom bombardment mass spectrometry - J E Evans, A Ghosh, B A Evans and
M R Natowicz, Biol. Mass Spectrom., 22 (1993) 331-337.
Page 102
References
26. 3-Hydroxydicarboxylic and 3-Ketodicarboxylic Aciduria in Three Patients:

Evidence for a New Defect in Fatty Acid Oxidation at the Level of 3-
Ketoacyl-CoA Thiolase - M J Bennett and W G Sherwood, Clin.Chem., 39
(1993) 897-901.
27. Dietary Supplement L-Carnitine: Analysis of Different Brands to Determine

Bioavailability and Content - D S Millington and G Dubay, Clin. Res. Reg.
Affairs, 10 (1993) 71-80.
28. Electrospray Tandem Mass Spectrometry in the Analysis of Organic

Acidemias - M S Rashed, P T Ozand, M E Harrison, P J F Watkins and S
Evans, Rapid Commun. Mass Spectrom., 8 (1994) 129-133.
29. Neonatal Screening for Inborn Errors of Metabolism by Automated Dynamic

Liquid Secondary Ion Tandem Mass Spectrometry - D H Chace and D S
Millington, in New Horizons in Neonatal Screening, J-P Farriaux and J-L
Dhondt, Eds, Elsevier (1994) 373-376.
30. Inborn Errors of Bile Acid Metabolism - K D R Setchell and N C O'Connell in

Liver Disease in Children, F J Suchy, Ed, (1994) 835-851.
31. Diagnosis of Metabolic Disease - D S Millington, D H Chace, S L Hillman, N

Kodo and N Terada, in Biological Mass Spectrometry: Present and Future, T
Matsuo, Y Seyama, R M Caprioli and M L Gross, Eds, Wiley (1994) 559-579.
32. ∆4-3-Oxosteroid 5β-reductase deficiency causing neonatal liver failure and

hemochromatosis - B L Shneider, K D R Setchell, P F Whitington, K A Nellson
and F J Suchy, J. Pediatr., 124 (1994) 234-238.
33. A new cause of progressive intrahepatic cholestasis: 3β-hydroxy-C27-steroid

dehydrogenase / isomerase deficiency - E Jacquemin, K D R Setchell, N C
O'Connell, A Estrada, G Maggiore, J Schmitz, M Hadchouel and O Bernard, J.
Pediatr., 125 (1994) 379-384.
34. Tiglylglycine Excreted in Urine in Disorders of Isoleucine Metabolism and the

Respiratory Chain Measured by Stable Isotope Dilution GC-MS - M J Bennett, S
Powell, D J Swartling and K M Gibson, Clin. Chem., 40 (1994) 1879-1883.
35. Inherited Defects of Fatty Acid Oxidation - C R Roe and M A Nada, in Fatty
Acids and Lipids: Biological Aspects, G C Simopolous and E Tremoli, Eds,
Karger (1994) 22-25.
36. Automated ESI-MS/MS using 96-Well Microplate Batch Processing for Rapid
High-Throughput Metabolic Profiling of the Butyl Derivatives of Free
Carnitine, Acylcarnitines and Amino Acids. - M Rashed, M Bucknall, A
Awad, M Jacob, D Little, M Al-Amoudi and P Ozand, Presented at the 3rd
International Symposium on Applied Mass Spectrometry in the Health
Sciences, Barcelona, June 1995.
37. Diagnosis of Inborn Errors of Metabolism from Blood Spots by Acylcarnitines

and Amino Acids Profiling using Automated Electrospray Tandem Mass
Spectrometry - M S Rashed, P T Ozand, M P Bucknall and D Little, Pediatr.
Res., 38 (1995) 324-331.
38. Glycine and L-Carnitine Therapy in 3-Methyl-crotonyl-CoA carboxylase

Deficiency - S L Rutledge, G T Berry, C A Stanley, J L K vanHove and D
Millington, J. Inher. Metab. Dis., 18 (1995) 299-305.
Page 103
References
39. Rapid Diagnosis of Maple Syrup Urine Disease in Blood Spots from
Newborns by Tandem Mass Spectrometry - D H Chace, S L Hillman, D S
Millington, S G Kahler, C R Roe and E W Naylor, Clin. Chem., 41 (1995) 62-
68.
40. The Role of Tandem Mass Spectrometry in Reducing the Number of False
Positive and False Negative Results in the Diagnosis of Metabolic Disease
from Dried Blood Spots - D H Chace, D S Millington and S L Hillman, in
Early Hospital Discharge: Impact on Newborn Screening, K A Pass and H L
Levy, Eds, (1995) 272-283.
41. High-performance Liquid Chromatographic - Electrospray Mass

Spectrometric Analysis of Bile Acids in Biological Fluids - A Roda, A N
Gioacchini, C Cerrè and M Baraldini, J. Chromatogr. B, 665 (1995) 281-294.
42. Inborn Errors of Metabolism Diagnosed in Sudden Death Cases by

Acylcarnitine Analysis of Postmortem Bile - M S Rashed, P T Ozand, M J
Bennett, J J Barnard, D R Govindaraju and P Rinaldo, Clin Chem., 41 (1995)
1109-1114.
43. Evidence for Intermediate Channeling in Mitochondrial β-Oxidation - M A

Nada, W J Rhead, H Sprecher, H Schulz and C R Roe, J. Biol. Chem., 270
(1995) 530-535.
44. ∆22-ursodeoxycholic Acid, a Unique Metabolite of Administered

Ursodeoxycholic Acid in Rats, Indicating Partial β-Oxidation as a Major
Pathway for Bile Acid Metabolism - K D R Setchell, H Yamashita, C M P
Rodrigues, N C O'Connell, B T Kren and C J Steer, Biochemistry, 34 (1995)
4169-4178.
45. Investigation of β-Oxidation Intermediates in Normal and MCAD-Deficient

Human Fibroblasts using Tandem Mass Spectrometry - M A Nada, D H
Chace, H Sprecher and C R Roe, Biochem. Molec. Medicine, 54 (1995) 59-66.
46. Medium Chain Acyl-CoA Dehydrogenase Deficiency in Pennsylvania:

Neonatal Screening Shows High Incidence and Unexpected Mutation
Frequencies - R Ziadeh, E P Hoffman, D N Finegold, R C Hoop, J C Brackett,
A W Strauss and E W Naylor, Pediatr. Res., 37 (1995) 675-678.
47. Rapid Diagnosis of Homocystinuria and other Hypermethioninemias from

Newborns' Blood Spots by Tandem Mass Spectrometry - D H Chace, S L
Hillman, D S Millington, S G Kahler, B W Adam and H L Levy, Clin. Chem.,
42 (1996) 349-355.
48. Disorders of Bile Acid Synthesis and Metabolism - K D R Setchell, in

Pediatric Gastrointestinal Disease, Pathophydiology, Diagnosis,
Management, W A Walker, P R Durie, J R Hamilton, J A Walker-Smith and J
A Watkns, Eds, (1996) 1205-1233.
49. Newborn Screening by Tandem Mass Spectrometry (MS-MS) - L Sweetman,

Clin. Chem., 42 (1996) 345-346.
50. Rapid Diagnosis of MCAD Deficiency: Quantitative Analysis of

Octanoylcarnitine and Other Acylcarnitines in Newborn Blood Spots by
Tandem Mass Spectrometry - D H Chace, S L Hillman, J L K van Hove and E
W Naylor, Clin. Chem., 43 (1997) 2106-2113.
Page 104
References
51. Additive Hypocholesterolemic Effect of Psyllium and Cholestyramine in the

Hamster: Influence in Fecal Sterol and Bile Acid Profiles - B P Daggy, N C
O'Connell, G R Jerdack, B A Stinson and K D R Setchell, J. Lipid res., 38
(1997) 491-502.
52. Screening Blood Spots for Inborn Errors of Metabolism by Electrospray

Tandem Mass Spectrometry with a Microtitre Plate Batch Process and a
Computer Algorithm for Automated Flagging of Abnormal Profiles - M S
Rashed, M P Bucknall, D Little, A Awad, M Jacob, M Alamoudi, M Alwattar
and P T Ozand, Clin. Chem., 43 (1997) 1129-1141.
53. A Method for the Quantitation of Conjugated Bile Acids in Dried Blood Spots
using Electrospray Ionisation Mass Spectrometry - K A Mills, I Mushtaq, A W
Johnson, P D Whitfield and P T Clayton, Pediatr. Res., 43 (1998) 361-368.
Page 105
References
Page 106
Appendix A
Appendix A - Data Compendium

The data shown in the following pages are for illustrative purposes only. The
intention of these inclusions is to give the reader examples of the types of data that
may be seen during the analysis of neonatal blood spots. This compendium is by no
means comprehensive.
The data were acquired as part of a demonstration for Dr Mohamed Rashed of the
King Faisal Specialist Hospital and Research Centre, Riyadh, and were acquired on a
Quattro LC mass spectrometer coupled to an HP1100/Gilson 215 combination.
Selected acylcarnitine profiles
Function type: Parents of m/z 84.80
Mass range: m/z 215 to m/z 510
Cone Voltage: 30 V
Collision Energy: 25.0 eV
Collision gas pressure: 1.4 x 10-3 mbar Argon
Scan time: 3.0 secs (0.05 sec inter-scan delay)
d3-free carnitine
d3-C16 carnitine
d3-C3 carnitine
d3-C8 carnitine
Typical acylcarnitine profile from a healthy patient.
Page 107
Appendix A
Normal trace vs patient with significant elevation of free carnitine
Normal patient vs patient with significant elevation of C3-carnitine. Indicative of

propionic acidaemia or methyl malonic acidaemia.
Page 108
Appendix A
Normal patient vs patient with elevated medium chain (C6, C8, C10, C10:1)
acylcarnitines. The patient from the lower trace was diagnosed with MCAD.
Normal patient vs patient showing elevations of a broad spectrum of acylcarnitines.

Indicative of multiple acyl-CoA dehydrogenase deficiency or glutaric acidaemia type
II.
Page 109
Appendix A
Normal patient vs patient with elevated isovaleryl cartnitine.
Normal patient vs patient showing elevation of the very long chain (C14-C18)
acylcarnitines. Diagnosis of long-chain acyl coA dehydrogenase deficiency is not as
clear cut from tandem mass spectra as it is with some other disorders. Further testing
would be indicated.
Page 110
Appendix A
Selected neutral / acidic amino acid profiles
Function type: Neutral Loss of 102.10 Da
Cone Voltage: 25 V
Typical neutral / acidic amino acid profile from a healthy patient.
Page 111
Appendix A
Normal patient vs patient with elevated methionine (m/z 206).
Normal patient vs patient with elevated Leu/Ile (m/z 188). Note that it is not
possible to differentiate the leucine isomers in a simple tandem mass spectrometry
experiment - additional chormatographic separation would be necessary for absolute
determinations.
Page 112
Appendix A
Normal patient vs patient with severely elevated Leu/Ile (m/z 188) from an
uncontrolled maple syrup urine disease (MSUD). Note the difference between this
spectrum and the one above showing the different levels of quantification that can be
achieved in these experiments.
Normal patient vs patient with slightly elevated Phe (m/z 222).
Page 113
Appendix A
Normal patient vs patient with severely elevated Phe (m/z 222) in uncontrolled
phenylketonuria (PKU).
Normal patient vs patient with elevated Ala (m/z 146).
Page 114
Appendix A
Normal patient vs patient with elevated levels of Tyr (m/z 238). Absolute
determination of elevated levels of tyrosine is difficult as a number of cases have
been reported in which transient neonatal tyrosinaemia has been observed.
Normal patient vs patient with elevated citrulline (m/z 215). Although citrulline is a
basic amino acid, it can often be detected in the neutral / acidic amino acid scan
function under the scanning conditions used.
Page 115
Appendix A
Selected basic amino acid profiles
Cone Voltage: 30 V
Normal basic amino acid profile from a healthy patient.
Normal patientvs patient with elevated levels of ciitrulline (m/z 232). This spectrum
was acquired in the same experiment as shown in the elevated citrulline example
above.
Page 116
Appendix A
Alternative scan function for Glycine and Alanine
Cone Voltage: 25 V
Collision Energy: 8 eV
Normal patient vs patient with elevated levels of glycine (m/z 132) and alanine (m/z
146). Note the presence of the analytes and the isotopically labelled internal
standards used for quantification.
Page 117
Appendix A
Page 118
Appendix B
Appendix B - Quantification
The use of multiple reaction monitoring (MRM) for the detection of metabolic
disorders in neonatal bloodspots has been shown to be comparable to the use of
full-scan data employed in a number of laboratories around the world. The use of
MRM analysis allows for faster flow rates to be used for sample introduction,
considerably reducing the inter-sample analysis time. In MRM analysis only specific
metabolites may be monitored following validation of mass spectrometric methods
for specific metabolites.
Multiple reaction monitoring is widely used in the pharmaceutical industry,

especially during clinical trials, for the quantitative monitoring of drug metabolites.
It seems logical to extend those methodologies to the problems encountered in
neonatal screening, as the absolute levels of endogenous metabolites present in an
abnormal sample are likely to be of use for the consulting physician. While the use
of relative mass spectral peak intensity ratios yields useful information about the
relative levels of endogenous metabolites to the spiked internal standards, this is not
truly representative of the total amount of samples injected into the system. MRM
data may be used to gain both absolute quantitative information, via integration of
the individual chromatographic traces, and relative quantitative information by
simply combining the 'spectra' across a chromatographic peak and comparing peak
intensity ratios.
The table below represents a summary of the different quantification methods

available using the MassLynx and NeoLynx software suite. The MassLynx Quantify
program uses classical chromatographic peak integration and area ratios to calculate
the analyte concentrations. The individual analyte chromatogram traces are
smoothed and integrated in order to determine the peak area used in subsequent
calculations. NeoLynx uses a comparison of individual peak intensities, derived
from combining and background subtracting the 'spectra' across the peak, rather than
a true integration in time.
This comparison was effected using a series of eight MRM acquisitions in which a
total of 52 species were detected. The data was then analysed using the two different
processes, and the results are presented in the Table below. The comparison column
shows the absolute difference in the analyte/internal standard ratios and as a
percentage of the ratio derived from the Quantify program. The average deviation
and standard deviation were calculated for a total of eight separate acquisitions.
This comparative analysis to test the validity of the dual processing of the MRM data
showed that the differences in the determined analyte/internal standard ratios only
varied by a few percent between the two methods suggesting that the use of
combined data from an MRM acquisition may be used for elementary spectral
analysis. As would be expected, the largest differences between the two methods is
apparent when the peaks relating to the analytes are quite small - a significant
number of the analytes measured are only present in minimal quantities in samples
from healthy patients.
A comparison between the concentrations derived from MCA and MRM data is
better illustrated in Example 4 in Appendix C.
Page 119
Appendix B
Acylcarnitines
FROM FROM NEOLYNX COMPARISON DEVIATION

QUANTIFY
# Name IS# Mass Area Ratio Intensity Ratio d(ratio) %(quan) Ave % SD
1 C2 carnitine 2 260.3 5620 1.324 7587 1.309 -0.015 -1.1 -0.48 1.55
2 d3-C2 carnitine 2 263.3 4245 1.000 5796 1.000
3 C3 carnitine 4 274.3 1071 1.376 1445 1.389 0.013 1.2 1.48 1.55
4 d3-C3 carnitine 4 277.3 778 1.000 1040 1.000
5 C4-carnitine 4 288.3 251 0.322 345 0.332 0.010 3.1 -0.90 3.72
6 C5-carnitine 4 302.3 364 0.468 495 0.476 0.008 1.7 -0.06 2.48
7 C6-carnitine 4 316.3 77 0.099 121 0.116 0.017 17.1 -0.43 13.60
8 C8:1-carnitine 10 342.4 387 0.130 496 0.123 -0.007 -5.4 -1.04 2.28
9 C8-carnitine 10 344.4 185 0.062 268 0.066 0.004 6.5 -0.39 6.71
10 d3-C8-carnitine 10 347.4 2971 1.000 4039 1.000
11 C10:1-carnitine 10 370.4 236 0.079 327 0.081 0.002 2.5 3.20 8.63
12 C10-carnitine 10 372.4 314 0.106 418 0.103 -0.003 -2.8 -3.65 2.26
13 C4DC-carnitine 10 374.4 1042 0.351 2486 0.615 0.264 75.2 83.54 10.19
14 C5DC-carnitine 10 388.4 132 0.044 148 0.037 -0.007 -15.9 24.18 42.57
15 C12-carnitine 10 400.4 170 0.057 229 0.057 0.000 0.0 -6.19 13.60
16 C14:2-carnitine 22 424.5 159 0.036 212 0.035 -0.001 -2.8 1.90 18.39
17 C14:1-carnitine 22 426.5 151 0.035 172 0.029 -0.006 -17.1 -0.48 11.67
18 C14-carnitine 22 428.5 217 0.050 252 0.042 -0.008 -16.0 -3.10 5.86
19 C14-Ohcarnitine 22 444.5 22 0.005 44 0.007 0.002 40.0 -3.93 33.71
20 C16:1carnitine 22 454.5 126 0.029 175 0.029 0.000 0.0 0.54 7.68
21 C16-carnitine 22 456.5 1712 0.391 2321 0.385 -0.006 -1.5 0.34 2.04
22 d3-C16-carnitine 22 459.5 4375 1.000 6030 1.000
23 C16-OH-carnitine 22 472.5 60 0.014 99 0.016 0.002 14.3 4.75 55.04
24 C18:2-carnitine 22 480.5 974 0.223 1307 0.217 -0.006 -2.7 -1.37 2.39
25 C18:1-carnitine 22 482.5 4215 0.963 5731 0.950 -0.013 -1.3 0.25 1.88
26 C18-carnitine 22 484.5 1465 0.335 1977 0.328 -0.007 -2.1 -0.45 1.80
27 C18:1-OH-carnitine 22 498.5 69 0.016 94 0.016 0.000 0.0 -0.16 9.83
Page 120
Appendix B
Amino Acids
FROM QUANTIFY FROM NEOLYNX COMPARISON DEVIATION
# Name IS# Mass Area Ratio Intensity Ratio d(ratio) %(quan) Ave % SD
1 Glycine 2 132.3 40 0.097 79 0.142 0.045 46.4 25.83 37.01
2 13C-15N-Glycine 2 134.3 407 1.000 555 1.000
3 Alanine 4 146.3 8064 3.690 11170 3.657 -0.033 -0.9 -0.49 2.10
4 d4-Alanine 4 150.3 2186 1.000 3054 1.000
5 Valine 6 174.3 9099 2.522 12620 2.553 0.031 1.2 1.45 1.50
6 d8-Valine 6 182.3 3607 1.000 4944 1.000
7 Glutamine 9 186.3 9582 0.587 13990 0.618 0.031 5.3 4.24 2.58
8 Leucine+Isoleucine 9 188.3 34140 2.093 47520 2.099 0.006 0.3 -0.04 0.74
9 d3-Leucine 9 191.3 16311 1.000 22630 1.000
10 Methionine 11 206.3 3137 0.285 4426 0.288 0.003 1.1 1.19 2.62
11 d3-Methionine 11 209.3 10996 1.000 15360 1.000
12 Citrulline 11 215.3 2662 0.242 3624 0.236 -0.006 -2.5 -2.31 1.58
13 Phenylalanine 14 222.3 27248 0.828 37980 0.833 0.005 0.6 -0.01 0.78
14 d5-Phenylalanine 14 227.3 32897 1.000 45600 1.000
15 Tyrosine 16 238.3 19266 1.281 26490 1.284 0.003 0.2 -0.31 1.35
16 d4-Tyrosine 16 242.3 15043 1.000 20630 1.000
17 Aspartate 19 246.3 16509 1.753 22930 1.768 0.015 0.9 0.36 1.30
18 Glutamate 19 260.3 16340 1.735 22430 1.729 -0.006 -0.3 0.14 1.39
19 d3-Glutamate 19 263.3 9418 1.000 12970 1.000
Page 121
Appendix B
Page 122
Appendix C
Appendix C - Examples Of Data

Processing Using NeoLynx
Data Files Supplied
Standard01-Standard10 For Example 1
Blood01-Blood10 For Example 2
Sample01-Sample08 For Example 3
MCA Test 01 - MCA Test 11 For Example 4
MRM Test 01 - MRM Test 11 For Example 4
Before processing each batch, check that the processing parameters are appropriate
for the type of data you are analysing. The user is expected to prepare the Sample
Lists in an appropriate format, and to prepare appropriate NeoNatal Rule sets.
Information relating to the appropriate mass / compound relationship may be found

elsewhere in the NeoLynx Research Manual.
The data provided for this exercise is intended for tutorial purposes only, and no
absolute information may be inferred from the profiles observed.
Page 123
Appendix C
EXAMPLE 1: CHECK PEAK INTENSITIES TO VERIFY PRESENCE OF A

COMPOUND
Objective: The objective of this exercise is to automatically analyse the

spectra provided and to determine which compounds are not
present (and thus by default, which compounds are present) from
the list provided.
The samples STANDARD01-STANDARD10 were mixtures of standard compounds

that had been butylated and reconstituted in 50% aqueous acetonitrile.
The samples were analysed using a five-function MCA acquisition:
1) Parents of m/z 85 for acylcarnitines
2) Neutral loss of 102 Da for neutral and acidic amino acids
3) Neutral loss of 119 Da for basic amino acids
4) Neutral loss of 56 Da for glycine and alanine
5) Neutral loss of 161 Da for arginine
The standard compounds used for this experiment were:
Free carnitine, Phenylalanine, Methionine, Leucine, Ornithine, Citrulline, Glycine

and Arginine
• Manually check the spectra and decide on appropriate criteria for the presence
or absence of each compound in question.
• Using NeoLynx, automatically process the data to check for minimum peak
intensities (Rule 0). Examine the results for the 10 Sample files, and record
which compounds are present in each mixture.
Page 124
Appendix C
EXAMPLE 1: WORKING TABLE
Test Name Rule Func Min Max Mass 1 Mass 2 Msg Msg Msg IS Conc
Ratio Ratio Low High OK
EXAMPLE 1: CONCLUSIONS
SAMPLE COMPOUNDS PRESENT
Standard 01
Standard 02
Standard 03
Standard 04
Standard 05
Standard 06
Standard 07
Standard 08
Standard 09
Standard 10
Page 125
Appendix C
EXAMPLE 1: Function 1
Unknown
Standard10 1 (1.306) Sm (SG, 2x1.00); Sb (30,10.00 ) 1: Parents of 85ES+
218.3 1.29e6
100
% 219.3
0
206.3 2.82e4
100
218.4
% 514.0
231.0 247.4 281.9 305.4 345.9 351.5 377.4 410.9 439.8 463.5 498.3
0
206.1 2.79e4
100
% 218.3 229.2
287.9 324.3 449.6 458.8 499.2 517.9
260.9 300.6 338.3 371.4 394.8 411.7 527.9
0
218.3 1.86e6
100
% 219.3
0
218.3 1.67e6
100
% 219.2
0 m/z
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540
Unknown
218.3 1.98e6
100
% 219.3
0
218.2 2.27e4
100
% 219.3
285.8 423.2 441.9 542.0
208.4 231.5 276.1 310.1 333.4 354.5 373.7 399.3 470.1 477.9 509.0 515.6
0
218.3 2.45e6
100
% 219.3
0
218.3 1.61e6
100
% 219.3
0
218.3 1.93e6
100
% 219.2
0 m/z
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540
Page 126
Appendix C
Unknown
Standard10 1 (1.306) Sm (SG, 2x1.00); Sb (30,10.00 ) 2: Neutral Loss 102ES+
222.2 3.18e6
100
188.2 206.1
% 162.2 223.2
189.2
163.2 215.2
0
222.2 5.73e6
100 206.1
% 207.2 223.2
188.2
0
222.2 5.39e6
100
188.2 206.1
% 189.2 223.2
215.2
0
222.2 4.24e6
100
206.1
% 162.2 223.2
188.2 207.2
0
188.2 206.1 2.19e6
100 162.2
% 163.2 189.2
132.3 215.2 222.2
0 m/z
130 140 150 160 170 180 190 200 210 220 230 240 250 260 270
Unknown
222.2 4.43e6
100
188.2
% 162.2 223.2
189.2
0
222.2 8.00e6
100
188.2
% 223.2
189.2 215.1
0
222.2 5.14e6
100
% 162.2 223.2
163.2
0
222.2 3.99e6
100
162.2 188.2
% 223.2
189.2 215.1
0
222.2 4.15e6
100
% 162.2 223.2
215.1
0 m/z
130 140 150 160 170 180 190 200 210 220 230 240 250 260 270
Page 127
Appendix C
Unknown
232.2 6.61e5
100
% 162.3
189.2 206.1 231.3 233.2
222.2
0
189.2 2.28e5
100 206.2
% 222.2
190.3 207.2 231.3 232.3
162.3
0
232.2 1.12e6
100
% 231.3 233.2
206.1 222.2
0
162.3 3.90e5
100
% 189.2 206.2
190.2 222.2 231.3
0
232.2 8.27e5
100
162.3
% 233.3
206.2
0 m/z
150 160 170 180 190 200 210 220 230 240 250
Unknown
162.3 4.64e5
100
% 163.2 189.2
190.3 222.2
0
232.2 1.40e6
100
% 231.3 233.2
222.2
0
162.3 4.62e5
100
% 189.2
163.3 222.2 231.3 232.3
190.2
0
232.2 8.96e5
100
162.3
% 231.3 233.2
222.2
0
232.2 9.08e5
100
% 162.3
231.3 233.2
189.2 222.2
0 m/z
150 160 170 180 190 200 210 220 230 240 250
Page 128
Appendix C
Unknown
132.0 2.16e5
100
% 133.0
122.8 159.0
0
132.0 5.28e5
100
% 131.2
0
131.9 6.45e3
100
158.0
% 127.3 132.8 134.7 142.6 145.7 155.3
122.2 124.3 124.8 138.0 144.4 147.2 151.9 153.9
0
132.0 1.07e4
100
159.1
% 158.0
123.6 127.5 130.4 135.5 140.9 142.4 144.5 147.8 150.7 153.4155.2
0
132.0 2.51e5
100
% 133.0
159.1
0 m/z
120 125 130 135 140 145 150 155 160
Unknown
132.0 3.38e5
100
%
123.1
0
132.0 1.13e4
100
% 133.1
121.6 123.8 127.2 130.0 136.6 138.8 144.0 150.0 156.4 159.1
0
132.0 1.94e4
100
159.0
% 131.0 133.2
155.8
0
132.0 2.62e5
100
% 131.2 133.0
0
132.0 3.96e5
100
%
131.2
0 m/z
120 125 130 135 140 145 150 155 160
Page 129
Appendix C
Unknown
218.2 231.1 2.46e5
100
% 219.2 232.1
206.1
0
231.1 6.00e5
100
% 232.2
218.3
0
231.1 3.17e5
100
% 232.1
218.2
0
218.2 231.1 3.57e5
100
% 217.4 219.2 232.2
0
218.2 3.17e5
100
% 219.2
206.1
0 m/z
200 205 210 215 220 225 230 235 240 245 250
Unknown
218.2 3.68e5
100
% 217.3 219.2
231.2
0
231.1 5.75e5
100
% 230.4 232.2
0
218.2 231.1 5.24e5
100
% 219.2 232.1
200.6
243.2
0
218.2 231.1 3.11e5
100
% 217.4 219.1 232.2
0
218.2 231.1 3.50e5
100
% 219.2 232.1
204.5
0 m/z
200 205 210 215 220 225 230 235 240 245 250
Page 130
Appendix C
EXAMPLE 2: EXAMINE SPECTRA FROM SPIKED BLOOD SPOTS
Objective: The purpose of this exercise is to prepare a rule table to

automatically detect which compounds have been spiked into the
blood samples.
The samples BLOOD01 to BLOOD10 were prepared and analysed. A standard

dried blood sample paper was divided into 10 separate pieces, and some were spiked
with methanolic solutions of pure amino acids. The samples were then extracted and
derivatised in the normal way.
The samples were analysed in a five-function MCA acquisition:
3) Neutral loss of 119 Da for basic amino acids
4) Neutral loss of 56 Da for glycine and alanine
5) Neutral loss of 161 Da for arginine
• BLOOD01data is from unadulterated blood.
• Examine the spectra to see what has been added to the blood sample.
• Using the simple ratio method (Rule 1), ratio the following compounds to the
C2 carnitine (m/z 260), Proline (m/z 172) and Glutamine (m/z 203) peaks in
functions 1 to 3, respectively: Carnitine, Phe, Leu, Met, Orn, Cit. Normal ratios
may be calculated from the sample Blood01 which has not been altered.
Abnormal levels were arbitrarily set at 3 times the normal peak intensity ratio
for inclusion as the Maximum Threshold in the Neonatal rule table.
• Which samples have been adulterated, and with what?
• Report the abnormal ratios.
Page 131
Appendix C
EXAMPLE 2: BLOOD 01
Blood Sample
blood01 1 (1.304) Sm (SG, 2x1.00); Sb (30,10.00 ) 1: Parents of 85ES+
260.3 1.17e5
100
261.2
218.5 456.8
274.2
484.8
200.9 482.6
228.1236.0 275.1
304.2 353.8 371.7 428.7 480.7
0 m/z
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540
Blood Sample
blood01 1 (1.304) Sm (SG, 2x1.00); Sb (30,10.00 ) 2: Neutral Loss 102ES+
172.1 7.37e5
100
188.1
146.1
162.0 260.1
222.1
186.0 238.0
160.1 190.1
206.0 212.1
176.1
0 m/z
130 140 150 160 170 180 190 200 210 220 230 240 250 260 270
Blood Sample
203.1 1.05e5
100
238.1
204.2
189.2
174.3 176.1 188.2 239.3
160.2 190.1 232.2
0 m/z
150 160 170 180 190 200 210 220 230 240 250
Page 132
Appendix C
Test Rule Func Min Ratio Max Mass 1 Mass 2 Msg Msg High Msg IS
Ratio Low OK Conc
SAMPLE COMPOUNDS ADDED (AMOUNT)
Blood 01
Blood 02
Blood 03
Blood 04
Blood 05
Blood 06
Blood 07
Blood 08
Blood 09
Blood 10
Page 133
Appendix C
Blood10 1 (1.306) Sm (SG, 2x1.00); Sb (30,10.00 ) 1: Parents of 85ES+

100 260.3 1.74e5
261.2
% 218.2 228.2 456.5
274.2 417.3 484.6
0
100
260.3 1.80e5
% 218.2 227.9 456.8

274.3 413.9 482.8
0
100 260.3 1.88e5
261.3
% 218.2 228.3 428.7 456.3
274.2 304.6 413.5 484.8
0
100
260.3 1.77e5
261.1
% 218.1 228.2 456.8
274.3 413.5 484.8
0
100 260.3 1.95e5
% 218.3 261.2
243.2 456.6
228.3 274.3 318.5 342.9 482.8
0
Blood04 1 (1.306) Sm (SG, 2x1.00); Sb (30,10.00 ); Sm (SG, 2x1.00); Sb (30,10.00 ) 1: Parents of 85ES+
100 260.3 1.55e5
261.3
% 218.4 228.0 456.6
274.1 338.4 388.4 428.6 482.6 502.8
0 m/z
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

100 260.3 1.80e5
261.4
%
218.3 228.2 456.7 482.7
274.4 304.4 424.4
0
100 260.3 1.55e5
261.3
% 456.6
218.4 227.9 482.7
274.1 338.3 388.4 428.6 502.8
0
100 260.3 1.98e5
261.3
% 456.5
218.3 228.4 482.7
274.2 298.4 316.3 428.6
0
100 260.4 1.40e5
% 261.3
218.3 228.2 456.6
274.3 294.2 381.8 408.6 482.6 486.9
0
100 260.3 1.17e5
261.2
%
218.5 228.1 274.2 456.8
304.2 353.8 371.7 428.7 484.8
0 m/z
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540
Page 134
Appendix C
Blood10 1 (1.306) Sm (SG, 2x1.00); Sb (30,10.00 ) 2: Neutral Loss 102ES+

100 222.1 1.55e7
%
223.1
172.1
0
100 222.1 4.95e6
% 188.1
172.1 223.1
146.1 189.1 260.1
0
100 172.1 8.87e5
% 188.1 260.1
146.1 206.1 222.1 238.0
160.1 162.1 186.1
189.0 212.0 246.1 261.1
0
100 206.1 1.04e6
172.1
% 188.1 260.1
146.1 222.1 238.1
160.1 162.0 186.1 189.1 212.1 246.0
0
100 172.1 1.01e6
% 188.1
146.1 189.1 206.1 212.0 222.1 260.1
162.1 186.1 238.0
160.1 246.1 261.2
0 m/z
130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

100 188.1 2.96e6
% 172.1
186.1 189.1
146.1 162.1 212.1 222.1 238.0 260.1
0
100 172.1 7.64e5
% 188.1 222.1 260.1

146.1 162.1 206.1 238.1
160.1 186.1 189.1 212.1 223.1 246.1 261.2
0
100 222.1 3.89e6
%
172.1 223.0
146.1 162.0 188.1 238.0 260.1
0
100 172.1 6.56e5
% 146.1 188.1 206.0 222.1 238.1 260.1

160.1 162.1 186.1 190.1 212.1 246.1
0
100 172.1 7.37e5
% 188.1
146.1 162.0 206.0 222.1 260.1
160.1 186.0 190.1 212.1 238.0
0 m/z
130 140 150 160 170 180 190 200 210 220 230 240 250 260 270
Page 135
Appendix C

100 222.2 2.73e5
% 203.2
223.1
189.2 204.0 238.0
0
100 203.2 1.26e5
% 222.1 232.2
174.0 204.3 223.3 238.2
160.2 189.2 192.3 249.2
176.2 188.1 212.1 239.0
0
100 203.1 2.00e5
%
204.3
160.2 174.1 176.1 189.3 222.2 232.0 238.1
0
100 203.1 1.89e5
%
206.1
174.2 176.3 188.0 189.2 222.1 232.1 238.1 247.3
0
100 203.2 2.22e5
%
204.2 238.2
160.4 174.2 176.3 189.2
0 m/z
150 160 170 180 190 200 210 220 230 240 250

100 203.2 1.02e5
%
204.1 238.2
174.3 176.3 188.2189.1 194.2 212.1 218.1 220.1 232.0
0
100 203.1 232.2 1.46e5
%
204.1 238.1
160.3 176.1 189.2 190.3 222.1 248.2
0
100 203.1 1.74e5
% 222.1
189.1 203.9
174.2 232.2 238.2
0
100 203.2 8.83e4
%
189.2 204.2 249.2
174.1 176.1 188.1 192.3 232.2 238.2 246.9
0
100 203.1 1.05e5
%
204.2
160.2 174.3 176.1 188.2 189.2 190.1 238.1 239.3
232.2
0 m/z
150 160 170 180 190 200 210 220 230 240 250
Page 136
Appendix C

100 132.0 2.67e5
146.0
%
132.9 147.1
128.8
0
100 132.0 2.47e5
146.0
%
133.0 147.0
129.0
0
100 132.0 2.42e5
146.0
%
130.0 143.9
0
100 132.0 2.57e5
146.0
%
133.0
130.0
0
100 131.9 2.35e5
146.0
%
129.2 144.1
0 m/z
120 125 130 135 140 145 150 155 160

100 131.9 2.50e5
146.0
%
133.0 146.9
130.0 144.0
0
100 131.9 2.00e5
146.0
%
133.0 147.0
0
100 131.9 2.86e5
146.0
%
147.0
0
100 132.0 1.68e5
146.0
%
132.9 147.0
0
100 132.0 1.93e5
146.0
%
132.9
0 m/z
120 125 130 135 140 145 150 155 160
Page 137
Appendix C

100 218.2 1.01e4
% 200.0 219.2 231.0 245.1

229.1 247.6 249.5
205.1 207.4 210.3 213.2 216.0 220.5 226.4 234.7 237.5 238.6
0
100 245.0 4.74e3
202.2
%
218.0 229.1 229.9 231.1
214.1 215.8 228.4 237.4 243.2 247.2
205.3 209.4 221.4 224.5 233.5 236.2 240.8
0
100 218.1 4.97e3
245.1 247.1
% 241.9
219.3 248.4
206.9 231.3
202.5 208.8 213.6 215.9 222.8 227.4 231.9 236.2 238.5 243.4
0
100 218.0 1.15e4
245.1
% 200.0 217.3 219.0
212.7 231.0 232.1 239.2 242.1 243.7
204.0 207.0 211.0 222.6 227.6 237.1 248.1
0
100 218.0 1.09e4
% 200.0 217.1
231.1 245.1
204.8 208.4 215.1 222.3 224.9 228.7 232.0 235.4 238.8 241.1 247.2 248.1
0 m/z
200 205 210 215 220 225 230 235 240 245 250

100 218.1 1.25e4
200.0
% 219.2
204.1 207.1 216.3 244.9 247.1
211.7 222.5 225.0 232.3 238.1 241.0 248.9
0
100 218.1 245.2 5.89e3
200.0
% 216.1
221.1
244.3
205.0 214.9 225.1 227.2 230.8
203.6 209.5 234.2 235.0 240.3 247.0
0
100 218.0 7.31e3
200.0202.2 217.1
% 231.1
207.0 212.0 219.2 232.0 244.8245.2 248.7
208.3 213.2 224.0 228.7 234.2 236.8 239.8 247.6
0
100 218.2 6.11e3
200.0 210.2 214.9 216.1 219.4 243.9 245.1
% 203.9 205.0 221.5 245.8
227.0 231.5 235.9 239.4 247.9
0
100 200.0 218.1 3.08e3
244.1 245.1
% 217.0 242.1
201.1203.3 205.7
209.9 214.9 220.6 224.5 225.7 228.4 233.6 234.9 237.9 238.8 249.0
0 m/z
200 205 210 215 220 225 230 235 240 245 250
Page 138
Appendix C
EXAMPLE 3: PROCESS SAMPLES CONTAINING INTERNAL STANDARDS

TO YIELD QUANTIFIED RESULTS
Objective: The purpose of this exercise is to quantify some metabolites of

interest in eight patient samples from information given about the
concentration of internal standards.
Samples SAMPLE01 to SAMPLE08 were prepared from affected neonatal blood

spots and were acquired using isotopically labelled internal standards, the
concentrations of which are known. Process the 8 data files NEO01 to NEO08 given
the following information:
The samples were analysed using a two-function MCA acquisition:
The following internal standards and concentrations were used:
d3-Free carnitine 10µM/L

d3-C2-carnitine 10uM/L
d5-phenylalanine 200uM/L
d3-leucine 400uM/L
d3-methionine 150uM/L
d4-tyrosine 400uM/L
Normally, the most accurate quantification is achieved if each analyte has it's own
isotopically labelled internal standard. This is not financially viable for
acylcarnitines, so the following approximation may be used:
Use d3-C2-carnitine as the internal standard for short-chain (C2-C5) acylcarnitines.
Use d3-C8 carnitine as the internal standard for the medium-chain (C6-C12)
acylcarnitines.
Use d3-C16 carnitine as the internal standard for the long-chain (>C12)
acylcarnitines.
The sample in NEO01 is normal.
• Examine the spectra manually.
• Determine the appropriate peak intensity ratios for the compounds and internal
standards of interest for the normal samples. Note the internal standards have
been added to each sample at the nominal cut-off level for the amino acids, and
at 10uM/L for the acylcarnitines. Use discretion in determining acylcarnitine
cut-off levels.
• Use NeoLynx to calculate the concentrations of the acylcarnitines and amino

acids in the abnormal samples.
Page 139
Appendix C
• Determine the [Phe]/[Tyr] concentration ratio automatically for the abnormal

samples.
EXAMPLE 3: SAMPLE 01
Sample 1 MeCN/H2O Res17/15 15/16 IE .5/1 3.0 E-3 Arg 10 µl injected from 40µl
sample01 1 (2.222) Sm (SG, 2x1.00); Sb (30,10.00 ) 1: Parents of 85ES+
260.1 1.09e5
100
459.4
218.1
%
263.2
347.2
460.5
456.4
221.1
274.1
482.5
0 m/z
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540
Sample 1 MeCN/H2O Res17/15 15/16 IE .5/1 3.0 E-3 Arg 10 µl injected from 40µl
sample01 1 (2.222) Sm (SG, 2x1.00); Sb (30,10.00 ) 2: Neutral Loss 102ES+
172.0 2.48e5
100
191.0
227.1
134.0
% 150.0
146.0
188.0
242.0
209.0 260.1
182.1 263.1
132.0 222.0 238.0
173.0 192.1
162.0 212.0 243.0
0 m/z
120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
Page 140
Appendix C
Test Rule Func Min Max Mass 1 Mass Msg Msg Msg IS
Ratio Ratio 2 Low High OK Conc
SAMPLE ABNORMAL MASSES / COMPOUND PRSENT (CONCENTRATION) - INFERENCE
Sample 01
Sample 02
Sample 03
Sample 04
Sample 05
Sample 06
Sample 07
Sample 08
Page 141
Appendix C
Manual Examination of Spectra for Example 3
EXAMPLE 3: Spectra for Sample 01
Sample01 1 (2.222) Sm (SG, 2x1.00); Sb (30,10.00 ) 1: Parents of 85ES+

260.1 1.09e5
100
459.4
218.1
%
263.2
347.2
460.5
456.4
221.1
274.1
482.5
0 m/z
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540
Sample01 1 (2.222) Sm (SG, 2x1.00); Sb (30,10.00 ) 2: Neutral Loss 102ES+

172.0 2.48e5
100
191.0
227.1
134.0
% 150.0
146.0
188.0
242.0
209.0 260.1
182.1 263.1
132.0 222.0 238.0
162.0 173.0 192.1
212.0 243.0
0 m/z
120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
Page 142
Appendix C

263.0 5.81e4
100
218.1
260.1
347.2
459.5
460.4
221.1
348.2
274.0 456.2 482.4

344.4 374.2 403.5
304.3
0 m/z
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

188.0 4.66e5
100
% 172.0
191.0
227.1
134.0 150.0
145.9 242.0
132.0 182.1 209.0 222.0 228.1 263.1

161.9 260.1
0 m/z
120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
Page 143
Appendix C

260.1 6.56e4
100
218.0 459.4
263.0
%
347.2
456.4
460.3
326.4 482.3
220.9
274.1 348.2 480.2
454.3 483.3
366.1 384.4 403.1 428.0
0 m/z
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

222.0 2.89e5
100
172.0
191.0
%
227.1
134.0 150.0
146.0
188.0
242.0
184.9 209.0 260.1
263.1
132.0 238.0
161.9 192.0 243.1
0 m/z
120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
Page 144
Appendix C

344.2 1.04e5
100
459.5
260.1
218.0
%
263.1
347.4
456.6
316.1 460.4
221.1 372.3
288.2
482.5
274.1
400.3 403.5 428.4
0 m/z
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

172.0 4.10e5
100
146.0
191.1
150.0 227.1
134.0 188.0
242.0
182.2 222.0 238.0 260.1 263.0
132.0 209.0
162.0
0 m/z
120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
Page 145
Appendix C

274.1 8.08e4
100
%
459.4
347.3
263.2
260.1
218.1
221.1
275.3
403.4 456.5
348.2 400.1
207.8 482.5
0 m/z
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

172.0 1.95e5
100
191.1
188.0
%
227.1
150.0
134.1
146.0
242.1
263.0
132.0 182.1
260.1
174.0 222.0
192.2 209.0 238.0
162.0
0 m/z
120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
Page 146
Appendix C

260.1 1.67e5
100
218.1
263.1
459.4
456.4
347.2
482.4
221.1 274.1
400.4 426.4 454.4
0 m/z
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

172.0 1.58e5
100
191.1
134.0 150.0 227.1

%
146.0
188.0
242.1
185.0 209.0 260.1

132.0 263.1
238.0
222.0
162.1 173.0 192.1 243.1
0 m/z
120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
Page 147
Appendix C

218.1 4.83e4
100
260.0
263.1
456.5
318.1
347.2
482.4
221.1
274.1 460.6 484.6
348.3 400.4
344.0
291.3 302.3 372.3 426.3 454.3 480.1
0 m/z
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

172.0 3.60e5
100
146.0
188.0 191.1
150.0
227.1
134.0
173.0 242.0 260.1

132.1 209.0 222.0 263.1
0 m/z
120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
Page 148
Appendix C

260.1 7.14e4
100
459.4
263.2
218.1
347.3
%
221.2 456.3
482.5
264.1 348.1 403.4
484.2
342.1 480.5
222.2 274.0 291.2 428.5
0 m/z
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

222.0 4.47e5
100
172.0
191.1
188.0 227.1
134.1 150.0
242.1
182.1 209.0 260.1 263.2
161.9
0 m/z
120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
Page 149
Appendix C
EXAMPLE 4: PROCESS SAMPLES ACQUIRED IN A 2-FUNCTION MRM

EXPERIMENT
Objective: To input the appropriate parameters to automatically process data

acquired in Multiple Reaction Monitoring mode using the
NeoLynx processing.
A series of test samples were prepared using blood samples that had been spiked
with analytes of interest, and appropriate amounts of internal standards. The samples
were first analysed in MCA mode (MCA TEST 01 - MCA TEST 11), diluted with
mobile phase and re-analysed using MRM mode (MRM TEST 01 - MRM TEST
11).
The following internal standard compounds were used for these particular analyses.
In this instance, the levels of the internal standards added are quoted nominally at
10uM/L as the purpose of the exercise is to notice trends in the data files.
d9-Free carnitine 10µM/L

d8-valine 10uM/L
d5-phenylalanine 10uM/L
d3-leucine 10uM/L
d3-methionine 10uM/L
d4-tyrosine 10uM/L
• Examine the MRM chromatograms manually, and determine appropriate

spectrum combine and background subtract parameters.
• Examine a 'typical' reconstructed spectrum and determine the peak intensity

ratios that should be calculated.
• Automatically process the MRM data files and examine the Concentration
Summary reports to look for trends.
• Record the trends.
• Using the same set of rules, automatically process all the MCA and MRM files
in a single Sample List.
• Check that the same trends are observed for each (MRM vs MCA) data set.
Page 150
Appendix C
WORKINGS TO EXAMPLES
The workings provided here are an example of one way of processing the data, and
are used for illustration only. The tables defined below were those used to achieve
the results given and agree with the manual interpretation.
For MCA data (Examples 1 to 3), the Mass Measure parameters used were:
For the MRM data (Example 4), the Spectrum Combine parameters used were:
For all the processing, the Peak Detection parameters:
Page 151
Appendix C
WORKED EXAMPLE 1:
The following rule table (NeoLynx_Standard.ntf) was used:
The peak intensities are examined in each mass spectral function, and messages are
yielded if the peak intensity for a particular compound is below than the minimum
specified threshold (see column).
Note that a high Maximum Threshold value must be set for the rules to work.
The following shows the sample compositions:
Standard01 Carnitine, Phe, Cit, Orn, Gly, Arg Missing: (Leu, Met)
Standard02 Carnitine, Phe, Cit, Orn, Gly, Arg Missing: (Met, Orn)
Standard03 Carnitine, Phe, Orn, Arg Missing: (Leu, Met, Cit, Gly)
Standard04 Phe, Leu, Cit, Arg Missing: (Carnitine, Met, Orn, Gly)
Standard05 Carnitine, Phe, Leu, Orn, Gly Missing: (Met, Cit, Arg)
Standard06 Carnitine, Leu, Met, Cit, Gly Missing: (Phe, Orn, Arg)
Standard07 Carnitine, Phe, Met, Orn, Arg Missing: (Leu, Cit, Gly)
Standard08 Phe, Leu, Met, Cit, Arg Missing: (Carnitine, Orn, Gly)
Standard09 Phe, Met, Orn, Gly, Arg Missing: (Carnitine, Leu, Cit)
Standard10 Carnitine, Phe, Leu, Met, Orn, Cit, Gly, Arg
Using the rules, the summary report should show the other compounds (bracketed) as
being absent.
Page 152
Appendix C
WORKED EXAMPLE 2:
The following table (NeoLynx_Blood.ntf) was used. Note that the minimum
threshold is not used, as we are only checking for high peak intensity ratios.
The blood samples were doctored in the following way:
Blood01 Normal
Blood02 Normal
Blood03 Added 5uL Phe solution
Blood04 Added 10uL Cit solution
Blood05 Added 10uL Leu solution
Blood06 Normal
Blood07 Added 10uL Met solution
Blood08 Normal
Blood09 Added 5uL of Phe, Leu and Cit solutions
Blood10 Added 20uL Phe solution
Note that comparing the results from Blood03 and Blood10, the reported Phe
concentration is approximately 4 times higher for Blood10, as would be expected
from the addition.
Page 153
Appendix C
WORKED EXAMPLE 3:
The following table (NeoLynx_Sample.ntf) was used.
Note that there is no Minimum Threshold used in this analysis, hence no Message
Low.
The following inferences may be drawn:
SAMPLE01 Normal
SAMPLE02 High Leu/Ile, 1064uM/L; MSUD patient
SAMPLE03 High Phe, 457uM/L; probable HyperPhe patient
SAMPLE04 C4, C6, C8, C10, C10:1 carnitines high; MCAD
SAMPLE05 High C3 carnitine; possible propionic acidaemia or MMA
SAMPLE06 Normal
SAMPLE07 High C5-OH carnitine; C4 carnitine not found (OK?); C16

carnitine high?
SAMPLE08 High Phe, 1264uM/L. [Phe]/[Tyr] also high, 14.5. Note use of IS
ratio.
The summary report for the Sample01-Sample08 files in the NeoLynx generic
formats are shown below in an Excel interpretation.
Page 154
Appendix C
WORKED EXAMPLE 4
The most important thing to note in this example is that a peak appearing in the
MRM spectrum will appear in the MCA trace, but a peak appearing in an MCA trace
will only appear in the MRM result if it has been programmed.
The following rule tables, NeoLynx_Test_MRM.ntf and NeoLynx_Test_MCA.ntf,

were used (different signal intensity thresholds from the MRM and MCA
experiments require different NeoLynx test files to be used in a single sample list):
Note that only a nominal Maximum Threshold has been used in this example as the
results should be viewed from the summary file generated - see below for the report
styles. If the results are examined using the CONC1.rep style, the following trends
should be noted:
Page 155
Appendix C
TEST 01 Blank sample - should be returned as the internal standard levels

being too low. This rule has not been included in the above table,
but should really be considered to differentiate real samples from
blanks - see Example 1.
TEST 02 Endogenous blood sample
TEST 03-06 Increasing levels of Phe and Tyr should be observed, with the
additions being in the proportion, 2, 4, 8, 12. Met, Leu/Ile and Val
levels should remain reasonably constant.
TEST 07-11 Increasing levels of some acylcarnitines should be observed.

Additions have been made for C2, C3, C5, C6, C8, C10, C12, C14,
C16 and C18. A general trend only should be recorded as
increasing with increasing file name number.
Page 156
Appendix D
Appendix D - Importing Worksheets

Overview
Information can be imported into a MassLynx Sample List in a number of ways. The
Sample Lists chapter in the MassLynx NT User's Guide contains a full explanation
of all methods. This chapter describes the simplest way – defining data in a
spreadsheet, saving it as a tab-delimited text file and importing the text file.
Text file data can be imported in one of the following ways.
Import Worksheet This requires an index column and a specific format for the first
row in the imported file.
Import Data This does not require an index field or a formatted first row but does
need to be imported into a formatted sample list
Import Worksheet
Load the required sample list format (Samples, Load Format). This defines the
appropriate columns for the analyses to be performed.
Select File, New.
The Field ID of the columns must be used on the first line of the file created. To
determine the Field IDs press the toolbar button or select Customize Display
from the Field option on the Samples menu to invoke the Customize Field Display
dialog.
Page 157
Appendix D
The name in brackets is the Field ID.
Another way of determining the Field ID is to click with the right mouse button on
column header and select Properties from the pop up menu displayed. The Field
Properties dialog is displayed with the Field ID shown on the top line. In the
example shown the Field ID is 'FILE_TEXT'.
Prepare the data in an external spreadsheet package as shown below.
Note: the first cell should contain the text 'INDEX' the following cells in the row
should contain the Field ID text.
When a file has been prepared (e.g. in Excel) save it as a tab-delimited text file
(*.txt). This file may then be directly imported as a Worksheet without modification.
Page 158
Appendix D
From the MassLynx top level, select File, Import Worksheet and select Tab
Delimited from the Files of type box.
Select the file to be imported and press Open. The example file shown above will
be imported into the sample list as.
This Sample List may then be saved and run in the normal way.
Page 159
Appendix D
Import Data
This method works in a similar way to that described above.
Load the required sample list format (Samples, Load Format). This defines the
appropriate columns for the analyses to be performed.
Select File, New.
Prepare the data in an external spreadsheet package as shown below.
Note: The order of the data in spreadsheet must match the order of the columns in
the sample list.
Count the number of row containing data (13 in this case) and ensure that the Sample
List has the same number. If the sample list is prepared with too few rows, some
sample data will not be imported and the samples will not analysed. If a sample list
is prepared with too many rows some rows will be blank and the sample list will not
run.
When a file has been prepared (e.g. in Excel) save it as a tab-delimited text file
(*.txt).
From the MassLynx top level, select File, Import Worksheet and select Tab
Delimited from the Files of type box.
Page 160
Appendix D
Select the file to be imported and press Open. The example file shown above will
be imported into the sample list as.
The Sample List may now be saved and run in the normal way.
Page 161
Appendix D
Page 162
Appendix E
Appendix E - NRS Configurations

ELECTRONIC SUMMARY REPORT INFORMATION
This section shows the layout of the six Electronic Reports generated using the
NeoLynx Results File (Sample_001.nrf) and the NeoLynx Results Scheme
(NeoLynx.nrs). It is intended to act as a starting point for defining reports and as a
reference to the default settings for restoring the original report in case inappropriate
values have been entered.
The NeoLynx.nrs scheme is supplied with the NeoLynx software and is designed to
produce reports of a similar format to previous versions of NeoLynx.
For all reports the sample list name can be included in the Report Name by entering
%Sample% in the Report Name field. This will make reports unique to a sample
list. E.g. if the sample list name is Blood.spl then the reports will be
Blood_Conc1.rep, Blood_Conc2.rep etc.
To make the result summaries below fit easily on the page, the word 'Carnitine' has
been omitted from the appropriate cells.
Page 163
Appendix E
Sample_Conc1.rep
The left column contains the filename from the sample list. The top row contains
name of test, and the body of the table contains the calculated analyte concentrations.
The report has been split into two sections for ease of viewing, with the filename
column being repeated for each section. The file is tab-delimited.
If viewed in a spreadsheet package accepting tab-delimited files, Sample_Conc1.rep

will have the appearance:
Page 164
Appendix E
C3 C4 C5:1 C5 C6 C5-OH C8 C10:1 C10

Carnitine Carnitine Carnitine Carnitine Carnitine Carnitine Carnitine Carnitine Carnitine
Sample01 1.51 0.12 0.04 0.06 0.3 0.35 0.53 0.68 0.37
Sample02 0.89 0.17 0.24 0.11 0.17 0.31 0.53 0.46 0.6
Sample03 2.07 0.39 0.25 0.29 0.23 0.44 0.3 0.42 0.38
Sample04 1.68 2.73 0.08 0.24 6 1.18 34.51 3.39 4.18
Sample05 23.55 0.34 0.27 0.33 0.23 0.38 0.31 0.15 0.37
Sample06 2.25 0.41 0.09 0.49 0.21 1.07 0.97 0.81 0.68
Sample07 1.63 0 0.34 0.5 0.46 11.83 1.68 1.46 1.67
Sample08 0.7 0.22 0.08 0.14 0.21 0.41 0.38 0.17 0.41
Sample101 1.51 0.12 0.04 0.06 0.3 0.35 0.53 0.68 0.37
Sample102 0.89 0.17 0.24 0.11 0.17 0.31 0.53 0.46 0.6
Sample103 2.07 0.39 0.25 0.29 0.23 0.44 0.3 0.42 0.38
Sample104 1.68 2.73 0.08 0.24 6 1.18 34.51 3.39 4.18
Sample105 23.55 0.34 0.27 0.33 0.23 0.38 0.31 0.15 0.37
Sample106 2.25 0.41 0.09 0.49 0.21 1.07 0.97 0.81 0.68
Sample107 1.63 0 0.34 0.5 0.46 11.83 1.68 1.46 1.67
Sample108 0.7 0.22 0.08 0.14 0.21 0.41 0.38 0.17 0.41
C14:1 C16 Leu/Ile Met Phe Tyr Phe/Tyr

Carnitine Carnitine
Sample01 0.17 1.42 155.06 12.03 41.7 127.64 0.33
Sample02 0.25 0.93 1064.37 6.34 46.1 81.75 0.56
Sample03 0.22 3.36 181.58 10.45 457.43 103.22 4.43
Sample04 0.23 3.26 174.43 14.33 61.78 176.63 0.35
Sample05 0.59 1.57 304.99 15.78 35.96 56.81 0.63
Sample06 1.81 8.03 183.48 12.99 37.3 168.52 0.22
Sample07 0.89 10.09 412.48 15.63 65.74 109.45 0.6
Sample08 0.2 2.98 312.09 13.83 1264.24 87.25 14.49
Sample101 0.17 1.42 155.06 12.03 41.7 127.64 0.33
Sample102 0.25 0.93 1064.37 6.34 46.1 81.75 0.56
Sample103 0.22 3.36 181.58 10.45 457.43 103.22 4.43
Sample104 0.23 3.26 174.43 14.33 61.78 176.63 0.35
Sample105 0.59 1.57 304.99 15.78 35.96 56.81 0.63
Sample106 1.81 8.03 183.48 12.99 37.3 168.52 0.22
Sample107 0.89 10.09 412.48 15.63 65.74 109.45 0.6
Sample108 0.2 2.98 312.09 13.83 1264.24 87.25 14.49
Page 165
Appendix E
Sample_Conc2.rep
The information in Samples_CONC1.REP is reformatted to show one sample per

line. The line contains the file name followed by each test name and the associated
analyte concentration. This report has been split into three sections for ease of
viewing, with the filename being repeated for each section. The file is tab-delimited.

Page 166
Appendix E
Sample01 C3 1.51 C4 0.12 C5:1 0.04 C5 0.06 C6 0.3
Sample02 C3 0.89 C4 0.17 C5:1 0.24 C5 0.11 C6 0.17
Sample03 C3 2.07 C4 0.39 C5:1 0.25 C5 0.29 C6 0.23
Sample04 C3 1.68 C4 2.73 C5:1 0.08 C5 0.24 C6 6
Sample05 C3 23.55 C4 0.34 C5:1 0.27 C5 0.33 C6 0.23
Sample06 C3 2.25 C4 0.41 C5:1 0.09 C5 0.49 C6 0.21
Sample07 C3 1.63 C4 0 C5:1 0.34 C5 0.5 C6 0.46
Sample08 C3 0.7 C4 0.22 C5:1 0.08 C5 0.14 C6 0.21
Sample101 C3 1.51 C4 0.12 C5:1 0.04 C5 0.06 C6 0.3
Sample102 C3 0.89 C4 0.17 C5:1 0.24 C5 0.11 C6 0.17
Sample103 C3 2.07 C4 0.39 C5:1 0.25 C5 0.29 C6 0.23
Sample104 C3 1.68 C4 2.73 C5:1 0.08 C5 0.24 C6 6
Sample105 C3 23.55 C4 0.34 C5:1 0.27 C5 0.33 C6 0.23
Sample106 C3 2.25 C4 0.41 C5:1 0.09 C5 0.49 C6 0.21
Sample107 C3 1.63 C4 0 C5:1 0.34 C5 0.5 C6 0.46
Sample108 C3 0.7 C4 0.22 C5:1 0.08 C5 0.14 C6 0.21
Sample01 C5-OH 0.35 C8 0.53 C10:1 0.68 C10 0.37 C14:1 0.17 C16 1.42
Sample02 C5-OH 0.31 C8 0.53 C10:1 0.46 C10 0.6 C14:1 0.25 C16 0.93
Sample03 C5-OH 0.44 C8 0.3 C10:1 0.42 C10 0.38 C14:1 0.22 C16 3.36
Sample04 C5-OH 1.18 C8 34.51 C10:1 3.39 C10 4.18 C14:1 0.23 C16 3.26
Sample05 C5-OH 0.38 C8 0.31 C10:1 0.15 C10 0.37 C14:1 0.59 C16 1.57
Sample06 C5-OH 1.07 C8 0.97 C10:1 0.81 C10 0.68 C14:1 1.81 C16 8.03
Sample07 C5-OH 11.83 C8 1.68 C10:1 1.46 C10 1.67 C14:1 0.89 C16 10.09
Sample08 C5-OH 0.41 C8 0.38 C10:1 0.17 C10 0.41 C14:1 0.2 C16 2.98
Sample101 C5-OH 0.35 C8 0.53 C10:1 0.68 C10 0.37 C14:1 0.17 C16 1.42
Sample102 C5-OH 0.31 C8 0.53 C10:1 0.46 C10 0.6 C14:1 0.25 C16 0.93
Sample103 C5-OH 0.44 C8 0.3 C10:1 0.42 C10 0.38 C14:1 0.22 C16 3.36
Sample104 C5-OH 1.18 C8 34.51 C10:1 3.39 C10 4.18 C14:1 0.23 C16 3.26
Sample105 C5-OH 0.38 C8 0.31 C10:1 0.15 C10 0.37 C14:1 0.59 C16 1.57
Sample106 C5-OH 1.07 C8 0.97 C10:1 0.81 C10 0.68 C14:1 1.81 C16 8.03
Sample107 C5-OH 11.83 C8 1.68 C10:1 1.46 C10 1.67 C14:1 0.89 C16 10.09
Sample108 C5-OH 0.41 C8 0.38 C10:1 0.17 C10 0.41 C14:1 0.2 C16 2.98
Page 167
Appendix E
Sample01 Leu/Ile 155.06 Met 12.03 Phe 41.7 Tyr 127.64 Phe/Tyr 0.33
Page 168
Appendix E
Sample_Conc3.rep
In this format, each line contains a single filename, a single test name and the
calculated concentration. The report has been split into columns for ease of viewing.
The file is tab-delimited.

Page 169
Appendix E
Sample01 C3 Carnitine 1.51 Sample03 Phe 457.43
Sample01 C4 Carnitine 0.12 Sample03 Tyr 103.22
Sample01 C5:1 Carnitine 0.04 Sample03 Phe/Tyr 4.43
Sample01 C5 Carnitine 0.06 Sample04 C3 Carnitine 1.68
Sample01 C5-OH Carnitine 0.35 Sample04 C5:1 Carnitine 0.08
Sample01 C10:1 Carnitine 0.68 Sample04 C6 Carnitine 6
Sample01 C10 Carnitine 0.37 Sample04 C5-OH Carnitine 1.18
Sample01 C14:1 Carnitine 0.17 Sample04 C8 Carnitine 34.51
Sample01 C16 Carnitine 1.42 Sample04 C10:1 Carnitine 3.39
Sample01 Leu/Ile 155.06 Sample04 C10 Carnitine 4.18
Sample01 Met 12.03 Sample04 C14:1 Carnitine 0.23
Sample01 Phe 41.7 Sample04 C16 Carnitine 3.26
Sample01 Tyr 127.64 Sample04 Leu/Ile 174.43
Sample01 Phe/Tyr 0.33 Sample04 Met 14.33
Page 170
Appendix E
Sample07 C4 Carnitine 0 Sample101 Tyr 127.64
Page 171
Appendix E
Sample104 C6 Carnitine 6 Sample107 C4 Carnitine 0
Sample106 C5 Carnitine 0.49
Sample106 C5-OH Carnitine 1.07
Sample106 C10:1 Carnitine 0.81
Sample106 C14:1 Carnitine 1.81
Sample106 Leu/Ile 183.48
Sample106 Met 12.99
Sample106 Phe 37.3
Sample106 Tyr 168.52
Sample106 Phe/Tyr 0.22
Page 172
Appendix E
Sample_Conc4.rep
In this format, each line contains a single filename, a single test name, the value in
the Spare1 column in the sample list, the value from the Spare2 column in the
sample list (in this case the column is blank) and calculated concentration. The
report has been split into columns for ease of viewing. The file is comma-delimited.
If viewed in a spreadsheet package accepting comma-delimited files,

Sample_Conc4.rep will have the appearance:
Sample01 C3 0 1.51 Sample01 C5-OH 0 0.35
Sample01 C4 0 0.12 Sample01 C8 0 0.53
Sample01 C5:1 0 0.04 Sample01 C10:1 0 0.68
Sample01 C5 0 0.06 Sample01 C10 0 0.37
Sample01 C6 0 0.3 Sample01 C14:1 0 0.17
Page 173
Appendix E
Sample01 C16 0 1.42 Sample04 C10:1 0 3.39
Sample01 Leu/Ile 0 155.06 Sample04 C10 0 4.18
Sample01 Met 0 12.03 Sample04 C14:1 0 0.23
Sample01 Phe 0 41.7 Sample04 C16 0 3.26
Sample01 Tyr 0 127.64 Sample04 Leu/Ile 0 174.43
Sample01 Phe/Tyr 0 0.33 Sample04 Met 0 14.33
Sample02 C3 0 0.89 Sample04 Phe 0 61.78
Sample02 C4 0 0.17 Sample04 Tyr 0 176.63
Sample02 C5:1 0 0.24 Sample04 Phe/Tyr 0 0.35
Sample02 C5 0 0.11 Sample05 C3 0 23.55
Sample02 C6 0 0.17 Sample05 C4 0 0.34
Sample02 C5-OH 0 0.31 Sample05 C5:1 0 0.27
Sample02 C8 0 0.53 Sample05 C5 0 0.33
Sample02 C10:1 0 0.46 Sample05 C6 0 0.23
Sample02 C14:1 0 0.25 Sample05 C8 0 0.31
Sample02 C16 0 0.93 Sample05 C10:1 0 0.15
Sample03 C5 0 0.29 Sample06 C3 0 2.25
Sample03 C6 0 0.23 Sample06 C4 0 0.41
Sample03 C5-OH 0 0.44 Sample06 C5:1 0 0.09
Sample03 C8 0 0.3 Sample06 C5 0 0.49
Sample03 C10:1 0 0.42 Sample06 C6 0 0.21
Sample03 C14:1 0 0.22 Sample06 C8 0 0.97
Sample03 C16 0 3.36 Sample06 C10:1 0 0.81
Sample04 C5 0 0.24 Sample07 C3 A 1.63
Sample04 C6 0 6 Sample07 C4 A 0
Sample04 C5-OH 0 1.18 Sample07 C5:1 A 0.34
Sample04 C8 0 34.51 Sample07 C5 A 0.5
Page 174
Appendix E
Sample07 C6 A 0.46 Sample102 C4 0.17
Sample07 C5-OH A 11.83 Sample102 C5:1 0.24
Sample07 C10:1 A 1.46 Sample102 C6 0.17
Sample07 C10 A 1.67 Sample102 C5-OH 0.31
Sample07 C16 A 10.09 Sample102 C10:1 0.46
Sample07 Leu/Ile A 412.48 Sample102 C10 0.6
Sample07 Met A 15.63 Sample102 C14:1 0.25
Sample07 Phe A 65.74 Sample102 C16 0.93
Sample07 Tyr A 109.45 Sample102 Leu/Ile 1064.37
Sample07 Phe/Tyr A 0.6 Sample102 Met 6.34
Sample08 C3 A 0.7 Sample102 Phe 46.1
Sample08 C4 A 0.22 Sample102 Tyr 81.75
Sample08 C5:1 A 0.08 Sample102 Phe/Tyr 0.56
Sample08 C5-OH A 0.41 Sample103 C5:1 0.25
Sample08 C10 A 0.41 Sample103 C5-OH 0.44
Sample08 C16 A 2.98 Sample103 C10:1 0.42
Sample08 Leu/Ile A 312.09 Sample103 C10 0.38
Sample08 Met A 13.83 Sample103 C14:1 0.22
Sample08 Phe A 1264.24 Sample103 C16 3.36
Sample08 Tyr A 87.25 Sample103 Leu/Ile 181.58
Sample08 Phe/Tyr A 14.49 Sample103 Met 10.45
Sample101 C3 1.51 Sample103 Phe 457.43
Sample101 C4 0.12 Sample103 Tyr 103.22
Sample101 C5:1 0.04 Sample103 Phe/Tyr 4.43
Sample101 C5 0.06 Sample104 C3 1.68
Sample101 C5-OH 0.35 Sample104 C5:1 0.08
Sample101 C10:1 0.68 Sample104 C6 6
Sample101 C10 0.37 Sample104 C5-OH 1.18
Sample101 C14:1 0.17 Sample104 C8 34.51
Sample101 C16 1.42 Sample104 C10:1 3.39
Sample101 Leu/Ile 155.06 Sample104 C10 4.18
Sample101 Met 12.03 Sample104 C14:1 0.23
Sample101 Phe 41.7 Sample104 C16 3.26
Page 175
Appendix E
Sample104 Tyr 176.63 Sample107 C4 0
Sample104 Phe/Tyr 0.35 Sample107 C5:1 0.34
Sample105 C5:1 0.27 Sample107 C5-OH 11.83
Sample105 C6 0.23 Sample107 C10:1 1.46
Sample105 C5-OH 0.38 Sample107 C10 1.67
Sample105 C8 0.31 Sample107 C14:1 0.89
Sample105 C10:1 0.15 Sample107 C16 10.09
Sample105 C10 0.37 Sample107 Leu/Ile 412.48
Sample105 C14:1 0.59 Sample107 Met 15.63
Sample105 Leu/Ile 304.99 Sample107 Tyr 109.45
Sample105 Met 15.78 Sample107 Phe/Tyr 0.6
Sample105 Phe 35.96 Sample108 C3 0.7
Sample105 Tyr 56.81 Sample108 C4 0.22
Sample105 Phe/Tyr 0.63 Sample108 C5:1 0.08
Sample106 C5:1 0.09 Sample108 C5-OH 0.41
Sample106 C6 0.21 Sample108 C10:1 0.17
Sample106 C5-OH 1.07 Sample108 C10 0.41
Sample106 C8 0.97 Sample108 C14:1 0.2
Sample106 C10:1 0.81 Sample108 C16 2.98
Sample106 C10 0.68 Sample108 Leu/Ile 312.09
Sample106 C14:1 1.81 Sample108 Met 13.83
Sample106 Leu/Ile 183.48 Sample108 Tyr 87.25
Sample106 Met 12.99 Sample108 Phe/Tyr 14.49
Sample106 Phe 37.3
Sample106 Tyr 168.52
Sample106 Phe/Tyr 0.22
Sample107 C3 1.63
Page 176
Appendix E
Sample_Anon.rep
This report contains the same information as Samples_CONC1.REP without the file
names. This format is intended for statistical use in population screening. The
report has been split into two sections for ease of viewing. The file is tab-delimited.
If viewed in a spreadsheet package accepting tab-delimited files, Sample_Anon.rep

Page 177
Appendix E
C3 Carnitine C4 Carnitine C5:1 C5 Carnitine C6 Carnitine C5-OH C8 Carnitine C10:1

Carnitine Carnitine Carnitine
1.51 0.12 0.04 0.06 0.3 0.35 0.53 0.68
0.89 0.17 0.24 0.11 0.17 0.31 0.53 0.46
2.07 0.39 0.25 0.29 0.23 0.44 0.3 0.42
1.68 2.73 0.08 0.24 6 1.18 34.51 3.39
23.55 0.34 0.27 0.33 0.23 0.38 0.31 0.15
2.25 0.41 0.09 0.49 0.21 1.07 0.97 0.81
1.63 0 0.34 0.5 0.46 11.83 1.68 1.46
0.7 0.22 0.08 0.14 0.21 0.41 0.38 0.17
1.51 0.12 0.04 0.06 0.3 0.35 0.53 0.68
0.89 0.17 0.24 0.11 0.17 0.31 0.53 0.46
2.07 0.39 0.25 0.29 0.23 0.44 0.3 0.42
1.68 2.73 0.08 0.24 6 1.18 34.51 3.39
23.55 0.34 0.27 0.33 0.23 0.38 0.31 0.15
2.25 0.41 0.09 0.49 0.21 1.07 0.97 0.81
1.63 0 0.34 0.5 0.46 11.83 1.68 1.46
0.7 0.22 0.08 0.14 0.21 0.41 0.38 0.17
C10 Carnitine C14:1 C16 Carnitine Leu/Ile Met Phe Tyr Phe/Tyr
Carnitine
0.37 0.17 1.42 155.06 12.03 41.7 127.64 0.33
0.6 0.25 0.93 1064.37 6.34 46.1 81.75 0.56
0.38 0.22 3.36 181.58 10.45 457.43 103.22 4.43
4.18 0.23 3.26 174.43 14.33 61.78 176.63 0.35
0.37 0.59 1.57 304.99 15.78 35.96 56.81 0.63
0.68 1.81 8.03 183.48 12.99 37.3 168.52 0.22
1.67 0.89 10.09 412.48 15.63 65.74 109.45 0.6
0.41 0.2 2.98 312.09 13.83 1264.24 87.25 14.49
0.37 0.17 1.42 155.06 12.03 41.7 127.64 0.33
0.6 0.25 0.93 1064.37 6.34 46.1 81.75 0.56
0.38 0.22 3.36 181.58 10.45 457.43 103.22 4.43
4.18 0.23 3.26 174.43 14.33 61.78 176.63 0.35
0.37 0.59 1.57 304.99 15.78 35.96 56.81 0.63
0.68 1.81 8.03 183.48 12.99 37.3 168.52 0.22
1.67 0.89 10.09 412.48 15.63 65.74 109.45 0.6
0.41 0.2 2.98 312.09 13.83 1264.24 87.25 14.49
Page 178
Appendix E
Sample_Summary.rep
The Sample_Summary.rep report is probably the most commonly used report format.
It shows a summary of all the Tests that have fallen outside the defined limits
together with information relating to the sample under analysis. The style of this
report has changed slightly from V3.3, but the information content is now
considerably more flexible.
If viewed in a spreadsheet package accepting tab-delimited files,

Sample_Summary.rep will have the appearance:
Page 179
Appendix E
File Name Function Well Test Name Rule Name Result High Calc Units
Description Conc Conc
Sample02 2:Neutral AA 1,1:2 Leu/Ile 1:2 Peak Ratio High Leu/Ile 400 1064.37
Sample03 2:Neutral AA 1,1:3 Phe 1:2 Peak Ratio High Phe 200 457.43
Sample03 2:Neutral AA 1,1:3 Phe/Tyr 2:4 Peak Ratio High Phe/Tyr 1.25 4.43
Sample04 1:Acylcarnitines 1,1:4 C4 Carnitine 1:2 Peak Ratio High C4 2 2.73
Sample04 1:Acylcarnitines 1,1:4 C6 Carnitine 1:2 Peak Ratio High C6 2 6
Sample04 1:Acylcarnitines 1,1:4 C10:1 Carnitine 1:2 Peak Ratio High C10:1 2 3.39
Sample07 1:Acylcarnitines 1,1:7 C5-OH Carnitine 1:2 Peak Ratio High C5-OH 2 11.83
Sample104 1:Acylcarnitines 2,1:4 C6 Carnitine 1:2 Peak Ratio High C6 2 6
Sample104 1:Acylcarnitines 2,1:4 C10:1 Carnitine 1:2 Peak Ratio High C10:1 2 3.39
Sample107 1:Acylcarnitines 2,2:7 C5-OH Carnitine 1:2 Peak Ratio High C5-OH 2 11.83
Page 180
Appendix F
Appendix F - Internal Standard

Methodology
By far the most efficient way to enhance the accuracy of an assay is by the addition
of an internal standard to the entire sample. The sample is then mixed to assure
homogeneity during aliquotting. The internal standard is chosen such that identical
or very similar behavior during the various phases of the analytical process is
guaranteed. Isotopically labeled analogues make the most ideal internal standards
and mass spectrometers allow ready differentiation from the natural compound.
Concentration calculations are then based on the relative response for the analyte to
that of the internal standard and contributions due to matrix, recovery or aliquoting
will cancel. The relative response, of the analyte to the internal standard must be
known and is usually expressed in terms of a response factor or determined in situ.
The response factor is defined as that number by which the response of the analyte
must be multiplied to equal the response of the internal standard, present in equal
concentration.
If the response factor used has been determined externally, then the accuracy of the
final result depends on the certainty with which the concentration of the internal
standard in the analytical mixture is known. Response factors can be determined in
situ by the use of calibration lines or single point calibrations, if linearity is assured.
In that case, accuracy of the calculated results only depends on the error in
aliquoting the internal standard; the actual concentration of the internal standard
cancels during the mathematical manipulations. The latter method is preferred in
situations where internal standard preparations have to be frequently replenished.
The internal standard is prepared at the desired approximate concentration and then
aliquoted into each sample with a high precision devise. Again, the exact volume
dispensed is not critical as long as the volumes delivered are identical for each
dispensation used for calibrators and for samples.
Equations governing internal standard methodology.
[ ] = concentration
R = response
U = unknown or analyte
IS = internal standard
RF = response factor
Equation. 1: [U] = RU/RIS x [IS] x RFU and
Equation. 2: RFU = RIS/RU x [U]/[IS]
Determination of response factor:
Prepare several samples of different analyte concentration. To each sample add an

equal aliquot of internal standard and mix to homogeneity. Analyze each sample in
Page 181
Appendix F
triplicate and plot the response ratio, RU/RIS vs. the concentration of the analyte, [U].
Analyze the plot for linearity.
In linear systems, calculate response factor. RF is the inverse of the slope of the line
divided by the internal standard concentration.
Equation 1 can be restated as:
Equation. 3: [U] = RU/RIS x constant
If the response factor is determined from in-run calibrators and the line goes through
zero, then
Equation. 4: [U]Sample = { (RU/RIS) Sample / (RU/RIS) Calibrator } x [U] Calibrator
Hence the internal standard concentration cancels.
Typical Response of A and IS
100
90
80
70
60
50
40
30
20
10
0
1 5 9 13 17 21 25 29
Page 182
Index
Index
A
Analysis Of Acylcarnitines, 4
Analysis Of Amino Acids, 7
Basic, 9
Bile Acids, 12
Glycine and Arginine, 10
Neutral and Acidic, 7
C
Combine, 61
Converting V3.2 and V3.3 Rule Files, 41
D
Data Acquisition, 23
Data Compendium, 108
Data Processing, 37
Data Processing Examples, 124
F
Function Names Table, 47
I
Importing Worksheets, 158
M
Mandatory Mass Table, 49
Mass Measure, 59
Modes Of Data Acquisition, 14
N
NeoLynx Browser, 65
Electronic Reports, 85
Report Schemes, 86
Toolbar, 67
View Options, 68
NeoLynx Report Scheme Configurations, 164
Neonatal Rules Editor, 39
P
Peak Detection, 63
Print Control, 64
Processing, 59
Processing Options, 44
Q
Quantification, 120
Page 183
Index
R
References, 102
Report Schemes, 86
Rule Definition Table, 52
S
Sample Introduction, 20
Sample List, 25
Sample Preparation, 15
Scan Function Editor, 29
Sequential Samples, 71
T
Test Editor Toolbar, 40
Test File Editor, 39
Test Modification History, 43
Tests
Adding, 55
Deleting, 56
Modifying, 55
Tuning, 21, 23
Page 184
Index
Page 185

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NeoLynx Research Application Manager

NeoLynx Research 3.5

No use of this product as a diagnostic tool is implied or should be inferred.

NeoLynx Research Application Manager Users Guide

No use of the NeoLynx Research Application Manager as a diagnostic device is

Copyright (C) 1993-1999 Micromass Ltd. All Rights Reserved.

Micromass ® is a registered trade mark of Micromass Limited

MassLynx and NeoLynx are registered trademarks of Micromass Ltd.

Windows is a trademark of Microsoft Corporation. Other product names mentioned

Sample Preparation and Data Acquisition 15

Data Processing with NeoLynx 39

The Results Summary Pane 80

Appendix A - Data Compendium 105

Appendix B - Quantification 117

Appendix C - Examples Of Data Processing Using NeoLynx121

Appendix D - Importing Worksheets 155

Appendix E - NRS Configurations 161

bold Anything you must type exactly as it appears

ALL Directory names, filenames and acronyms.

Key combinations and key sequences appear in the following formats.

2. Definition of Tune page, Inlet and Scanning Method files.

3. Definition of NeoLynx Test Files.

4. Creation of a list of samples using the Sample List Editor.

6. Acquisition of sample data.

7. Processing of acquired data and production of reports using the NeoLynx

A data compendium of samples from a number of patients with inherited metabolic

The function of a tandem mass spectrometer is to:

1. Produce ions from the compounds in the sample under analysis.

• determine the metabolite concentrations of interest

• compare them to a standard set of rules

• produce reports detailing both normal and abnormal results.

In a typical experiment the mobile phase pumped from an LC system, or an infusion

High mass biopolymers, for example peptides, proteins and oligonucleotides,

Electrospray is a concentration dependant technique, and it is this characteristic that

L-carnitine (3-hydroxy-4-aminobutyrobetaine) plays a vital role in the mitochondrial

Figure 1.2 Butylation and fragmentation of acylcarnitines to yield a common

MS1 (Scanning) Collision cell MS2 (Static)

normal01 1 (2.902) 1: Parents of 85ES+

Notation Common Name Mass of butyl ester /Da

Analysis Of Amino Acids

A number of inherited diseases result in the disruption of the metabolism and

In phenylketonuria a deficiency in phenylalanine hydroxylase inhibits the conversion

An elevation of methionine in blood may be indicative of two inherited amino acid

Maple syrup urine disease is an inherited disorder characterised by the accumulation

Neutral and Acidic Amino Acids

An analysis where the molecular species of interest lose an uncharged common

The fragmentation for this transition is shown in Figure 1.5.

MS1 (Scanning) Collision cell MS2 (Scanning)

normal01 1 (2.902) 3: Neutral Loss 102ES+

Basic Amino Acids

MS1 (Scanning) Collision cell MS2 (Scanning)

normal01 1 (2.902) 4: Neutral Loss 119ES+

176.2 189.1 204.1 212.2 231.1 245.2

Glycine and Arginine

MS1 (Static) Collision cell MS2 (Scanning)

Figure 1.11 A schematic representation of the daughter or product ion scan in

daughters of 132.2 15/15, 15/15, collision 15, cone 20

30.4 41.3 48.3

Arginine is another anomally from the normal fragmentations described above.

Amino Acid Sigma # Mass of ester +H Preferred loss

mass spectrometer tuning .dbf (TDAT) or .ipr (Ethernet)

mass spectrometer acquisition .mdb (TDAT) or .exp (Ethernet)