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LC/MS: AN ESSENTIAL TOOL IN DRUG DEVELOPMENT

Article in International Journal of Advances in Pharmaceutical Analysis · December 2012

DOI: 10.7439/ijapa.v1i2.10

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 International Journal of Advances in Pharmaceutical Analysis
IJAPA Vol. 1 Issue 2 (2011) 24-37
Journal Home Page http://www.ijapa.ssjournals.com

LC/MS: AN ESSENTIAL TOOL IN DRUG DEVELOPMENT

Pavan Kumar Baluguri*, Srikanth Nama, Babu Rao Chandu Bhargavi sakala
Donbosco College Of Pharmacy,5thMile, Guntur. Andhra Pradesh, India

ABSTRACT
The combination of high-performance liquid chromatography and mass spectrometry (LC/MS) has
had a sign cant impact on drug development over the past decade. Continual improvements in LC/MS
interface technologies combined with powerful features for structure analysis, qualitative and
quantitative, have resulted in a widened scope of application. These improvements coincided with
breakthroughs in combinatorial chemistry, molecular biology, and an overall industry trend of
accelerated development. The use of high-performance liquid chromatography combined with mass
spectrometry (HPLC–MS) or tandem mass spectrometry (HPLC–MS–MS) has proven to be the
analytical technique of choice for most assays used in various stages of new drug discovery. A
summary of the key components of HPLC–MS systems, as well as an overview of major application
areas that use this technique as part of the drug discovery process, will be described here. This review
will also provide an introduction into the various types of mass spectrometers that can be selected for
the multiple tasks that can be performed using LC–MS as the analytical tool. The strategies for
optimizing the use of this technique and also the potential problems and how to avoid them will be
highlighted.

Keywords: LC-MS analyticaltool, HPLC-MS, HPLC-MS-MS, LC-MS

1. Introduction: properties (e.g. physiological solubility,

Current trends in drug development emphasize permeability and chemical stability) of these
high volume approaches to accelerate lead new chemical entities (NCEs) are assessed and
candidate generation and evaluation. Drug HPLC–MS is often used for these assays
discovery-based technologies that involve
bimolecular screening and combinatorial 2. Principles of LC–MS: The elements of an
chemistry paved the way, resulting in shortened LC–MS system include the auto sampler, the
timelines and the generation of more HPLC system, the ionization source (which
information for more drug candidates. The interfaces the LC to the MS) and the mass
impact on the overall drug development cycle spectrometer. Ideally, these elements are all
has been sign cant, creating unprecedented under the control of a single computer system.
opportunities for growth and focus, particularly HPLC is a common technique, so it will not be
in the analytical sciences The use of high- described here. It should be noted that to
performance liquid chromatography combined interface HPLC with MS, there are some
with mass spectrometry (HPLC–MS) or tandem restrictions on the flow rate and mobile phases
mass spectrometry (HPLC–MS–MS) has proven that can be used .Typical reversed phase HPLC
to be the analytical technique of choice for most systems connected to MS would use some
assays used in various stages of new drug combination of water and either methanol or
discovery 1.New drug discovery can be defined acetonitrile as the mobile phase. There are
as the process whereby compound libraries are limitations on the mobile phase modifiers; for
screened, then hits are selected and modified to example, in most cases the modifiers have to be
become leads that are optimized until a volatile. Mobile phase modifiers are chemicals
compound emerges that can be developed into a added to the mobile phase that are used
drug candidate. HPLC–MS and HPLC–MS–MS primarily to improve the chromatography of the
are used for the analysis of newly synthesized Ana lutes of interest. Typical mobile phase
compounds that become part of a compound modifiers would include ammonium acetate,
library. These assays check that the correct acetic acid and formic acid. There are multiple
compound has been synthesized and that the articles that focus on the HPLC parameters that
purity is sufficient to be used in the library. In a are important in LC–MS assays.
second stage, various physical and chemical 3. Types of mass spectrometers:

Corresponding Author*: [email protected] 24

Review Article Baluguri et al/2011

There are many types of mass spectrometers capable of generating well over 2,000
available for interfacing with HPLC2 , one of the compounds per year, using a variety of
more common systems used for HPLC–MS is automated synthesis technologies. If traditional
the single quadruple mass spectrometer; this approaches to analytical support were
system will provide a mass spectrum for each maintained, then analysts would outnumber
chromatographic peak that elutes from the LC chemists by nearly 40 to 1!
column and is analysed by the MS system. The * An earlier availability of information leads to
second type of system shown is the time-off faster decision making.
light (TOF) mass spectrometer, which has the * Integration of instrumentation with
added capability of providing a higher mass information network is a popular approach for
resolution spectrum from each component that is combining high throughput analytical
assayed. The third system shown is the triple information generation with Drug-candidate
quadruple MS–MS system, which is most often screening.
used for bio analytical assays but can also be * Software is a powerful resource for the
used for metabolite identification assays 3. The coordination of analysis events and the
fourth MS system is called an ion-trap mass management and visualization of data.
spectrometer and has the unique capability of 4.2. Partnerships and Acceptance: What has
producing MS data that are important when happened in the pharmaceutical industry during
performing structural elucidation assays. In this relatively short time span is truly
addition to these four types of mass remarkable. With the advent of advanced
spectrometers, there are a growing number of technologies responsible for increasing the rate
additional types, including hybrid systems that of sample generation, there is strong motivation
have unique capabilities. Hybrid mass to respond with LC/MS-based analysis
spectrometers combine two of the basic types of Techniques. The understanding of principles,
mass spectrometer to make a specialty system; fundamentals, operation, and maintenance
an example of a hybrid mass spectrometer is the enabled researchers to improve analytical
‘Q-TOF’ MS–MS system, which combines a performance. Here, the power of `seeing is
quadruple mass spectrometer with a TOF mass believing'' led to lower barriers of acceptance as
spectrometer. well as to a new breed of practitioners

4. Integration of LC/MS into Drug 5. Systematic LC/MS Metabolite

Development Identification in Drug Discover:
Liquid chromatography/mass spectrometry The study of how a drug is absorbed, distributed,
(LC/MS)-based techniques provide unique metabolized, and eliminated by the body is a
capabilities for pharmaceutical analysis. LC/MS vital but costly and time-consuming step in the
methods are applicable to a wide range of drug discovery process. Metabolism can dictate
compounds of pharmaceutical interest, and they the rate of absorption into the body, lead to the
feature powerful analytical gores of merit production of new and possibly toxic species, or
(sensitivity, selectivity, speed of analysis, and activate the drug. For example, morphine’s
cost effectiveness). These analytical features effect is primarily due to one of its glucuronide
have continually improved, resulting in easier to conjugate metabolites .Until recently, metabolite
use and more reliable instruments. These identification only took place once a compound
improvements were timely and coincided with had been chosen for drug development.
the aforementioned developments in the However, the discovery of a toxic metabolite
pharmaceutical industry. can set a research program back significantly.
4.1. Emerging Analytical Needs: Perhaps a As a result, many pharmaceutical companies are
major cause of these opportunities is the fact that now conducting metabolite identification studies
the rate of sample generation far exceeded the in the early phases of drug candidate selection.
rate of sample analysis. To put this factor in But this is no easy task. The metabolism of a
perspective, consider the following example that drug within a test animal can be extremely
deals with combinatorial chemistry. Prior to the complex, involve multiple enzymatic pathways,
advent of combinatorial chemistry technologies, and lead to a range of compounds with varying
a single bench chemist was capable of concentrations (7). Other drugs have one or two
synthesizing ca. 50 ®nil compounds per year, major metabolic pathways that dominate their
depending on the synthesis. Today, chemists are metabolism, but several minor pathways can

25
Review Article Baluguri et al/2011

produce at least one metabolite. We have seen metabolites form from a single compound.
>30 detectable, and theoretically identifiable,

Figure1: Diagram showing how the Automated Legend Identification System (ALIS) system
uses MS for high throughput activity screening.
5.1. Chemistry-library synthesis: In a typical the analysis of Caco-2samples 34. In one
arrangement, an HPLC–MS system will be used example, Fung et al.35 described a higher
to provide information on compound identity throughput assay for Caco-2 samples that was
and purity as a first step in building a discrete capable of handling 100 compounds per week,
compound library. A common system would based on HPLC–MS–MS using a triple
have an in-line UV detector for the purity quadruple MS system. One of the tools required
assessment, as well as the MS system for the to assay this many compounds was an MS
compound identity confirmation. The mass method development tool, provided as part of
spectrometer is often either a single quadruple or the software package by the instrument vendor;
a TOF system. These systems are often highly this tool is important for applications that require
automated; for example, Isbell et al. 24 describe a the system operator to develop discrete MS–MS
high throughput procedure that combines an transitions for each compound that is assayed.
automated compound purification procedure Another way in which Fanged al. 32 improved
with the compound analysis step. This process is the assay efficiency was by reducing the number
based on a combination of HPLC–MS with of samples that had to be injected through the
software-controlled fraction collectors that are elimination of a calibration curve. The Caco-2
triggered on the basis of the observed or results for a given compound (permeability
expected m/z response of the compound of calculation) are based on the ratio of two
interest to make the whole process highly samples; therefore, they demonstrated that the
automated. ratio of the MS responses of the two samples
5.2. In vitro screening: One of the more could be used instead of the ratio of the
commonly used in vitro screens is the human concentrations of the two samples, thereby
colon adrenal carcinoma cell line (Caco-2), eliminating the need for a calibration curve for
which is used for the measurement of the each compound. Another higher throughput in
permeability potential of a compound – one of vitro assay is the one used to assess a potential
the aspects of the absorption process 31.There are of a compound to inhibit of one or more of the
several reports on how LC–MScan be used for human cytochrome P450 isoforms (CYPs); this

26
Review Article Baluguri et al/2011

step is important to determine a compound’s automated assay based on a single quadruple

potential for drug–drug interactions. In this case, LC–MS system that used an automated data
the assay can be optimized to be high throughput analysis system and could test 75 compounds
because the analysis does not measure the per week for metabolic stability. Wring et al. 36
compound that is being tested. There have been described a system for metabolic stability that
several reports in recent years describing how included automated liquid handling and an LC–
HPLC–MS–MS can be used for providing MS assay based on a triple quadruple mass
higher throughput assays to support various spectrometer that was capable of handling 50
CYP screens for enzyme inhibition .In a recent compounds per week. More recently, Xuet al. 30
example, Pang et al. described a high throughput reported a highly automated system based on
assay based on HPLC–MS–MS to screen for robotic sample preparation of the test compound
five important CYP ribozymes – CYP 3A4, plates, as well as the human liver microsomal
CYP2D6, CYP2C9,CYP2C19 and CYP1A2. incubations with three time points selected
5.3. High-throughput screening: Although (5,15and 30 min) in addition to the time zero
HTS is usually performed using various point. All samples were measured in triplicate to
fluorescence procedures to look for compounds improve the reliability of the results. The assay
that have the desired in vitro activity, there have was based on a single quadruple LC–MS system
been some examples where mass spectrometry that included an eight-probe auto sampler and
has been used for this step in the new drug eight HPLC columns in a parallel mode. This
discovery process As discussed by Flab and system was able to assay 240 samples per hour,
Jindal 26, the strategy uses mixtures of which enabled up to 176 test compounds to be
compounds plus a target protein to identify evaluated per day. In addition to the high
potential lead compounds for a therapy based on throughput assay, the authors described
compound so that interact with the target automated data processing tools that enabled the
protein. This high throughput screen uses analysis of this data in a short time frame.
HPLC–MS to identify the ligand compounds; in
this case the mass spectrometry system is a TOF. 7. LC/MS Metabolite Identification in Drug
5.4. ADME–PK screening: Perhaps the most Discovery:
common use for LC–MS in new drug discovery MS has emerged as an ideal technique for the
is for the various ADME studies that make up identification of such structurally diverse
the majority of the effort provided by the drug metabolites. When coupled with on-line HPLC,
metabolism and pharmacokinetic (DMPK) the technique is extremely robust, rapid,
groups in their participation in the process 27–30. sensitive, and easily automated 11. Not
There are several in vitro ADME screens, surprisingly, LC/MS and LC/MS/MS have
followed by various in vivo preclinical ADME– become the methods of choice for
PK screens. These screens are almost always pharmacokinetic studies, yielding concentration
supported by LC–MS assays. versus time data for drug compounds from in
vivo samples such as plasma 2.Instruments and
6. In vitro screen is the metabolic stability software packages. Furthermore, just as in a
assay: police investigation Nevertheless, identifying
The goal of this assay is to provide a prediction metabolites remains a time-consuming process
of the in vivo intrinsic clearance of a compound. because arrange of instrumental techniques and
Aniseed and Thacker recently reviewed the area software applications are needed to obtain the
of metabolic stability assays and also provided a appropriate data. The analyst must be quite
good summary of the relative importance of the experienced to handle these various the data is
major CYP isoforms for human metabolism. rarely obvious or completely conclusive, but
Generally, the metabolic stability assays are rather requires previous experience to unravel it.
based on the incubation of the compound in the For all these reasons, interpreting the data is
presence of either human liver microtomes or typically the largest bottleneck in metabolite
human hepatocytes; in either case, the samples identification.
are typically assayed using a compound-specific In this report, we present the choices and
analysis based on HPLC–MS–MS (usually with decisions involved in metabolite identification
a triple quadruple system). Several examples of and how they can be merged into a systematic
high throughput metabolic stability assays based approach. The discussion covers the tools and
on LC–MS have been reported. In an early techniques available to the mass spectra merits,
example, Korfmacher et al. 44 described an the complementary natures of different types of

27
Review Article Baluguri et al/2011

mass spectrometers, and innovative software 9.1. Tandem MS: Tandem MS is the
that reduces the size of data sets. This new cornerstone of metabolite identification
12,14
approach greatly increases sample throughput .Tandem MS actually covers a variety of
and turnaround time for useful information. A scanning techniques including product ion,
complete metabolite identification study is also precursor ion, and neutral-loss scanning.
described, which uses all of the techniques and Tandem mass spectrometers usually contain an
instrumentation discussed. isolation stage and a fragmentation stage within
the same device. Although many different ways
The flow chart in the basic approach to exist to complete a tandem MS experiment, all
metabolite identification: of them follow the same basic series of events.
First, the ion of interest is isolated on the basis
of its m/z ratio and then passed into the collision
cell, a region of local high pressure. (In trapping
instruments, the isolation and fragmentation
normally take place within the same space
between electrodes, and the stages are separated
by time rather than space.) The collision cell is
filled with an inert gas such as argon or helium,
and a voltage is applied. Energized ions collide
with the target gas, and each collision imparts a
small amount of energy to the ion until sufficient
energy is deposited to cleave an internal bond or
bonds. The resulting ion fragments pass out of
the cell and into the detector.
9.2. Targeted product ion analysis: In drug
discovery, each new compound normally arises
from a small alteration to an initial lead template
8. Systematic approach to metabolite that was discovered in a receptor-binding assay.
identification and characterization: The final compound may bear very little
The approach assumes that it is possible to similarity to that initial lead, but it has a lineage
predict numerous common alterations to the that stretches back to the initial compound.
drug such as oxidation and oxidative Consequently, after the first one or two
conjugation. Typically, the compounds under compounds have been fully assayed, any
investigation in a single drug discovery project structural regions or 'soft spots' within the
is structurally very similar because a prospective compounds in a drug series, which are highly
lead compound's structure is 'fine-tuned' for susceptible to metabolism, are determined.
better selectivity and potency toward the Common metabolic alterations can be predicted,
receptor of interest. Therefore, the mass and a list of expected metabolites can be
spectrometric that analyses compounds in an compiled, on the basis of a previously analysed
analogous series quickly learns the most series. By combining this list with a list of
common metabolic alterations to the parent suspected metabolites identified by precursor
structure and any novel modifications. This and neutral-loss scan data, a series of ions can be
experience and information allows for a guided targeted for production analysis.
analysis with targeted searches for expected Any type of mass spectrometer capable of
metabolites. (To ensure that novel metabolites product ion scanning can be used at this point,
caused by less-common metabolic pathways are including a triple quadruple. Thus, all of the
detected precursor ion scanning is available.) experiments discussed so far can be completed
using a single instrument. However, to localize
9. Techniques and instrumentation: alterations within a molecule to a specific site,
An exhaustive description of all available MS additional stages of tandem MS (MS) require a
instruments and their modes of operation is more specialized mass spectrometer.
beyond the scope of this article and not 9.3. Determining sites of modification:
necessary to appreciate the strategies being Multiple stages of MS can provide large
applied here. However, a basic knowledge of a amounts of structural information regarding each
few types of major systems and their important analyse, thereby allowing for a more detailed
modes of operation is required and included. characterization of the metabolites15,16.

28
Review Article Baluguri et al/2011

Completing MS experiments requires a mass h urine sample. No further sample preparation

spectrometer that can capture and store ions 13, 15, was performed. Minimal sample clean-up is
16
. While the ions are stored, they can be used because the nature, number, and
subjected to excitation and collisional concentrations of metabolites present are
fragmentation. The trapping instrument can then unknown, and it is therefore impossible to
capture the resultant fragment ions, which can determine if any will be lost during a sample
then be forced to undergo further fragmentation. preparation procedure.
The second-generation mass spectrums will now 9.4.1. Step 1: Collecting precursor ion scan
give structural information regarding the isolated and neutral-loss data: After sample
fragment, allowing easier characterization of preparation, a list of potential metabolites in the
that ion. Because this procedure can be applied bile and urine sample needs to be compiled from
to each of the initial parent ion fragments, precursor ion and constant neutral-loss analyses
detailed structural information can be acquired completed on a triple quadruple mass
rapidly. This technique often allows the isolation spectrometer. If the compound had been radio
of a small region of the parent ion molecule that labeled, an online radioactivity detector would
has been modified and, in some cases, even the have also been used to mark time points where
individual atom that are different. At present, the the compound elutes within the chromatographic
Fourier transform (FT) and the quadruple ion run. Figure A demonstrates the type of data
trap mass spectrometers are the only trapping recorded in a precursor scan. At least eight
mass spectrometers available FTMS instruments apparent metabolites are evident in a single
are not yet capable of high throughput on a experiment, solely on the basis of the fragment
regular basis because they are expensive and ions' similarity to those produced by the parent
require skilled operators. Thus, cheaper and compound standard. An additional five possible
simpler quadruple ion trap mass spectrometers metabolites were found in the urine via
are typically used for these types of trapping precursor ion scanning data (data not shown).
experiments. The total analysis took 44 min and required one
9.4. Identification of a dosed compound: With bile and one urine injection to detect all 13
the basics of instrumentation and techniques metabolites. Typically, more than a single series
described, a full metabolite identification study of fragment ions are scanned, corresponding to
of a compound dosed in vivo can be examined. characteristic fragments from the top, middle,
Sprague-Daley male rats were dosed and lower portions of the parent compound.
intravenously with Schering-Plough discovery Common alterations to these fragments, such as
compound SCH X at 2 mg/kg body weight and hydroxylation, are monitored by looking for
orally at 10 mg/kg. Urine and bile were collected precursors of the analyse at the native fragment
over 24 h at regular time intervals. Before mass and fragments
analysis, the individual time point samples for Corresponding to the metabolic hydroxylations,
each animal were pooled for each dosing region which are 16 Da higher.
to generate one 0- to 24-h bile and one 0- to 24-

29
Review Article Baluguri et al/2011

Figure.2: Precursor ion and constant neutral-loss scan experiments.

(a) Precursor ion scan and (b) constant neutral-loss (176 Da) scan experiments using rat bile. In both cases, the
data are shown as an individual mass trace. Each trace is labeled with the putative metabolite’s potential
identity on the basis of their detected mass
9.4.2. Step 2: Product ion analysis of included+16 Da (hydroxylation), '14 Da (DE
potential metabolites: The product ion data in methylation), and +32 Da (hydroxylation). In
the next step were acquired on the Q-TOF addition, any putative metabolites identified
mass spectrometer, but a triple quadruple or by the precursor ion and neutral-loss scans
quadruple ion trap mass spectrometer could also underwent product ion analysis. The rapid
also have been used. The Q-TOF instrument scanning abilities of the Q-TOF instrument
was used to examine a list of 'expected' allow several product ion experiments to be
metabolites that had been previously observed performed in rapid succession in a single LC
for analogy from this compound series and run.

30
Review Article Baluguri et al/2011

Figure.3. Product ion scan data used to identify several putative metabolites first detected in
Figure 2. (a) Mass chromatograms for each of the examined metabolites. The individual traces are labeled with
the potential identity of the metabolite on the basis of information from Figure 2 experiments. (b) Tandem MS
spectra from peaks Ian II.

10. Structural elucidation of metabolites by to determine the type of metabolite modification

MS.: Because of constraints due to space, and indicate which parts of the molecule were
expense, and complexity, the quadruple ion trap changed. In most cases however, especially for
is the instrument of choice for MS experiments molecules that only break into a few large
in a typical metabolite identification laboratory. fragments, MS4 or evenMS5 may be required to
For an MS experiment, the masses of the intact locate the site of modification, an O-glucuronide
metabolite and a related fragment ion are metabolite had to undergo MS5 before locating
required. Because this information comes from the site of oxidation. In each trace, the fragment
the MS/MS experiments described in Step 2, ion that will undergo additional fragmentation is
MS3 experiments can be set up directly on the highlighted. Thus, this technique quickly
instrument without any further pinpoints a very small area that has been altered.
experimentation.MS3 often gives data sufficient

Figure.4.MS1 through MS5 data from the analysis of an O-glucuronide metabolite.

Each trace shows the next level of tandem MS in the sequential cleavage observed in the metabolite

31
Review Article Baluguri et al/2011

10.1. Accurate mass measurement: The power software to fewer structural possibilities.
of accurately determining the mass of a Furthermore, the operator normally has prior
metabolite lies in being able to determine a list knowledge of the parent compound's structure,
of possible empirical formulae. Obviously, the which limits the number and types of atoms the
more accurate the mass measurements, the fewer software should take into consideration. Until
degrees of freedom are available to the software recently, accurate mass measurement required
calculating a formula, and the shorter the list of double-sector instruments or Fourier transform
possibilities. Accurate mass product ion data ion cyclotron resonance instruments, which are
further limits the number of possible formulae very large, complex, and expensive. However,
by providing data that are even more specific to the advent of less complex and expensive bench
the empirical formulae calculator. Product ion top TOF instruments and hybrid instruments
experiments narrow the site of modification to such as Q-TOF has allowed the application of
small portion of the molecule and an accurate accurate mass measurement in the metabolite
mass determination of this fragment limits the identification laboratory.

Figure.5.: Accurate mass data from monkey bile.

a) Raw data and (b) accurately Measured data after the external reference mass calibration was applied. The
partial chemical structure shows the two most likely alterations that may have occurred

11. Still searching: greater of the overall radioactivity detected in

The strategies detailed in this report are the that experiment. However, inevitably, other
result of more than two years of work. They are low-level metabolites are automatically
not meant to exhaustively identify every detected using the prescribed strategies.
metabolite present within in vivo samples, but 12. 10 years of MS instrumental
rather to rapidly and accurately identify sites of developments – Impact on LC–MS/MS in
metabolic liability that might affect clinical chemistry:
pharmacokinetic measurement results and Within the past decade mass spectrometry (MS)
detect the formation of possibly toxic has entered the clinical laboratory and is now
metabolites. Therefore, only major metabolites being used for a wide range of applications.
are identified, but the definition of a major The technique can be considered essential for
metabolite differs widely among researchers. the determination of many clinically relevant
Because ~50%of our samples are radioactive, analyses in combination with either gas
our laboratory definition of 'major' includes any chromatography (GC) or liquid
resolved radioactive peak that constitutes 5% or chromatography (LC). The power of MS,

32
Review Article Baluguri et al/2011

especially when coupled to LC, is recognized Research scientists especially set their focus on
by clinical laboratories worldwide and the various desorption techniques. The
growing versatility of these systems puts combination of LC with matrix-assisted laser
clinical laboratories in a position where they desorption ionization (MALDI) started to
can provide a rapid response to changing emerge in the mid-1990s 18,19 and continuous
clinical needs. Even though it requires some effort was undertaken to further improve the
effort much needed assays can be developed in technique 20–25. Very recently LC–MS methods
the laboratory instead of waiting for a were described using desorption electro spray
manufacturer to respond. Furthermore it is ionization (DESI) 26 and direct analysis in real
undoubted that these techniques pro-vide a time (DART) 27,28 interfaces.
higher level of sensitivity and specificity in 12.2. Improvements in tandem mass
many cases compared to other analytical spectrometry technology: Since LC–MS/MS
techniques and that pateend care has benefited technology is increasingly used for quantization
from their use. Besides specificity and in clinical science, as well as in other fields of
sensitivity the ability of these techniques to science, there is a need for on-going
measure multiple analyses simultaneously is a improvements of the technology. A triple
tremendous benefit of LC coupled to MS quadruple instrument in SRM mode is the
methods since many other techniques are instrument-of-choice in routine and high-
limited to determine one analyse at a time. throughput quantitative clinical analysis.
Especially these multi-component methods can Commercial triple quadruple MS with
make the purchase of a liquid chromatography atmospheric pressure ionization (API) sources
tandem mass spectrometry (LC–MS/MS) are widely used nowadays. In the case of triple
instrument cost-effective. Improvements in quadruple instruments the most commonly
automation and software help clinical requested improvements were defined by
laboratories to deal with staffing and service Bennett to be: greater sensitivity, dynamic
issues. linear range, mass resolution, wider mass
12.1. Ion source developments: Coupling MS range, faster acquisition cycle time and reduced
to LC was a very important motivation in the cost of ownership. Advances in triple quadruple
development process of atmospheric pressure technology are challenging and focus remains
ion sources. Systems where the samples are in the source and interface regions to improve
introduced via a liquid stream achieved wide ruggedness and reduce matrix effects. Some
acceptance and commercial importance. Electro minor improvements in quadruple
spray ionization (ESI) and atmospheric manufacturing processes and RF power supply
pressure chemical ionization (APCI) are the stabilities enabled the production of a
liquid introduction ion sources which had the commercial system with enhanced mass
most commercial success and enormous resolution without significant losses in ion
improvements were made in the first 15 years transmission 51,52. On the other hand significant
after their invention during the mid-1980s. Two instrumental developments were achieved in
excellent reviews were published dealing with the last 10 years in the fields of other mass
the evolution of these ionization sources 16,17. analysers like linear ion traps and HRMS like
Based on these developments multimode ion quadruple QTOF and FT-MS based
sources were introduced on the market by instruments. Therefore the future of triple
various manufacturers. In addition the quadruples will be determined on the variable
atmospheric pressure photo ionization (APPI) how extensively the clinical field adopts to high
ion source was developed and improved within resolution, high mass accuracy instruments into
the last 10 years. Therefore more detailed their workflows and analytical requirements.
information about multimode and APPI ion Up to now, in case of triple quadruple
sources will be presented in the following instruments, mass resolution was typically
chapters and their suitability regarding their ignored in favour of the outstanding linearity
application to clinical chemistry will be and increased sensitivity due to the selectivity
discussed. Apart these commercially successful offered by tandem MS. Now new tasks are
LC–MS ion sources very creative approaches gaining more and more interest where
were investigated for the hyphenation of LC improved selectivity and full-scan data at low
with MS using other available ion sources. duty cycle times are crucial.

33
Review Article Baluguri et al/2011

13. Schematic of a quadruple linear ion trap and description:

Figure.6:TheTriplet OF MS technology features diagrammed.

(a) A detailed illustration of the major platform features. (b) An image of the machined Triplet OF MS
instrument platform.

Ion mobility-mass spectrometry: Interfacing al. 1 there was a considerably increase in

ion mobility spectrometry (IMS) with MS can interest within this research area. During the
provide significant advantages. The potential last 10 years instruments became
was understood early in the development of commercially available and both applications
IMS, and the coupling of the two techniques is and instrumental designs of IMMS are now
virtually as old as IMS itself. So ion mobility- one of the most rapidly growing areas of MS.
mass spectrometry (IMMS) cannot be As a matter of fact numerous articles, reviews
regarded as new, but after the demonstration and even books} are dealing with the topic of
of protein conformer separation by Calmer et IMMS.

34
Review Article Baluguri et al/2011

Schematic of an ambient-pressure IMS(toff)MS.

14. Progress in automation of LC-MS in standardization and quality assurance of small

laboratory medicine: molecule analytical routine methods the
Standard techniques of analyse detection in introduction of GC–MS as a reference method
clinical chemistry rely on indirect characteristics was an essential progress, in particular for
of an analytic, e.g. its absorption of light, endocrinology. However, for several reasons the
chemical reactivity or physical interaction with application of GC–MS remained restricted to
macro-molecules. In mass spectrometric few specialized institutions in laboratory
methods, in contrast, analyses are detected medicine (mainly toxicological laboratories,
directly from molecular characteristics as metabolism centres, and reference laboratories).
molecular mass and molecular disintegration The handling and maintenance of GC–MS
patterns. Thus, mass spectrometric techniques instruments is very demanding and time-
are very attractive for the quantification of consuming; sample preparation is very laborious
biomarkers or xenobiotic in the context of and includes sample extraction and analytic
diagnostic procedures, since those techniques reprivatisation; the analytical run times are long
can enable analyses of much higher specificity with a typical sample throughput of less than 50
compared to standard technologies such as samples per day
photometry or ligand binding tests. With gas
chromatography–mass spectrometry (GC–MS), 15. Future of LC-MS/MS application in
first mass spectrometric methods were laboratory medicine:
introduced to laboratory medicine about 40 At present only rather few clinical laboratories
years ago. GC–MS allowed the highly specific worldwide are equipped with LC-MS/MS
and sensitive quantification of thermo-stable systems, and in these laboratories this
molecules below a molecular weight of about technology typically makes up for less than 1%
500. Thus, GC–MS became a key technology, of all analyses. The success of efforts to
e.g. in toxicology. With respect to substantially improve the practicability and

35
Review Article Baluguri et al/2011

robustness of LC-MS/MS application by 5. Allanson JP, Biddlecombe RA, Jones AE,

automation will be crucial for a more Pleasance S. 1996.The use of automated
widespread application of this technology in the solid phase extraction in the `96well' format
future. But is application of LC-MS/MS in for high throughput bioanalysis using
laboratory medicine beyond the status quoi liquidchromatography coupled to tandem
really reasonable? Indeed LC-MS/MS can close mass spectrometry.RapidCommun Mass
substantial gaps in the parameter portfolio of Spectrom 10:811±816.
laboratory medicine 6. Allen MC, Shah TS, Day WW. 1998. Rapid
LC-MS/MS can allow routine analyses on a determination oforal pharmacokinetics and
reference method level of accuracy plasma free fraction usingcocktail
(incorporating isotope dilution technology) for approaches: methods and applicationPharm
important analysts for which immunoassays Res15:93±97.
offer critically limited accuracy or cannot be 7. Niessen, W.M. (2003) Progress in
applied at all (e.g., steroid hormones , plasma liquidchromatography-mass
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dimethyl arginine, microbial antigens). A. 1000,413–436
LC-MS/MS can allow really comprehensive 8. Korfmacher, W.A. et al. (1997) HPLC-
therapeutic drug monitoring (including API/MS/MS:a powerful tool for integrating
assessment of metabolisation) for a personalized drug metabolisminto the drug discovery
drug therapy; this is of utmost importance in the process. Drug Discov.Today2, 532–537
context of recent findings of pharmacokinetics 9. Hopfgartner, G. et al. (2002) High-
for a large variety of drugs. The availability of throughputquantification of drugs and their
companion testing will probably also becomes a metabolites inbiosamples by LC-MS/MS
key issue in the licensing of new drugs. and CE-MS/MS:possibilities and
LC-MS/MS enables highly multiplexed limitations. Ther. Drug Monit.24, 134–143
metabolic profiling which probably holds 10. White, R.E. (2000) High-throughput
substantial potentials for disease monitoring. screeningin drug
LCMS/ MS will probably be the analytical metabolismandpharmacokineticsupport of
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