R-Biopharm Rhne

GB02/V2

Mycotoxin
Technical Manual

R-Biopharm Rhne Mycotoxin Technical Manual

No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any
means, electronic, mechanical, photocopying, recording or otherwise, without prior permission of the publisher
(R-Biopharm Rhne Ltd, Block 10, Todd Campus, West of Scotland Science Park, Acre Road, Glasgow, Scotland,
G20 0XA).
Limit of Liability / Disclaimer of Warranty:
While the publisher and author have used their best efforts in preparing this manual, they make no representations
or warranties with respect to the accuracy or completeness of the contents and specifically disclaim any implied
warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales
representatives or written sales materials. The advice and strategies contained herein may not be suitable for your
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any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or
other damages.
For general information on our products and services or for technical support, please contact:
R-Biopharm Rhne Ltd
Block 10 Todd Campus
West of Scotland Science Park
Acre Road, Glasgow
Scotland G20 0XA
Tel: +44 (0) 141 945 2924
Fax: +44 (0) 141 945 2925
[email protected]
www.r-biopharm.com

Contents

1.

Introduction to Mycotoxins

2.
2.1
2.2
2.3
2.4
2.5
2.6
2.7

Testing for Mycotoxins

Legislation of Mycotoxins
Sampling
Detection of Mycotoxins
Principle of Immunoaffinity Columns
Principle of Derivatisation
Derivatisation of Aflatoxins
Products Available from
R-Biopharm Rhne

6
6
6
7
7
8
8

3.

3.1

3.2
3.3
3.4
3.5

Introduction to Terminology Used

in Mycotoxin Detection
10
Abbreviations Used in Mycotoxin
Detection
10
Mycotoxin Levels
10
Weights
11
Volumes
11
Dilutions
12

4.
4.1
4.2

Handling Mycotoxins
Hazards
Decontamination

13
13
13

5.
5.1

5.2

5.3

5.4

5.5

5.6

Calibration Curves
An Example Calibration Curve for
Aflatoxin Detection
An Example Calibration Curve for
Deoxynivalenol Detection
An Example Calibration Curve for
Fumonisin Detection
An Example Calibration Curve for
Ochratoxin Detection
An Example Calibration Curve for
T-2 & HT-2 Detection
An Example Calibration Curve for
Zearalenone Detection

14

18

6.

Reporting Results

19

7.

7.1
7.2
7.3

Determining the Concentration of

Toxin Present
AFLAPREP An Example
DONPREP An Example
EASI-EXTRACT AFLATOXIN
An Example

R-Biopharm Rhne Mycotoxin Technical Manual

15
16

7.4

7.5

7.6
7.7

EASI-EXTRACT T-2 & HT-2

An Example
EASI-EXTRACT ZEARALENONE
An Example
FUMONIPREP An Example
OCHRAPREP An Example

8.
8.1
8.2
8.3
8.4
8.5
8.6

8.7

Validating a Method
Precision
Repeatability (r)
Reproducibility (R)
Limit of Detection
Limit of Quantification
Determination of LOD and LOQ of
an HPLC System
Accuracy

23
23
23
23
24
24

9.

How to Spike a Sample

25

10.

General Guidelines on
Improving Recoveries

26

11.

11.1
11.2
11.3

11.4
11.5
11.6

General Guidelines on
Improving Chromatograms
High Baseline Noise
Mobile Phase Contamination
Contamination Downstream from
the Pump
Poor Reproducibility for Peak Areas
Peak Broadening and Peak Splitting
Ghost Peaks

21
22
22
22

24
24

27
27
27
27
27
27
28

17
17
18

20
21
21

12. HPLC Troubleshooting

29
12.1 Introduction
29
12.2 When to Adjust the Mobile Phase
30
12.3 Solvents and Mobile Phase
31
12.4 Pump
32
12.5 Column and Column Heater
34
12.6 Detector
34
12.7 Removing Bubbles from Detector Cell 36
12.8 Cleaning the Detector Flow Cell

Without Disassembly
36
12.9 Prevention of Air Bubbles
37
12.10 Maintenance of Fluorescence Detectors 37
13.

21

Glossary

38

Introduction to Mycotoxins

Mycotoxins are toxic chemical compounds

that are produced by certain fungi. They are
produced under specific conditions of moisture
and temperature and are generally associated
with diseased or mouldy crops. Not all fungi can
produce mycotoxins. Even those with the ability
to produce mycotoxins may not produce them all
of the time. Growth of the mycotoxin depends on
temperature, pH, humidity and the presence of
plant substrates.
There are many mycotoxins, however only a few
of them are regularly found in food and animal
feedstuffs. Most mycotoxins are chemically stable
so they tend to survive storage and processing
even at extreme temperatures, such as freezing.
Nevertheless, those that do occur in food have
great impact on the health of humans and can
cause significant economic losses in terms of plants
and livestock.

Mycotoxins are responsible for a diverse range

of toxic effects because their chemical structures
are very different from each other. Acute effects
require that high amounts of toxin are present in
the food being consumed and such incidents are
usually restricted to the less developed parts of
the world where resources for control are limited.
Chronic effects are caused by the accumulation
of a low level of toxin in the body over a long
period of time and can affect the long-term health
of the population. Some of the most common
mycotoxins are carcinogenic, genotoxic or may
target the kidney, liver or immune system.

Testing for Mycotoxins

2.1 Legislation of Mycotoxins

higher number of samples of lower weight as the

following pictures illustrate.

Legislation for mycotoxins is constantly changing

with more emphasis being placed on sampling,
method criteria and precision of results. The
number of commodities and mycotoxins that
are now covered under EU legislation is also
increasing. R-Biopharm Rhne has summarised all
legislation in an easy to follow booklet and poster
to help you understand current legislation, please
contact us for a copy or for further information.

2.2 Sampling
Mycotoxins are heterogeneously distributed
in naturally contaminated commodities. The
primary goal of sampling is to obtain a lab
sample that accurately represents the lot from
which it was taken. In order to obtain a sample
that is representative it is recommended to take a

Low Number of Samples = High Sample Variance

Sampling photographs supplied by:

Kim Esbensen
Applied Chemometrics, Analytical Chemistry, Applied
Biotechnology, Bioenergy & Sampling Research Group,
University of Aalborg Esbjerg, Denmark

R-Biopharm Rhne Mycotoxin Technical Manual

There are several possible sources of variability

when sampling a commodity for mycotoxins:
Sampling and sub sampling
Sample Preparation
Analysis
For further information on sampling plans see
http://eur-lex.europa.eu/en/index.htm.
There is also a Sampling Advice booklet for
mycotoxins available from the Food Standards
Agency in the UK, which provides a simple guide
to European Sampling of cereals, dried fruit, nuts,
spices, coffee, fruit juices and wines and offers
advice on how to record and interpret results.
This booklet is available from the Food Standards
Agency or from R-Biopharm Rhne on request.

High Number of Samples = Low Sample Variance

Testing for Mycotoxins

2.3 Detection of Mycotoxins

advantage in that they can be automated for largescale analysis of samples.

Mycotoxins occur in small quantities in foodstuffs

therefore their identification and quantitative
assessment requires sophisticated sampling,
preparation, extraction and analytical techniques
such as High pressure liquid chromatography (HPLC)
Immunoassays (ELISAs)
Lateral flow (LF) or dipsticks
Membrane cards
Mass spectroscopy (MS)
Thin layer chromatography (TLC)
Gas chromatography (GC)
Liquid chromatography with tandem
mass spectrometry (LC-MS/MS)

Following extraction of the toxin the sample

extract is filtered, diluted and passed through the
immunoaffinity column. Any toxin that is present
in the sample is retained by the antibody within
the gel suspension. The column is washed to
remove unbound material and the toxin is then
released by the antibody following elution with
solvent. The eluate can then be analysed by HPLC
or LC-MS/MS. The total extraction and clean-up
procedure takes approximately 20 minutes. The
result is improved clean-up and concentration
of the toxin from food and feed samples giving a
much cleaner chromatogram, therefore providing
more accurate and sensitive detection.

2.4 Principle of Immunoaffinity Columns

RBR mycotoxin immunoaffinity columns contain a
gel suspension of monoclonal antibodies specific
to the toxin of interest. The use of a monoclonal
antibody makes the test highly specific for a
target mycotoxin and offers improved sensitivity.
Immunoaffinity columns have the added
SAMPLE

R-Biopharm Rhne products are developed,

manufactured and dispatched under ISO 9001 and
ISO 13485 registered quality systems, guaranteeing
products of consistent quality that always meet our
performance criteria.

WASHING

Sample

The sample extract

(containing
the toxin)
The sample extract
(containing
toxin) is
is passedthe
through
passed
through the
the
column.
column
Mycotoxins

Washing

ELUTION
Elution

Passage of solvent
The antibodies isolate
andantibody
concentrate
the and Passage
throughofthe
column
The
isolates
pure
methanol
concentrates
the toxin
through the
toxin and retains
it inand denatures
the column
antibody
retains
it in the column denatures
the
column.
antibodythe
and
releasesand
the releases
toxin. the toxin
Other Material

Testing for Mycotoxins

2.5 Principle of Derivatisation

Some mycotoxins do not fluoresce naturally
therefore they require to be derivatised prior to
injection onto the HPLC system. Derivatisation
is the technique that transforms a chemical
compound into a product of similar chemical
structure for enhanced detection. Generally, a
specific functional group of the compound is
targeted during derivatisation. The resulting new
chemical compound is generally easier to detect
and can be used for quantification.

membrane. These layers are sandwiched between

rigid plastic housings. The KOBRA CELL is fitted
between the HPLC column and the detector and
generates the derivatisation agent, bromine, online from potassium bromide and nitric acid which
are present in the mobile phase. The derivatisation
results in a significant increase in the fluorescent
signals of the modified forms of aflatoxin B1
and G1 allowing improved detection using a
fluorescence detector.

2.6 Derivatisation of Aflatoxins

When testing for aflatoxins, only B2 and G2
fluoresce brightly under UV light and therefore can
easily be detected by HPLC with a fluorescence
detector. Aflatoxins B1 and G1 do not fluoresce to
such a high degree naturally and therefore must be
derivatised. The KOBRA CELL is a unique system,
which offers a popular derivatisation method when
testing for aflatoxins. The KOBRA CELL is an
electrochemical cell consisting of a platinum
working electrode and a stainless steel auxiliary
electrode separated from each other by a

Derivatisation without KOBRA CELL

R-Biopharm Rhne Mycotoxin Technical Manual

Derivatisation with KOBRA CELL

Testing for Mycotoxins

2.7 Products Available from

R-Biopharm Rhne
Immunoaffinity, solid phase and molecularly
imprinted polymer columns are available for the
quantitative analysis of the following mycotoxins
both individually or in combination when used in
conjunction with HPLC or LC-MS/MS Aflatoxin B1, B2, G1, G2 and M1
Deoxynivalenol
Fumonisin B1 and B2
Ochratoxin A
Patulin
T-2 & HT-2
Zearalenone
Standards and reference materials:
RBR manufacture and supply a range of mycotoxin
standards and reference materials for use with
HPLC. Please contact your local R-Biopharm
distributor for further information.
KOBRA CELL for derivatisation of
aflatoxins B1 and G1:
The only electrochemical cell recommended in EC
standard methods and used by key institutions,
food companies and government laboratories
worldwide.

Additional products for analysis

of mycotoxins:
RBR also provide a number of screening tests such
as cards and ELISA systems for the detection of a
range of mycotoxins.

Introduction to Terminology Used in Mycotoxin Detection

3.1 Abbreviations Used in Mycotoxin

Detection

micro

IAC

immunoaffinity column

nano

dH2O

distilled / deionised water

milli

MeOH methanol

litre

ACN

acetonitrile

gram

PBS

phosphate buffered saline

ppb

parts per billion

AA

acetic acid

ppm

parts per million

repeatability

ppt

parts per trillion

reproducibility

3.2 Mycotoxin Levels

When talking about mycotoxins, parts-per
notation is routinely used, which helps to describe
low value proportions in measured quantities.
Common parts-per notation that are used in
science are parts per million (ppm), parts per
billion (ppb) and parts per trillion (ppt).
To convert parts per million and parts
per billion:

Parts per million:

1 part per 1,000,000 parts or one part in 106

g / ml = mg / l
g / ml = mg / L
g / g = g / ml = ng / mg
1 g in 1,000 g or 1 kg = 1,000 ppm

For example, if we have 1 mg / g, this is 1000 x

greater than g / g; therefore it is equal to
1000 ppm.
Parts per billion:

1 part per 1,000,000,000 parts or one part in 109

g / L = ng / ml
ng / g = g / kg
1 g in 1,000 g or 1 kg = 1 ppb

R-Biopharm Rhne Mycotoxin Technical Manual

10

1 ppm = 1,000 ppb = 1,000,000 ppt

0.001 ppm = 1 ppb = 1000 ppt
0.000001 ppm = 0.001 ppb = 1 ppt
1 ppm = 1,000,000 ppt

Summary:
ppm --> ppb --> ppt
(each differing by a factor of 1,000)

Introduction to Terminology Used in Mycotoxin Detection

3.3 Weights
The gram is a unit of mass and is the most
commonly used unit of measurement for solid
material.
To convert grams and milligrams:

To convert milligrams to nanograms:

1 g = 1,000 mg

(g x 1,000 = mg)

1 mg = 1,000,000 ng

(mg x 1,000,000 = ng)

1 mg = 0.001 g

(mg / 1,000 = g)

1 ng = 0.000001 mg

(ng / 1,000,000 = mg)

To convert grams and micrograms:

To convert micrograms to nanograms:

1 g = 1,000,000 g

(g x 1,000,000 = g)

1 g = 1,000 ng

(g x 1,000 = ng)

1 g = 0.00001 g

(g / 1,000,000 = g)

1 ng = 0.001 g

(ng / 1,000 = g)

To convert grams and nanograms:

1 g = 1,000,000,000 ng

(g x 1,000,000,000 = ng)

1 ng = 0.000000001 g

(ng / 1,000,000,000 =g)

Summary:
g --> mg --> g --> ng
(each differing by a factor of 1,000)

To convert milligrams to micrograms:

1 mg = 1,000 g

(mg x 1,000 = g)

1 g = 0.001 mg

(g / 1,000 = mg)

3.4 Volumes
The litre is a unit of volume and is the most
commonly used unit of measurement for liquid
material.
To convert litres and millilitres:

To convert millilitres to micro litres:

1 L = 1,000 ml

(L x 1,000 = ml)

1 ml = 1,000 l

(ml x 1,000 = l)

1 ml = 0.001 L

(ml / 1,000 = L)

1 l = 0.001 ml

(l / 1,000 = ml)

To convert litres and micro litres:

Summary:

1 L = 1,000,000 l

(L x 1,000,000 = l)

L --> ml --> l
(each differing by a factor of 1,000)

1 l = 0.00001 L

(l / 1,000,000 = L)

11

Introduction to Terminology Used in Mycotoxin Detection

1 In 2 Dilution Or Doubling Dilution Or

Serial Dilutions

3.5 Dilutions
The following are examples of percentage solutions
used in mycotoxin testing:
1 % Sodium Bicarbonate Soution
1 g of sodium bicarbonate in 100 ml water.

A serial dilution can be used to accurately create

diluted solutions and is routinely used in producing
calibration curves. For example:
Vial 1: Measure 2 ml of 100 % methanol.

2 % Sodium Bicarbonate Solution

2 g of sodium bicarbonate in 100 ml water.
60 % Methanol
60 ml of methanol plus 40 ml of water.

Vial 3: Take 1 ml from vial 2 and add 1 ml of

water (equivalent to 25 % methanol).

80 % Methanol
80 ml of methanol plus 20 ml of water.

Vial 4: Take 1 ml from vial 3 and add 1 ml of

water (equivalent to 12.5 % methanol).

Methanol : Acetic Acid (98 : 2 (v/v))

98 ml of methanol plus 2 ml of acetic acid.

R-Biopharm Rhne Mycotoxin Technical Manual

Vial 2: Take 1 ml from vial 1 and add 1 ml of

water (equivalent to 50 % methanol).

12

Handling Mycotoxins

4.1 Hazards

4.2 Decontamination

Mycotoxins are very hazardous substances. Only

laboratories equipped to handle toxic materials
and solvents should perform analyses. Suitable
protective clothing, including gloves, safety glasses
and lab coats should be worn throughout the
analysis.

Prior to disposal, excess standard solutions should

be treated with at least one-tenth their volume of
5 % sodium hypochlorite. Labware and
contaminated waste should be immersed in 5 %
sodium hypochlorite solution for 30 minutes
followed by the addition of 5 % acetone for
30 minutes. Flush with copious amounts of water
before disposal. After decontamination labware
should be thoroughly washed. Incinerate waste if
regulations permit.

Flammable solvents should be stored in an

explosion-proof cabinet. Use a chemical hood and
protective equipment as applicable.
The columns contain 0.01 % (w/v) thimerosal. Skin
or eye splashes should be washed immediately
with quantities of water. Contact your local
R-Biopharm distributor for a Material Safety Data
Sheet for further information if required.

13

Calibration Curves

The sample components are eluted as Guassian

shaped peaks in the chromatogram. The
retention times provide the qualitative aspect
of the chromatogram. The retention time of a
compound should be the same under identical
chromatographic conditions. The peak height, or
peak area, is related to the quantity of analyte. For
determination of the actual amount of analyte, the
area or height is compared against standards of a
known concentration.
100 %

will produce a series of readings. For most analyses

a plot of response versus concentration will create
a linear relationship. The response of the unknown
can then be measured using the calibration curve.
The calibration curve when plotted should be
linear, and deviations from this straight line give
an indication about the precision of the result.
For example, a correlation of >98 % will indicate
that all standards produced create a good straight
line and with good certainty in the result. With a
correlation of <98 %, there will be less certainty
about the results.

Peak Height

W5 %

5%

LW

RW

A calibration curve is used to determine the

concentration of a substance in an unknown
sample by comparing the unknown to a set of
standard samples of known concentration.
The calibration curve is a plot of how the
instrument responds, the so-called analytical
signal, changes with changing concentration of
analyte. A series of standards across a range of
concentrations will be prepared, preferably with
serial dilutions, with the middle standard usually
matching the concentration of interest. Analysing
each of the standards using the chosen technique

R-Biopharm Rhne Mycotoxin Technical Manual

14

Certain criteria must be met in developing a

calibration curve:
It is recommended to run at least a 3 - 5 point
calibration curve.
In constructing a calibration curve the levels
of the calibration standards should bracket or
include the range of expected results /
measurements. For example, if the sample is
20 ppb the curve should be 5, 10, 20, 40 and
80 ppb.

Calibration Curves

5.1 An Example Calibration Curve for

Aflatoxin Detection
Take 5 ml methanol and place in a 5 ml amber
vial.
Remove 400 l.
Add 400 l of 1,000 ng/ml total aflatoxin
standard to give an 80 ng/ml solution.

HPLC Standard 4
Take 2.5 ml of the 80 ng/ml solution and put in
a 5 ml amber vial.
Add 2.5 ml water to give a 40 ng/ml standard.
This is equivalent to 1 ng per aflatoxin (4 ng
total) per 100 l injection.

HPLC Standard 2
Take 2.5 ml of standard 3 and put in a 5 ml
amber vial.
Add 2.5 ml 50 % methanol to give a 10 ng/ml
standard.
This is equivalent to 0.25 ng per aflatoxin
(1 ng total) per 100 l injection.

HPLC Standard 3
Take 2.5 ml of standard 4 and put in a 5 ml
amber vial.
Add 2.5 ml 50 % methanol to give a 20 ng/ml
standard.
This is equivalent to 0.5 ng per aflatoxin (2 ng
total) per 100 l injection.

HPLC Standard 1
Take 2.5 ml of standard 2 and put in a 5 ml
amber vial.
Add 2.5 ml 50 % methanol to give a 5 ng/ml
standard.
This is equivalent to 0.125 ng per aflatoxin
(0.5 ng total) per 100 l injection.

15

Calibration Curves

5.2 An Example Calibration Curve for

Deoxynivalenol Detection
Diluent solution 15 % methanol.
HPLC Standard 5
Take 5 ml of diluent solution and place in a 5 ml
amber vial.
Remove 400 l.
Add 400 l of 50 g/ml deoxynivalenol solution
to give a 4 g/ml (4 ppm = 4,000 ng/ml)
standard.
This is equivalent to 400 ng per 100 l injection.
HPLC Standard 4
Take 2 ml of standard 5 solution and put in a
5 ml amber vial.
Add 2 ml of diluent solution to give a 2 g/ml
(2 ppm = 2,000 ng/ml) standard.
This is equivalent to 200 ng per 100 l injection.
HPLC Standard 3
Take 2 ml of standard 4 and put in a 5 ml amber
vial.
Add 2 ml of diluent solution to give a 1 g/ml
(1 ppm = 1,000 ng/ml) standard.
This is equivalent to 100 ng per 100 l injection.

R-Biopharm Rhne Mycotoxin Technical Manual

16

HPLC Standard 2
Take 2 ml of standard 3 and put in a 5 ml amber
vial.
Add 2 ml of diluent solution to give a 0.5 g/ml
(0.5 ppm = 500 ng/ml) standard.
This is equivalent to 50 ng per 100 l injection.
HPLC Standard 1
Take 2 ml of standard 2 and put in a 5 ml amber
vial.
Add 2 ml of diluent solution to give a 0.25 g/ml
(0.25 ppm = 250 ng/ml) standard.
This is equivalent to 25 ng per 100 l injection.

Calibration Curves

5.3 An Example Calibration Curve for

Fumonisin Detection
Take 7.5 ml of acetonitrile : methanol : water
(25 : 25 : 50 (v/v/v)) and place in a 10 ml amber
vial.
Remove 200 l.
Add 200 l of 150,000 ng/ml fumonisin standard
to give a 4,000 ng/ml solution.
HPLC Standard 3
Take 500 l of the 4000 ng/ml solution and put
in a 5 ml amber vial.
Add 1.5 ml of 50 % methanol to give a
1,000 ng/ml standard.
This is equivalent to 100 ng per 100 l injection.

HPLC Standard 2
Take 1 ml of standard 3 and put in a 5 ml amber
vial.
Add 1 ml of 50 % methanol to give a
500 ng/ml standard.
This is equivalent to 50 ng per 100 l injection.
HPLC Standard 1
Take 1 ml of standard 2 and put in a 5 ml amber
vial.
Add 1 ml of 50 % methanol to give a 250 ng/ml
standard.

5.4 An Example Calibration Curve for

Ochratoxin Detection
Take 5 ml of methanol and place in a 5 ml amber
vial.
Remove 500 l.
Add 500 l of 1,000 ng/ml ochratoxin standard
to give a 100 ng/ml solution.
HPLC Standard 4
Take 100 l of the 100 ng/ml solution and put
in a 5 ml amber vial.
Add 1.4 ml of acetic acid : methanol (2 : 98 (v/v)).
Add 1.5 ml of water to give a 3.33 ng/ml
standard.
This is equivalent to 0.33 ng per 100 l
injection.
HPLC Standard 3
Take 1.5 ml of standard 4 and put in a 5 ml
amber vial.
Add 750 l of acetic acid : methanol (2 : 98 (v/v)).
Add 750 l of water to give a 1.67 ng/ml
standard.
This is equivalent to 0.167 ng per 100 l
injection.

17

HPLC Standard 2
Take 1.5 ml of standard 3 and put in a 5 ml
amber vial.
Add 750 l of acetic acid : methanol (2 : 98 (v/v)).
Add 750 l of water to give a 0.833 ng/ml
standard.
This is equivalent to 0.0833 ng per 100 l
injection.
HPLC Standard 1
Take 1.5 ml of standard 2 and put in a 5 ml
amber vial.
Add 750 l of acetic acid : methanol (2 : 98 (v/v)).
Add 750 l of water to give a 0.416 ng/ml
standard.
This is equivalent to 0.0416 ng per 100 l
injection.

Calibration Curves

5.5 An Example Calibration Curve for T-2

& HT-2 Detection
HPLC Standard 3
30 l of 10 g/ml T-2 & HT-2 standard should
be added to the glass tube.
Blow down to dryness, follow derivatisation as
per IFU. Reconstitute in 2 ml of mobile phase in
order to obtain 150 ng/ml solution.
This is equivalent to 15 ng/ml per 100 l
injection.

HPLC Standard 1
Take 1ml of standard 2 and put in a 5 ml amber
vial.
Add 1ml of mobile phase to give a 37.5 ng/m l
standard.
This is equivalent to 3.75 ng per 100 l
injection.

HPLC Standard 2
Take 1 ml of standard 3 and put in a 5 ml amber
vial.
Add 1 ml of 70 % acetonitrile to give a 75 ng/ml
standard.
This is equivalent to 7.5 ng per 100 l injection.

5.6 An Example Calibration Curve for

Zearalenone Detection
Take 3 ml of acetonitrile and place in a 5 ml
amber vial.
Remove 1.8 ml.
Add 1.8 ml of 1,000 ng/ml zearalenone
standard to give a 600 ng/ml solution.
HPLC Standard 4
Take 2 ml of the 600 ng/ml solution and put in
a 5 ml amber vial.
Add 2 ml of water to give a 300 ng/ml standard.
This is equivalent to 30 ng per 100 l injection.

HPLC Standard 2
Take 2 ml of standard 3 and put in a 5 ml amber
vial.
Add 2 ml of 50 % acetonitrile to give a
75 ng/ml standard.
This is equivalent to 7.5 ng per 100 l injection.

HPLC Standard 3
Take 2 ml of standard 4 and put in a 5 ml amber
vial.
Add 2 ml of 50 % acetonitrile to give a
150 ng/ml standard.
This is equivalent to 15 ng per 100 l injection.

HPLC Standard 1
Take 2 ml of standard 2 and put in a 5 ml amber
vial.
Add 2 ml of 50 % acetonitrile to give a
37.5 ng/ml standard.
This is equivalent to 3.75 ng per 100 l
injection.

R-Biopharm Rhne Mycotoxin Technical Manual

18

Reporting Results

All results should be expressed in parts-per

notation and in order to accurately report results
the analyst should account for any losses during
the method. It is recommended that a reference
sample of the same commodity type as the
material being tested is run through the complete
immunoaffinity column procedure. The
recoveries obtained with the spiked sample can
then be used to correct the results obtained with
the test sample.
For example, known reference sample is at 10 ppb,
results obtained from HPLC were 8 ppb. This is
equivalent to 80 % recovery. For all future samples,
similar to the reference standard you can add 20 %
onto the result in order to account for losses, i.e. if
you obtain a value of 2 ppb and you know this is
only 80 % recovery, adding 20 % gives a value of
2.5 ppb.

19

Current legislation states that for results to be

accepted recoveries should fall within the
following range:
Commodity

All Foods

Total
Acceptable
Contamination Range Of
Level
Recoveries
<1.0 ppb

50 - 120 %

1 - 10 ppb

70 - 110 %

>10 ppb

80 - 110 %

RBR products are developed, manufactured and

dispatched under an ISO 9001 and
ISO 13485 registered quality management
systems, guaranteeing products of consistent
quality that meet CEN specifications of 70 110 %
recovery.

Determining the Concentration of Toxin Present

When reporting results, they should be expressed

in parts per notation. However, with most HPLC
systems the value given will be expressed in
nanograms per injection. Therefore, you must
convert nanograms to the required parts-per
notation. In order to do this you must know the
gram equivalent of sample that was injected onto
the HPLC system. From there you can calculate a
multiplication factor to convert nanograms per
injection to parts-per notation.

In the example below 0.05 g of sample was

injected onto the HPLC system, which produced a
reading of 0.116 ng for aflatoxin G2. In order to
present this value as ppb (i.e. nanograms per gram
of sample) it is necessary to multiply the value
obtained for G2 (i.e. nanograms per 0.05 g of
sample injected) by a factor of 20 in order to
calculate how many nanograms would be present
in 1 g of sample. In this case, you would report the
contamination of the unknown sample as
0.1162 x 20 = 2.324 ppb aflatoxin G2.

Example printout from HPLC system:

Sample No.

Sample Name

Ret. Time
Min
G2
Fluorescence

Area
ml / *min
G2
Fluorescence

Height
mV
Fluorescence
G2

Amount
ng
G2
Fluorescence

STD 1
5 ng / ml

10.687

9.0839

18.98

0.2070

STD 2
10 ng / ml

10.340

11.7919

25.06

0.2687

STD 3
20 ng / ml

11.376

21.1858

42.71

0.4827

STD 4
40 ng / ml

10.987

43.6115

89.53

0.9937

unknown

10.139

5.1009

10.89

0.1162

unknown

9.998

4.2571

9.13

0.0970

Average:

10.207

10.845

21.515

0.236

Rel. Std. Dev:

6.222 %

127.789 %

125.188 %

127.789 %

R-Biopharm Rhne Mycotoxin Technical Manual

20

Determining the Concentration of Toxin Present

The following section works through some

examples for each toxin on how to determine the
multiplication factor for converting nanograms per
injection to parts per billion.

7.1 AFLAPREP An Example

7.3 EASI-EXTRACT AFLATOXIN

An Example

50 g of sample --> 500 ml of extraction buffer.

1 g of sample <-- 10 ml of filtrate taken and
passed through IAC.
Elute in total volume of 2 ml.
1 g of sample --> 2ml.
0.05 g <-- 100 l sample injected onto HPLC.
An estimated total of 0.05 g of sample would be
injected onto the system for analysis. However,
to report your results in ppb, i.e. ng/g of sample,
you should multiply the estimated ng of sample
detected by a factor of 20.
1 ng/g = 1 ppb.
1 g per 0.05 g = multiplication factor of 20.
Therefore, ng obtained x 20 = ppb.

50 g of sample <-- 100 ml of extraction buffer.

1 g of sample <-- 2 ml of filtrate taken and passed
through IAC.
Elute in total volume of 3 ml.
1 g of sample --> 3 ml.
0.033 g <-- 100 l sample injected onto HPLC.
An estimated total of 0.033 g of sample would be
injected onto the system for analysis. However,
to report your results in ppb, i.e. ng/g of sample,
you should multiply the estimated ng of sample
detected by a factor of 30.
1 ng/g = 1 ppb.
1 g per 0.033 g = 30.
Therefore, ng obtained x 30 = ppb.

7.2 DONPREP An Example

7.4 EASI-EXTRACT T-2 & HT-2 An Example

25 g of sample --> 200 ml of extraction buffer.

0.25 g <-- 2 ml of filtrate taken and passed

through IAC.
Elute in total volume of 1.5 ml.
Evaporate to dryness.
0.25 g of sample --> 1 ml reconstitution volume.
0.025 g <-- 100 l sample injected onto HPLC.

25 g of sample --> 200 ml of extraction buffer.

0.25 g <-- 2 ml of filtrate taken and passed
through IAC.
Elute in total volume of 1.5 ml.
Evaporate to dryness.
0.25 g of sample --> 1 ml reconstitution volume.
0.025 g <-- 100 l sample injected onto HPLC.

An estimated total of 0.025 g of sample would be

injected onto the system for analysis. However,
to report your results in ppb, i.e. ng/g of sample,
you should multiply the estimated ng of sample
detected by a factor of 40.

An estimated total of 0.025 g of sample would be

injected onto the system for analysis. However,
to report your results in ppb, i.e. ng/g of sample,
you should multiply the estimated ng of sample
detected by a factor of 40.

1 ng/g = 1 ppb.
1 g per 0.025 g = 40.
Therefore, ng obtained x 40 = ppb.

1 ng/g = 1 ppb.
1 g per 0.025 g = 40.
Therefore, ng obtained x 40 = ppb.

21

Determining The Concentration Of Toxin Present

7.5 EASI-EXTRACT ZEARALENONE

An Example

7.7 OCHRAPREP An Example

Example 1:

25 g of sample --> 125 ml of extraction buffer.

4 g <-- 20 ml of filtrate taken and diluted with PBS.
1 g <-- 25 ml of diluted filtrate taken and passed
through IAC.
Elute in total volume of 3 ml.
1 g of sample --> 3 ml.
0.033 g <-- 100 l injected onto HPLC.

50 g of sample --> 200 ml of extraction buffer.

1 g <-- 4 ml of filtrate taken and passed through
IAC.
Elute in total volume of 3 ml.
1 g of sample --> 3 ml.
0.033 g <-- 100 l injected onto HPLC.

An estimated total of 0.033 g of sample would be

injected onto the system for analysis. However,
to report your results in ppb, i.e. ng/g of sample,
you should multiply the estimated ng of sample
detected by a factor of 30.

An estimated total of 0.033 g of sample would be

injected onto the system for analysis. However,
to report your results in ppb, i.e. ng/g of sample,
you should multiply the estimated ng of sample
detected by a factor of 30.

1 ng/g = 1 ppb.
1 g per 0.033 g = 30.
Therefore, ng obtained x 30 = ppb.

1 ng/g = 1 ppb.
1 g per 0.033 g = 30.
Therefore, ng obtained x 30 = ppb.

Example 2:
7.6 FUMONIPREP An Example
25 g of sample --> 125 ml of extraction buffer.
2 g <-- 10 ml of filtrate taken and made up to 50 ml.
2 g --> 50 ml.
0.4 g --> 10 ml passed through IAC.
Elute in total volume of 3 ml.
0.4 g of sample --> 3 ml.
0.009 g <-- 70 l taken to derivatise.
0.009 g <-- 140 l total volume after derivatisation.
0.006 g <-- 100 l injected onto HPLC.
An estimated total of 0.006 g of sample would be
injected onto the system for analysis. However,
to report your results in ppb, i.e. ng/g of sample,
you should multiply the estimated ng of sample
detected by a factor of 166.67.
1 ng/g = 1 ppb.
1 g per 0.006 g = 166.67.
Therefore, ng obtained x 166.67 = ppb.

R-Biopharm Rhne Mycotoxin Technical Manual

22

10 g of sample --> 200 ml of extraction buffer.

0.25 g <-- 5 ml of filtrate taken and passed
through IAC.
Elute in total volume of 3 ml.
0.25 g of sample --> 3 ml.
0.0083 g <-- 100l injected onto HPLC.
An estimated total of 0.0083 g of sample would
be injected onto the system for analysis. However,
to report your results in ppb, i.e. ng/g of sample,
you should multiply the estimated ng of sample
detected by a factor of 120.
1 ng/g = 1 ppb.
1 g per 0.0083 g = 40.
Therefore, ng obtained x 120 = ppb.

Validating a Method

During the validation process it is important to

determine if the method is fit for purpose, i.e. do
the characteristics of the method meet with the
minimum characteristics required.
Validation is about establishing the analyte and
matrices to which the method can be applied,
establishing the LOD, LOQ, the concentration
range over which the method can be employed,
and the accuracy and precision of the method.
Once the characteristics of a method have been
established, they should be compared with the
method requirements for a particular purpose
to establish whether they are a good match. For
a method to be fit for purpose there should
be a good match between characteristics and
requirements.
In order to validate a method, reference material
or spiked samples should be used to determine
recoveries and the method should show good
precision, repeatability and reproducibility.
R-Biopharm Rhne would recommend that a
minimum of 10 columns should be used to
validate a method.

8.1 Precision
The precision of an analytical procedure expresses
the scatter between a series of measurements
obtained from multiple sampling of the same
sample under certain conditions. The precision is
expressed as % relative standard deviation (% RSD)
for a statistically significant number of samples,
e.g. >6.
% RSD = (STDEV / MEAN % RECOVERY) x 100
Precision is normally determined as repeatability (r)
and reproducibility (R)

23

8.2 Repeatability (r)

Expresses the precision under the same operating
conditions over a short interval of time. It is
therefore, a measure of precision using:
the same analyst.
the same apparatus.
close time interval between replicate analytes.
the same reagents.
the same laboratory, etc.
For example, validation of a method within a lab
would involve testing 10 columns on the same day.
During method validation, method repeatibility
and column repeatibility can be determined.
Column repeatibility is determined by running
the same extract through 10 columns. Method
repeatibility is determined by extracting the same
sample 6 times and passing each extract through
one column. RBR would recommend the % RSD is
<10 %.

8.3 Reproducibility (R)

Express the precision between different laboratories.
It is therefore, a measure of precision using:
different analysts.
different laboratories.
different equipment.
different sources of supply of reagents.
For example, large-scale validation as in a ring
trial would involve several labs testing the same
method.

Validating a Method

8.4 Limit of detection

8.6 Determination of LOD and LOQ of an

HPLC system

The limit of detection (LOD) for a given analyte

is the minimal amount needed to be able
to distinguish the analyte signal above the
background detector noise. A peak is at the LOD
when the signal value measured at its maximum is
3 times the noise signal.

The LOD / LOQ varies depending on method,

HPLC conditions, derivatisation technique and the
HPLC equipment used.

LOD = 3 x signal / noise ratio

LOQ = 3 x LOD

8.5 Limit of quantification

8.7 Accuracy
The limit of quantification (LOQ) of an analyte
is the lowest amount of that analyte in a sample
which can be accurately measured with reliability.
The LOQ is generally taken as being 3 times the
LOD.

R-Biopharm Rhne Mycotoxin Technical Manual

24

The accuracy of an analytical method expresses

the closeness of a measured level of an analyte to
the known true level of that analyte. Accuracy is
frequently reported as % recovery. The recovery
provides an insight into the extraction efficiency of
a method.

How to Spike a Sample

Reference material can be purchased, however

it can be difficult to source the matrix of interest
therefore samples can be spiked in order to
validate a method.
To spike a sample a known blank sample should
be taken and the required volume of sample
should be weighed out into a suitable container.
The calculated volume of standard should then be
added to the sample and left overnight in the dark.
If the sample is naturally contaminated it is advised
not to spike the sample as discrepancies in results
can occur. However, if you are required to spike a
contaminated sample, i.e. in FAPAS rounds, you
should spike the sample at approximately 10 times
the level of contamination.

40 ml of milk was taken and spiked at 25 ppt

using a 40 ng/ml Standard:

C1 x V1 = C2 x V2
40 ng/ml x V1 = 25 ppt (0.025 ppb) x 40 ml
40 ng/ml x V1 = 0.025 ng/ml x 40 ml
40 ng/ml x V1 = 1 ng
V1 = 1 ng per 40 ng/ml
V1 = 0.025 ml

25 g of turmeric was taken and spiked at 5 ppb

using a 1,000 ng/ml Standard:

C1 x V1 = C2 x V2
1,000 ng/ml x V1 = 5 ppb x 25 g
1,000 ng/ml x V1 = 5 ng/ml x 25 g
1,000 ng/ml x V1 = 125 ng
V1 = 125 ng per 1,000 ng/ml
V1 = 0.125 ml

C1 x V1 = C2 x V2

C1 = Concentration of standard to be used

for spiking
V1 = Volume of standard to be added to

sample
C2 = Required concentration of sample
V2 = Required volume of sample

25

5 g of maize was taken and spiked at 2 ppm

using a 1,000 ng/ml Standard:

C1 x V1 = C2 x V2
1,000 ng/ml x V1 = 2 ppm (2,000 ppb) x 5 g
1,000 ng/ml x V1 = 2,000 ng/ml x 5 g
1,000 ng/ml x V1 = 10,000 ng
V1 = 10,000 ng per 1,000 ng/ml
V1 = 10 ml

10

General Guidelines on Improving Recoveries

CEN specifications state that recoveries should

be between 70 - 110 %. If however, recoveries
obtained are below the acceptable 70 % check the
following
Check the pH of the sample lies within the
range 7.0 7.8. A pH out-with this range can
affect the performance of the antibody.

Use the recommended IFU or application notes.

If there is no specific application note available
contact R-Biopharm Rhne for assistance.

Check the volume applied to the column. The

sample should not take longer than 30 minutes
to pass through the column. If necessary filter
or centrifuge the sample after dilution with PBS
to remove precipitates. Exposing the antibody
to low levels of solvent over a prolonged period
of time can have an adverse effect.

Use KOBRA CELL for derivatisation of aflatoxins.

Check the concentration of solvent being passed

through the immunoaffinity column. Higher
solvent concentrations can decrease the
performance of the antibody. The following
solvent concentrations should not be exceeded
Aflatoxin

Ochratoxin
Zearalenone
Fumonisin

T-2 & HT-2

R-Biopharm Rhne Mycotoxin Technical Manual

30 % methanol
2.5 % acetonitrile
5 % acetonitrile
15 % acetonitrile
5 % methanol
5 % acetonitrile
18 % methanol

26

Use the standards provided by RBR.

Ensure that backflushing has been carried out
during the elution step. Backflushing increases
the amount of time the solvent is in contact with
the antibody gel, ensuring that all of the toxin is
eluted.

11

General Guidelines on Improving Chromatograms

Each of the following items needs to be optimised

in order to generate a satisfactory separation and
a chromatogram that is suitable for quantitative
purposes:

Mobile phase composition.

Columns and packing dimensions.

Injection volume.

Sample pre-treatment.

Mobile phase flow rate.

Column temperature.

Detector parameters.

11.1 High baseline noise

Stop the pump.
If noise disappears, the cause lies in the pump
(refer to section 12 or contact your HPLC
provider).
If the noise remains the cause lies in the detector
(refer to section 12 or contact your HPLC
provider).

11.2 Mobile phase contamination

Increase the detector wavelength.
If the drift decreases there is a problem with the
mobile phase.
Prepare fresh mobile phase. Use higher grade
solvents.

27

11.3 Contamination downstream

from the pump
Remove the device that appears to be
contaminated.
If baseline settles, clean the device.

11.4 Poor reproducibility for peak areas

When the retention time reproducibility is
normal the pump is most likely not the cause.
The source of the problem is likely to be found in
the auto sampler
OR
Due to deterioration of the sample itself.

11.5 Peak broadening and peak splitting

Problems with peak resolution indicate:
Improper mobile phase (adjust mobile phase).
Deteriorated column.
A failed fitting.
Excessive injection of sample.

11

General Guidelines on Improving Chromatograms

11.6 Ghost peaks

A ghost peak is a peak that has come from
a previous injection, or from contaminated
equipment or mobile phase.
Cause

Confirmation Method

What do to

Contamination of pump or
equipment upstream from pump.

Clean using an acid or alkaline

solution:
Acid : Nitric acid 4 - 6 N.
Alkaline : Methanol 5 - 10 % W/V.

Mobile phase contamination.

Use higher grade

solvents / water.

Contamination in auto sampler.

R-Biopharm Rhne Mycotoxin Technical Manual

Watch for the ghost peaks

while injecting mobile phase.

28

Clean equipment.

12

HPLC Troubleshooting

12.1 Introduction
Critical criteria for successful HPLC analysis are:

Correct mobile phase.

Analytical column and guard column in
good condition (change every 2 - 3 months).
Lamp for the detector in good condition
(<1,000 hours use).

Once the conditions for HPLC analysis have

been optimised the next critical step is sample
preparation. Hence, to optimise the clean-up effect
of the immunoaffinity column all samples must
be analysed using protocols recommended in
the application notes. This is especially critical for
samples with high pigmentation e.g. spices and
dried fruits.
Note: R-Biopharm Rhne only recommend the
following information as a general guide to
troubleshooting your HPLC system. For further
information we would advise you to contact your
HPLC provider.

Solvent Rack & Reservoires

Pump

Auto sampler

Column heater and column

Detector

29

12

HPLC Troubleshooting

12.2 When to Adjust the Mobile Phase

More water should be added:
If unable to get baseline separation
between peaks. This will allow complete
elution of the first toxin before elution of the
second toxin begins. The run time will increase
but good peak separation is essential for
accurate chromatography.

If toxins are eluted too quickly and merge with

the solvent front. The toxins will be retained on
the column for longer and allow the baseline to
settle before elution of the toxins.

If there is a contaminating peak from the matrix.

R-Biopharm Rhne Mycotoxin Technical Manual

30

More solvent should be added when:

Peaks are too broad (starting to merge).
Retention time is too long.
There is a contaminating peak from the matrix.
Adjusting the mobile phase is not always
successful. Sample preparation and clean up
must be optimised to improve HPLC
chromatography.

12

HPLC Troubleshooting

12.3 Solvents and Mobile Phase

Solvents that are not miscible will partition from
each other, resulting in incomplete removal of the
original solvent from the HPLC system. Therefore,
when changing the mobile phase the following
sequence of solvents should be used:

Aqueous Solvents
Buffer Solutions or Salt Solutions

Water

Ethanol, Isopropanol or Acetone

Organic Solvents
Acetonitrile or Methanol

Chloroform or Ethyl Acetate

n-Hexane or ISO-Octane

31

12

HPLC Troubleshooting

12.4 Pump
Abnormal operating pressure of the pump may be
observed and can either be:
Too high
Too low
Variable

Pump Pressure Too High

Cause

Confirmation Method

What To Do

Downstream side of pump

is clogged, e.g. clog in auto
sampler, column, filter,
tubing, fittings.

Remove and examine devices and /

or parts that may be clogged in the
flow route.

Refer to the appropriate

manual.

Line filter is clogged (behind

pump outlet).

Operate the pump without anything

connected to the outlet port. If the
pressure is 20 kg / cm2 or higher
when pumping at 10 ml / minute,
the line filter is clogged.

Replace the line filter.

Damper or tubing is clogged

inside the pump.

Remove the outlet port filter and

pump the solvent. If the pressure
is 20 kg / cm2 or higher when
pumping at 10 ml / minute, the
internal damper or internal tubing is
clogged.

Requires engineer or pump to

be returned for service.

R-Biopharm Rhne Mycotoxin Technical Manual

32

12

HPLC Troubleshooting

Pump Pressure Too Low

Cause

Confirmation Method

Pump head contains air

bubbles.
Inlet filter is clogged (use of
buffer solutions often leads
to clogging).

What To Do
Open the purge valve and
remove the air bubbles.

When disconnected solvent drips

rather than flows from inlet line.

Replace the inlet filter.

Plunger seal is leaking.

Tighten the pump head

securing screws.
Replace plunger seal.

Inlet tube fitting is leaking.

Tighten the fitting at the pump

inlet.
Refer to appropriate manual.

Leak at downstream side

of pump (auto sampler,
column, fittings).
Pump head deterioration.

Perform pressure test (put screw

into pump outlet, at 0.1 ml / min
pressure should reach 500 kg / cm
smoothly).

Clean pump head thoroughly

with acid or alkaline solution.

Confirmation Method

What To Do

Variations in Pump Pressure

Cause
Pump head contains
bubbles.

Open the purge valve and

remove the bubbles.

Inlet filter is clogged.

Replace the inlet filter.

Insufficient degassing
of the solvent.

Degas the solvent.

Inlet tube fitting is leaking.

Tighten the fitting.

Plunger seal is leaking.

Tighten the pump head

securing screws.
Replace the plunger seal.

Pump head deterioration.

Perform pressure test.

Clean the pump head

thoroughly using an acid or
alkaline solution.

Abnormal plunger
movement.

Sound of metal contacting metal

can be heard.

Loosen the pump head

securing screws slightly.

33

12

HPLC Troubleshooting

12.5 Column and Column Heater

Always use a column heater to prevent:
Periodic baseline drift.
Lengthening of retention time.
Shortening of retention time.
Poor reproducibility of retention time.

12.6 Detector
Dealing with noise:
Cause

Confirmation Method

What to do

Bubble in cell.

Monitor the baseline with

pump on and off. Bubbles
cause abnormally large baseline
variation when the flow rate
changes.

1. Remove air from the

solvent using a degasser.

Suspension in cell.

Use a magnifying glass to

carefully examine the cell.
Again monitor baseline as for
'Bubble in cell' method.

Clean the cell: wash through or

disassemble.

Fluid is leaking from cell.

Remove the cell from the

instrument. Examine the cell and
the inside of the instrument.

1. Clean and tighten

connections.

2. Install a back pressure coil.

2. Replace damaged cell.

Energy reduction.

If the output is 0.4 V at 250 nm,

the lamp or mirror has
deteriorated.

Solvent absorption or cell

window contamination.

Replace the lamp

(1000 hours) or mirror.
(5000 hours).
1. Use high grade solvents.
2. Check the wavelength.
3. Wash through cell.

R-Biopharm Rhne Mycotoxin Technical Manual

34

12

HPLC Troubleshooting

Electronic noise.

Ground the instrument.

1. Solvent absorption.

1. Use high grade solvents.

2. Cell window contamination.

2. Clean the cell.

3. Column equilibrium.

1. Increase in bubble size.

Use 'Bubble in cell' method.

1. Remove air from the

solvent using a degasser.

2. Column contamination.
2. Install a back pressure coil.
3. Reduce flow of solvent.
3. Wash / change column.
4. Check pump and / or
blocked tubing.

Decrease in bubble size.

Use 'Bubble in cell' method.

1. Remove air from the

solvent using a degasser.
2. Install a back pressure coil.

35

12

HPLC Troubleshooting

12.7 Removing Bubbles from

Detector Cell

12.8 Cleaning the Detector Flow Cell

Without Disassembly

Increase the pump flow.

If bubbles remain:
Connect the outlet port of the pump directly to
the inlet of the cell and pump at 2 ml per
minute.

Contamination of the cell wall can sometimes be

removed by passing the following organic solvents
or strong nitric acid through the cell. When
sufficient cleaning cannot be obtained using this
method, disassemble and clean the flow cell.

If bubbles remain:
Attach a stainless steel tube to the outlet of the
flow cell and cover with a teflon tube. Bubbles
may be compressed and pass through the cell
if pressure is applied within the cell. Apply
pressure on syringe and continually bend and
release the teflon tube. If the baseline moves in
the negative direction the bubbles in the cell are
decreasing in size. Continue until all bubbles
have passed through the cell.

Replace the solvent in the flow cell

with distilled water.
Pass concentrated nitric acid through the cell.
(4 N, 1 ml / minute for 30 minutes)
Pass distilled water through the cell.
(1 ml / minute for 30 minutes)
Pass acetone through the cell.
(1 ml / minute for 30 minutes)
Pass distilled water through the cell.
(1 ml / minute for 30 minutes)

Caution: Disconnect the column before running

this decontamination protocol.

R-Biopharm Rhne Mycotoxin Technical Manual

36

12

HPLC Troubleshooting

12.9 Prevention of Air Bubbles

12.10 Maintenance of Fluorescence

Detectors

Bubbles can be prevented by always using a

degasser.

The lamp should be changed every 1000 hours.

This can be done in-house and the maintenance
manual usually has a very good description of
how to change the lamp. If having difficulty
changing the lamp watch the engineer change
it or have the engineer supervise changing the
lamp.

Solvent Absorption can be prevented by

always using HPLC grade solvent.
Cell suspensions and tube blockages can be
prevented by flushing the system with water
following use of a mobile phase which contains
salt.
When using a methanol wash before or after a
mobile phase containing salt take care to ensure
the salt is soluble in methanol. If uncertain use
water before and after running the mobile phase.
Advisable washing regime would be 30 minutes
water at 1 ml per minute followed by 30 minutes
methanol at 1 ml per minute.
When dealing with organic mobile phase
replace the solvent inside the cell with a solvent
which is miscible with the organic mobile phase
and water, do not mix liquids which are not
miscible (e.g. chloroform and water). Use
methanol as a solvent which is miscible with
both.

37

Before changing the lamp set excitation wave

length to 400 nm and emission wavelength to
500 nm. Record the excitation and emission out
put (V) values. These values should increase
when the new lamp is installed.
The dust filters should be changed each time
the lamp is changed to prevent dust building
up inside the detector. When the air filter is
clogged the lamp cooling efficiency is reduced,
contributing to faster deterioration of the lamp.
The best way to clean the dust from the inside
of the detector is to use an air pump and blow
the dust away. This avoids touching any parts
and the risk of causing damage.

13

Glossary

Analyst:

An individual carrying out analyses on samples.

Analyte:

The substance which is to be separated during chromatography.

Antibody:

Are found in the blood and are used by the immune system to identify
foreign objects.

Antigen:

A substance that promotes the production of antibodies and can cause

an immune response.

Baseline:

Is the name given to the part of the chromatogram that represents any
time period during which only mobile phase is passing through the
detector.

Commodity:

A substance such as grain, spice, etc, to be tested. Interchangeable with

matrix.

Concentration:

Concentration is the measure of how much of a given substance there

is mixed with another substance.

Chromatogram:

The visual output of the chromatograph. In the case of an optimal

separation, different peaks or patterns on the chromatogram
correspond to different components of the separated mixture.

Chromatography:

IUPAC definition Chromatography is a physical method of

separation, in which the components to be separated are distributed
between two phases, one of which is stationary whilst the other moves
in a definite direction.

Detector noise:

A change in baseline that is not associated with eluted solute.

Extraction:

Method to separate compounds.

Gradient system:

Can deliver more than one solvent to the HPLC system.

HPLC:

High Performance Liquid Chromatography or High-Pressure Liquid

Chromatography is a form of chromatography applying high pressure
to drive the solutes through the column faster.

Isocratic system:

One solvent HPLC system.

Matrix:

Substance such as grain, spice, etc, to be tested. Interchangeable with

commodity.

R-Biopharm Rhne Mycotoxin Technical Manual

38

13

Glossary

Mobile phase:

The phase which moves in a definite direction transporting the sample

through the HPLC system.

Mycotoxin:

A toxin produced by a fungus.

Naturally
contaminated:

Toxin found naturally in a sample.

Pump:

Delivers a continuous pulse-free flow of mobile phase to the injector,

column and detector.

Precision:

Characterises the degree of mutual agreement among a series of

individual results.

Recovery:

The percentage of toxin detected which can be calculated from testing

a known sample.

Repeatability:

The variation in measurements taken by a single person or instrument

on the same item and under the same conditions.

Reproducibility:

The ability of a test or experiment to be accurately reproduced,

or replicated, by someone else working independently.

Retention time:

The characteristic time it takes for a particular analyte to pass through

the system, from the column inlet to the detector, under set conditions.

Sample:

The matter analysed in chromatography. It may consist of a single

component or it may be a mixture of components.

Solute:

Refers to the sample components in partition chromatography.

Solvent:

A liquid that dissolves a solid or liquid solute, resulting in a solution.

Spiked sample:

A sample that has been contaminated by the addition of a known

concentration of toxin.

Spike solution:

A solution with a known concentration of toxin that is added to a

sample to form a spike sample.

Standard curve:

A standard curve is a quantitative research tool. A method of plotting

assay data that is used to determine the concentration of a substance.

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R-

R-Biopharm Rhne Ltd

Block 10 Todd Campus
West of Scotland Science Park
Acre Road, Glasgow G20 0XA
Phone: +44 (0) 141 945 2924
Fax: +44 (0) 141 945 2925
www.r-biopharm.com
R-Biopharm Rhne Mycotoxin Technical Manual

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