Aflatoxin Analysis_Confermatory Detection Methods

Analysis of Aflatoxins -Confirmatory
methods
Thanuja Dissanayake
Research Scientist
Residue Analysis Laboratory
Industrial Technology Institute
Industrial Technology Institute 2023/06/30

Things to be done
 Why is it necessary to do confirmatory analysis?
 Sampling techniques for Aflatoxins
 Sample preparation for HPLC and LC-MS/MS method
 Laboratory safety
 Introduction to HPLC-FLD analysis?
 Introduction to LC-MS/MS analysis?
 Quantification
 Pros and cons of LC-MS/MS analysis
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Why is it necessary to do confirmatory analysis?
 False positive identifications of presumptive methods and ELISA
methods
 Stringent regulatory limits
 Decision making – batches, shipments and consignments

Test method
Sample extraction Sample cleanup
Detection
Sampling
Lot Sampler

Sampling techniques for Aflatoxins
Definitions
1. ‘Lot’ means an identifiable quantity of a food commodity delivered at
one time and determined by the official to have common
characteristics, such as origin, variety, type of packing, packer,
consignor or markings;
2. ‘Sublot’ means a designated part of a large lot in order to apply the
sampling method on that designated part; each sublot must be
physically separate and identifiable;
3. ‘Incremental sample’ means a quantity of material taken from a single
place in the lot or sublot;
4. ‘Aggregate sample’ means the combined total of all the incremental
samples taken from the lot or sublot;
5. ‘laboratory sample’ means a sample intended for the laboratory.

Sampling techniques for Aflatoxins Contd.
 Aflatoxins are very heterogeneously distributed in a lot, in
particular in a lot of food products with a large particle size
such as dried corn kernels and groundnuts.
 In order to obtain the same
representativeness, for batches with food products with large
particle size, the weight of the aggregate sample should be
larger than in case of batches with food products with a
smaller particle size.
 Since the distribution of mycotoxins in
processed products is generally less heterogeneous than in the
unprocessed cereal products, it is appropriate to provide for
simpler sampling provisions for processed products.

 Sampling shall be performed by an authorised person

 Each lot which is to be examined shall be sampled separately. Large
lots shall be subdivided into sublots to be sampled separately.
 In the course of sampling and preparation of the samples, precautions
shall be taken to avoid any changes,
 which would affect:
 the mycotoxin content, adversely affect the analytical determination
or make the aggregate samples unrepresentative;
 the food safety of the lots to be sampled.
 Also, all measures necessary to ensure the safety of the persons
taking the samples shall be taken.

Incremental samples
As far as possible incremental samples shall be taken at various places
distributed throughout the lot or sublot.
Preparation of the aggregate sample
The aggregate sample shall be made up by combining the incremental
samples.
Replicate samples
The replicate samples for enforcement, trade (defence) and reference
(referee) purposes shall be taken from the homogenised aggregate sample
Packaging and transmission of samples
Each sample shall be placed in a clean, inert container offering adequate
protection from contamination and against damage in transit. All necessary
precautions shall be taken to avoid any change in composition of the
sample, which might arise during transportation or storage.

 More details are given in EU commission regulation,
Commission Regulation (EC) No 401/2006 of 23 February 2006 laying
down the methods of sampling and analysis for the official control
of the levels of mycotoxins in foodstuffs (Text with EEA relevance)
Link
http://data.europa.eu/eli/reg/2006/401/2014-07-01

Laboratory Safety and cleaning
LAB WARE USED FOR NEGATIVE SAMPLES
 Prepare a solution consisting of dishwashing liquid and water.
Completely submerge the used glassware, funnels, beakers, etc.,
wash thoroughly, then rinse with clean water before reusing.
LAB WARE USED FOR POSITIVE SAMPLES

 Prepare a bleach solution consisting of 1 part bleach to 10 parts water
(e.g.,100 mL bleach to 1,000 mL water). Completely submerge the
used glassware, funnels, beakers, etc., and soak for at least 5
minutes. Remove items from the bleach/water solution, submerge in a
dishwashing liquid/water solution, wash thoroughly, and then rinse with
clean water before reusing.

DISPOSABLE MATERIALS
 Prepare a bleach solution consisting of 1 part bleach to 10 parts water
in a plastic container.
 Soak disposable materials, such as used columns, vials, filter, etc., for
at least 5 minutes. Pour off the liquid down the drain and place the
materials in a garbage bag and discard.

CLEANING AFLATOXIN SOLUTION SPILLS
• Perform the following procedures only while wearing disposable
impermeable gloves and chemical splash goggles.
• If hands become contaminated, wash immediately with undiluted
bleach followed by soap and water.
• Clean areas and materials contaminated by any aflatoxin solution or
positive (i.e., > 20 µg L-1) extraction solutions spills with bleach.
• The affected area should be completely covered with 5-6 percent
sodium hypochlorite (household bleach) dispensed from a plastic
wash bottle or spray bottle.
• Apply 10 parts of bleach to 1 part of spilled material and leave for at
least 5 minutes. Wipe up the bleach using an absorbent cloth or paper
towels. Place cleaning materials in a plastic waste bag, close tightly,
and discard.

Positive sample disposal
Negative Samples (< 20 µg L-1)
 Dispose of the excess sample extract solution in an approved waste
container. Place the sample slurry in a garbage bag for routine
disposal.
Positive Samples (>20 µg L-1)
 The sample extract and slurry left over after completion of the test
procedure must be decontaminated prior to disposal in a waste drum.
Once the sample analysis has been completed, using a plastic wash
bottle, add bleach to the slurry and allow to filter. When the filtration is
complete, dispose the slurry in a garbage bag.
 To decontaminate the sample extraction solution, add bleach equal to
one half of the volume remaining in the test tube. Pour the
decontaminated extraction solution into an approved waste container.

Sample preparation for HPLC and LC-MS/MS
method
 Quick, Easy, Cheap, Effective, Rugged and Safe –
QuEChERS
(Solid – Liquid Extraction, Dispersive solid phase
cleanup)
 Solid-Liquid extraction and Solid Phase Extraction cleanup-
SPE
 Solid-Liquid extraction and Immunoaffinity Cleanup – IAC

QuEChERS Method

QuEChERS Method

Solid-Liquid extraction and Solid Phase
Extraction cleanup- SPE
SPE is used for six main purposes in sample preparation:

• Removal of interferences
• Concentration or trace enrichment of the analyte
• Desalting
• Phase exchange
• In situ derivatization
• Sample storage and transport

Solid Phase Extractions
Interference retained
Analyte retained

Steps in SPE Process Symbol Legend
Sample Components
A = Analyte(s) of interest
W = Weakly retained, undesired
matrix component(s)
X = Intermediate undesired
matrix component(s)
Z = Strongly retained undesired matrix
component(s)
Solvents
C = Conditioning solvent
D = Equilibration solvent
L = Loading solvent
R = Rinsing (washing) solvent
E = Eluting solvent


Immunoaffinity Extraction/Cleanup
Immunoaffinity cleanup procedure
• Higher selectivity
• Higher sensitivity
• Low matrix interference

Introduction to HPLC

High Performance Liquid Chromatography
Injector
Mixer
Pumps
Column
Column
Detector
Detector
Waste
Solvents
Mobile phase

Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents

Mixer mAU
Pumps
Column
Detector
Solvents

Mixer mAU
Pumps
Column
Detector
Solvents

Mixer mAU
Pumps
Column
Detector
Solvents

Mixer mAU
Pumps
Column
Detector
Solvents

Mixer mAU
Pumps
Column
Detector
Solvents

Mixer mAU
Pumps
Column
Detector
Solvents

Mixer mAU
Pumps
Column
Detector
Solvents

Mixer mAU
Pumps
Column
Detector
Solvents

Mixer mAU
Pumps
Column
Detector
Solvents

Mixer mAU
Pumps
Column
Detector
Solvents

Mixer mAU
Pumps
Column
Detector
Solvents

Mixer mAU
Pumps
Column
Detector
Solvents

Mixer mAU
Pumps
Column
Detector
Solvents

Mixer mAU
Pumps
Column
Detector
Solvents

FLD Detectors
 High sensitivity for selective groups of compounds at ~fg level.
 By using a specific wavelength, analyte atoms are excited and then
emit light signal (fluorescence). The intensity of this emitted light is
monitored to quantify the analyte concentration.
40
Introduction to LCMS/MS

to - elution time of unretained peak
tR- retention time - determines sample identity
tR
Area or height is
proportional
tR to the quantity of analyte.
mAU
to
Injection time
Abundance at selected wavelength

Chromatogram + Mass Spectrum

Liquid Chromatography Mass Spectrometry
(LC-MS)

Comparison of Ionization Techniques
ElectroSpray

Triple Quadrupole MS
Schematic of a triple quadrupole mass spectrometer

MRM – Multiple Reaction Monitoring
Quantifier
Qualifier

MRM – Multiple Reaction Monitoring contd..

Detectors
1. Ion detection using an electron multiplier
CEM – Channel Electron Multiplier

2. Faraday cup

Derivatisation for HPLC/LCMS
 To permit analysis of compounds with inadequate stability
 To improve chromatographic behavior or detectability
 Can be done
 Pre column
 On-line
 Post column
 Derivatisation agents/ Fluorescence tags- OPA. Dancyl Chloride,
FMOC,
CHO
CHO
o-phthalaldhyde
(OPA)
Quantification

Pros and cons of HPLC & LC-MS/MS analysis
Pros
HPLC-FLD LC-MS/MS
Quantitative results can be obtained Quantitative results can be obtained
Can be consider as a confirmatory Gold standard Confirmatory
technique if IAC are used technique available
Multi-analyte analysis applications Ability of analyzing multi-analytes
are limited
Ability of working with multi-analyte
Muli-analyte standards can be used reference standard in different
only with the given conditions columns

Pros and cons of HPLC & LC-MS/MS analysis
Cons
HPLC-FLD  LC-MS/MS
Costly compare to presumptive tests Costly compare to presumptive tests
and HPLC
Required specific chemicals
Required specific chemicals
Required trained analysts
May give false-positive Required highly qualified & trained
analysts
identifications
Matrix effect can suppress the signal


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Analysis of Aflatoxins -Confirmatory

Industrial Technology Institute 2023/06/30

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Sample extraction Sample cleanup

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 Sampling shall be performed by an authorised person

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LAB WARE USED FOR POSITIVE SAMPLES

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SPE is used for six main purposes in sample preparation:

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Immunoaffinity cleanup procedure

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Start Injection time

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Abundance at selected wavelength

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