8. Ms Azel Swemmer - LC-MSMS, the new reference method ... - AFMA

LC-MS/MS, the “new” reference
method for mycotoxin analysis
What is necessary for the implementation
of the multi-residue mycotoxin method in
an analytical laboratory?
Azel Swemmer
azel@fdalab.co.za
AFMA Conference March 2009

Introduction
Aspects that will be covered:
• Analytical processes
• Requirements for ISO 17025
accreditation
– Manpower, equipment, techniques
and QA
• Detection, quantification of trace level
analytes

ANALYTICAL PROCESS
Sampling
Certificate of
Analysis
Data Manipulation
Sample Submission
Sample Extraction/
Clean-up
Instrumentation

Management
requirements
(section 4)
• ISO 9001:2008
Accreditation
ISO 17025 standard
Method
accreditation
Large batches, routine
matrices
Technical
requirements
(section 5)
Technique
accreditation
Small batches, non-routine
matrices

Technical Requirements
• Skilled manpower
• Sensitive equipment
And methodology
• An appropriate QA
system

ANALYTICAL PROCESS
Sampling
Certificate of
Analysis
Data Manipulation
Sample Submission
Sample Extraction/
Clean-up
Instrumentation

ANALYTICAL PROCESS
Sampling
Certificate of
Analysis
Data Manipulation
Sample Submission
Sample Extraction/
Clean-up
Instrumentation

Extraction and cleanup of the
samples
• Most critical step in the analytical process –
essential to control this step
– Selection of;
• Sub-sample size,
• Extraction solvents - dependant on the
matrix
• Cleanup step – liquid/liquid or SPE or no
cleanup?
• Concentration step to reach detection
limits
– How How can can we we control control this this step?
step?

QA of the extraction step
• Use of Certified Reference Materials
(CRM)
• Spiking of samples
• QC samples in batch
• Participation in proficiency testing
schemes

SOP must clearly spell out the
parameters to control during
sample extraction
• Laboratory temperature
• pH meter fluctuations
• Centrifugation speed and time
• Scale calibration
• Auto-pipette calibration
• Grade of solvents/reagents specified in the
method

ANALYTICAL PROCESS
Sampling
Certificate of
Analysis
Data Manipulation
Sample Submission
Sample Extraction/
Clean-up
Instrumentation

Sensitivity depends on instrumentation used
Potential for
interferences
MS-MS
LC/GC-MS
HPLC-F
HPLC-UV
ELISA
Microbiological
Ability to
discriminate
(Price increase)

Comparison of different techniques
Method Method Method Method of of of of Analysis
Analysis Analysis Analysis
Thin Thin Layer Layer Chromatography Chromatography (TLC)
(TLC)
Enzyme Enzyme Linked Linked Immunosorbant
Assay Assay (ELISA)
(ELISA)
High High Performance Performance Liquid
Liquid
Chromatography Chromatography (HPLC)
(HPLC)
Gas Gas chromatography chromatography (GC (GC-MS) (GC
MS)
LC LC-MS/MS LC MS/MS
Costs/ Costs/ Costs/ Costs/
group/sample
group/sample
group/sample
group/sample
(Rand) (Rand) (Rand) (Rand)
N=10 N=10 N=10 N=10 ---- 100 100 100 100
350
250
550
600
1250
Method Method Method Method characteristics
characteristics
characteristics
characteristics
High Sensitivity
Costs per single
group
Costs per single
group
Selected groups e.g.
Trichothenescenes
All groups

Mycotoxin Multi Residue Method
• The first first multi multi-methods multi
methods were published 1999 1999-2002 1999
2002
(Berger (Berger (Berger (Berger et et et et al., al., al., al., Razzazi----Fazeli Razzazi Razzazi Razzazi Fazeli Fazeli Fazeli et et et et al., al., al., al., Rundberget Rundberget Rundberget Rundberget et et et et al.,Spanjer al., al., al., Spanjer Spanjer Spanjer et et et et al. al. al. al.
etc.) etc.) etc.) etc.)
• At this time, time, LC LC-MS/MS LC
MS/MS was mainly a scientific approach and
a a lot lot of of maintenance maintenance was was necessary necessary to to run run samples samples within within a
a
broad broad range range of of commodities
commodities
• Since the instruments getting more robust,
LC LC-MS/MS LC
MS/MS MS/MS has has been been increasingly increasingly applied applied for for routine
routine
food food control.
control.
• Today, it seems that they are fit for purpose !

Instrumentation – LC MS/MS
Ion source
Column oven
Solvent delivery module MS/MS
Auto sampler

Advantages of LC-MS/MS
• Sensitive detection for not one; but
numerous mycotoxins in a single
analysis.
• Does not require tedious and expensive
sample preparation due to sensitive
tandem mass spectrometer – Results in
fast turn around time for client.
• Currently the method of choice in
analytical laboratories.

Time scale to perform a batch (12 – 24) of
analyses
Samples weighing – 1,5 hours
Samples are extracted with an extraction solvent – 2 hours
Samples are cleaned up on a SPE cartridge – 3 hours
Injection into LC-MS/MS – 8 hours
Data Analysis – 2 hour
Report – 0.5 hour

LC-MS/MS Results.
Blank samples
A) B)
Certified reference
material at 3.2
ug/kg
Blank feed sample (A) vs naturally contaminated deoxynivalenol wheat sample (B)

Quantitation of T2 and HT2
R = 1.000
A B
R = 0.9973
Figure 4: Typical Calibration Curves obtained for T2 Toxin (A) and HT2
Toxin (B). All data is calculated against matrix based calibration curves

Analysis performed against
certified reference material.
Day 1
Day 2
Day 3
Day 1
Day 2
Day 3
Deoxynivalenol Deoxynivalenol (DON) (DON)
(DON)
Added
amount
(µg/Kg)
265
265
265
3.2
3.2
3.2
Zearalenone
Zearalenone
Added
amount
(µg/Kg)
Value
obtained
(µg/Kg)
2.84
2.92
2.57
Value
obtained
(µg/Kg)
266.70
298.63
255.49
Day 1
Day 2
Day 3
Day 1
Day 2
Day 3
Aflatoxin B1
B1
Added
amount
(µg/Kg)
19
19
19
Aflatoxin B2
B2
Added
amount
(µg/Kg)
2.3
2.3
2.3
Value
obtained
(µg/Kg)
33.37
30.12
28.39
Value
obtained
(µg/Kg)
2.96
2.39
2.77

Conclusions
• Man-power must be skilled (VERY hard
to find in SA)
• Equipment is expensive
• Technique accreditation – most difficult
• QA requirements is laborious and difficult
to control

ANALYTICAL PROCESS
Sampling
Certificate of
Analysis
Data Manipulation
Sample Submission
Sample Extraction/
Clean-up
Instrumentation

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