LC-MS/MS, the new reference method for mycotoxin analysis

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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 [email protected]

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Introduction

Aspects that will be covered

:

• Analytical processes

• Requirements for ISO 17025

accreditation

– Manpower, equipment, techniques

and QA

• Detection, quantification of trace level

analytes

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Sample Extraction/

Clean-up

Sample Extraction/

Clean-up

Certificate of

Analysis

Certificate of

Analysis

Sampling

Instrumentation

ANALYTICAL PROCESS

Sample Submission

Data Manipulation

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Accreditation

ISO 17025 standard

Management requirements (section 4) Technical requirements (section 5)

• ISO 9001:2008

Method

accreditation Technique accreditation

Small batches, non-routine matrices

Large batches, routine matrices

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Technical Requirements

• Skilled manpower

• Sensitive equipment

And methodology

• An appropriate QA

system

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Sample Extraction/

Clean-up

Sample Extraction/

Clean-up

Certificate of

Analysis

Certificate of

Analysis

Sampling

Instrumentation

ANALYTICAL PROCESS

Sample Submission

Data Manipulation

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Sample Extraction/

Clean-up

Sample Extraction/

Clean-up

Certificate of

Analysis

Certificate of

Analysis

Sampling

Instrumentation

ANALYTICAL PROCESS

Sample Submission

Data Manipulation

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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

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QA of the extraction step

• Use of Certified Reference Materials

(CRM)

• Spiking of samples

• QC samples in batch

• Participation in proficiency testing

schemes

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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

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Sample Extraction/

Clean-up

Sample Extraction/

Clean-up

Certificate of

Analysis

Certificate of

Analysis

Sampling

Instrumentation

ANALYTICAL PROCESS

Sample Submission

Data Manipulation

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Sensitivity depends on instrumentation used

MS-MS

LC/GC-MS

HPLC-F

HPLC-UV

ELISA

Microbiological

Potential for

interferences

Ability to

discriminate

(Price increase)

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Comparison of different techniques

Selected groups e.g. Trichothenescenes

600

Gas chromatography Gas chromatographyGas chromatography

Gas chromatography (GC(GC(GC----MS)(GC MS)MS)MS) All groups

1250

LC LCLC LC----MS/MSMS/MSMS/MSMS/MS

Costs per single group

550

High Performance Liquid High Performance Liquid High Performance Liquid High Performance Liquid Chromatography (HPLC) Chromatography (HPLC)Chromatography (HPLC) Chromatography (HPLC)

Costs per single group

250

Enzyme Linked Enzyme Linked Enzyme Linked

Enzyme Linked ImmunosorbantImmunosorbantImmunosorbantImmunosorbant Assay (ELISA)

Assay (ELISA)Assay (ELISA) Assay (ELISA)

High Sensitivity

350

Thin Layer Chromatography (TLC) Thin Layer Chromatography (TLC)Thin Layer Chromatography (TLC) Thin Layer Chromatography (TLC)

Method characteristics Method characteristics Method characteristics Method characteristics Costs/ Costs/ Costs/ Costs/ group/sample group/sample group/sample group/sample (Rand) (Rand) (Rand) (Rand) N=10 N=10 N=10 N=10 ---- 100100100100 Method of Analysis Method of AnalysisMethod of Analysis Method of Analysis

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Mycotoxin Multi Residue Method

• The first multiThe first multiThe first multi----methods were published 1999The first multi methods were published 1999methods were published 1999methods were published 1999----2002200220022002

(Berger et al., Razzazi (Berger et al., Razzazi (Berger et al., Razzazi

(Berger et al., Razzazi----Fazeli et al., Rundberget et al.,Fazeli et al., Rundberget et al.,Fazeli et al., Rundberget et al.,Fazeli et al., Rundberget et al.,SpanjerSpanjerSpanjerSpanjer et al. et al. et al. et al. etc.)

etc.) etc.) etc.)

• At this time, LCAt this time, LCAt this time, LC----MS/MS was mainly a scientific approach and At this time, LC MS/MS was mainly a scientific approach and MS/MS was mainly a scientific approach and MS/MS was mainly a scientific approach and a lot of maintenance was necessary to run samples within a a lot of maintenance was necessary to run samples within a a lot of maintenance was necessary to run samples within a a lot of maintenance was necessary to run samples within a broad range of commodities

broad range of commoditiesbroad range of commodities broad range of commodities

• Since the instruments getting more robust,Since the instruments getting more robust,Since the instruments getting more robust,Since the instruments getting more robust,

LC LC LC

LC----MS/MS has been increasingly applied for routineMS/MS has been increasingly applied for routineMS/MS has been increasingly applied for routineMS/MS has been increasingly applied for routine food control.

food control. food control. food control.

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Instrumentation – LC MS/MS

Column oven

Solvent delivery module MS/MS

Ion source

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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.

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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

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LC-MS/MS Results.

Blank feed sample (A) vs naturally contaminated deoxynivalenol wheat sample (B)

A) _B)

Blank samples

Certified reference material at 3.2 ug/kg

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Quantitation of T2 and HT2

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

A _B

R = 1.000

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Analysis performed against

certified reference material.

2.57 3.2 Day 3 2.92 3.2 Day 2 2.84 3.2 Day 1 Value obtained (µg/Kg) Added amount (µg/Kg) Deoxynivalenol DeoxynivalenolDeoxynivalenol

Deoxynivalenol (DON)(DON)(DON)(DON)

255.49 265 Day 3 298.63 265 Day 2 266.70 265 Day 1 Value obtained (µg/Kg) Added amount (µg/Kg) Zearalenone Zearalenone Zearalenone Zearalenone 28.39 19 Day 3 30.12 19 Day 2 33.37 19 Day 1 Value obtained (µg/Kg) Added amount (µg/Kg) Aflatoxin Aflatoxin Aflatoxin Aflatoxin B1B1B1B1 2.77 2.3 Day 3 2.39 2.3 Day 2 2.96 2.3 Day 1 Value obtained (µg/Kg) Added amount (µg/Kg) Aflatoxin AflatoxinAflatoxin Aflatoxin B2B2B2B2