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

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1 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 AFMA Conference March 2009

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

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

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

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

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

7

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

9 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 can we control this step?

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

11 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

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

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

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

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

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

17 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.

18 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

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

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

21 Analysis performed against certified reference material. Day 1 Day 2 Deoxynivalenol (DON) Added amount (µg/kg) Value obtained (µg/kg) Day 1 Day 2 Aflatoxin B1 Added amount (µg/kg) Value obtained (µg/kg) Day Day Zearalenone Aflatoxin B2 Added amount (µg/kg) Value obtained (µg/kg) Added amount (µg/kg) Value obtained (µg/kg) Day Day Day Day Day Day

22 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

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

24 This is our policy to resolve complaints

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