Choosing Fit-For-Purpose Food Safety Methods
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- Lawrence Mitchell
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1 Choosing Fit-For-Purpose Food Safety Methods
2 Outline Method selection Screening & quantification methods Extraction, cleanup and chromatography Detection methods Method criteria validation Conclusions
3 What do we mean by a fit-for-purpose method? Need to understand the intended end-use of analytical measurement. Factors determining fit-for-purpose: Speed & cost constraints Required accuracy/precision & sensitivity Number of samples size of program Laboratory vs on-site testing Official purposes e.g. enforcement
4 How to select the best method for my purposes Check scope of method matrix applicability, LOD, etc. Identify method limitations Look at method robustness - capacity to remain unaffected by small but deliberate variations in method parameters (indication of reliability) Decide to follow a recipe or adapt a method
5 How to select the best method for melamine in fish? 23 methods listed in melamine review article Scope 3 methods applied to fish LODs given as ng/g Only two methods validated but both LC/MS/MS Only HPLC using DAD is for beef need to modify & validate Ref:Tittlemier (2010) Food Addit. Contam., 27:
6 Target and non-target analysis Target analysis Looking for anticipated contaminants/residues Untargeted analysis Looking for differences between normal and suspect food samples Food safety monitoring programs are targeted
7 Screening and Quantitative analysis Screening technique Gives Yes/No or above/below level Have failure rates - false negative/positive Quantitative method Identification definitive LOQ and precision performance data MAYBE
8 Approaches to screening Targeted screening e.g. LFDs, ELISA, dipsticks, microbiological screens Mostly only suitable for single targets e.g. aflatoxin B 1, chloramphenicol. Some compound class screens CALUX, antimicrobial (PrimiTest) Instrumental screening e.g. search against database of standards Can screen for large number of targets e.g. pesticides
9 Getting the analyte out of the foodstuff - extraction Liquid-liquid extraction Microwave-assisted extraction Accelerated solvent extraction (ASE) Supercritical fluid extraction (SFE) SPME & headspace On-line extraction - TurboFlow
10 Measuring efficiency of extraction (recovery) Blank samples Spiking the blank matrix - how Incurred samples mycotoxins, pesticides, veterinary drugs - Binding to matrix Naturally contaminated reference materials
11 Selecting the best sample clean-up technique IAC mycotoxins/vet drugs SPE vet drugs, pesticides SAX and CAX polar compounds QuEChERS pesticides SEC (GPC) TurboFlow
12 How to decide extent of necessary sample clean-up Balance between clean-up and specificity How many samples to be analysed Direct analysis of crude sample extracts Dilute-and-shoot
13 How to choose the right separation technique
14 Derivatization in GC and HPLC GC derivatization to increase volatility e.g. TMS derivatives for GC/MS analysis of trichothecenes
15 Derivatization in GC and HPLC HPLC derivatization to introduce chromophore e.g. post-column bromination to enhance aflatoxin fluorescence 25cm x 4.6 (5µm) m 50 x 4.6(3.5µm)
16 How to choose a detection technique Gas chromatography (GC) FID, ECD, FPD - screening MS, MS/MS, HRMS NPD ECD FID FPD PID PDD TCD High performance liquid chromatography HPLC) HPLC UV, DAD, Fluorescence MS, MS/MS, HRMS (Exactive)
17 What information do you need from the analysis? Quantification - precision, accuracy A sample Identification - selectivity (specificity) Quantitative Track Qualitative Track LC/MS SIM or Scan How much? (Cal. table) Yes Is there a target list? No What s there? Confirmation Spectral Match LC/MS or Orbitrap MRM Accurate Mass Ion Trap LC/MSMS Orbitrap
18 Performance of mass analyzers Ease of Use Scan speed Scan sensitivity Qualitative (identification, structural info) Sensitivity (specific detection) Quantification (accuracy, precision) LC/MS Exactive Ion Trap Orbitrap (MS/MS w/ accurate mass) LC/MSMS esp. dirty matrix, < 5 pg on-col. LC/MSMS Better Exactive Ion Trap Ion Trap Orbitrap Exactive Orbitrap Exactive unknowns (acc. mass) Ion Trap (MS n ) knowns LC/MS (SIM) Ion Trap (MRM) LC/MS Exactive LC/MSMS LC/MS LC/MS LC/MSMS Exactive (accurate mass) Ion Trap Orbitrap LC/MSMS LC/MSMS LC/MS Orbitrap Orbitrap
19 Using internal and external standards for calibration Structural analog similar structure, polarity, close retention time - pesticides Stable isotope analog IS IS Food Chemistry (2006) 97: Journal of Chromatography A (2007), 1149:
20 Direct analysis of crude extracts potential problems Ion suppression in MS Martix-matched calibration MS source contamination problems reduced sensitivity Chlorpyrifos-methyl in a complex matrix - cumin
21 Analytical Quality Assurance (AQA) Use of validated methods Traceability of standards Recovery checks Frequent running of blanks Analysis of TMs and CRMs
22 Official methods, criteria based approach Interlaboratory validated methods Minimum performance of methods must meet stipulated criteria European Commission & Codex approach
23 Single laboratory method validation Validation data generated usually in method originators laboratory Linearity, Precision (repeatability & run-to-run conditions) Recovery (spike & test materials) LOD, LOQ, MU Robustness IUPAC Harmonized Protocol Pure & Appl. Chem., 74 : (2002)
24 Inter-laboratory method validation IUPAC/AOAC/ISO Harmonized Protocol Acceptable data from a minimum of 8 laboratories (study usually undertaken by not less than 12 labs) Blind duplicates analysed - spikes, blanks, + naturally contaminated - minimum of three levels of contamination Method performance :- RSDr, RSDR, Recovery, LOD, LOQ
25 Method criteria for mycotoxins* *Taken from EC Regulation No 401/2006
26 Method criteria for identification of residues/contaminants* Maximum permitted tolerances for mass spectrometric ion intensities for scanned spectrum of target compared with standard *Taken from EC Directive 2002/657/EC
27 Method criteria for identification of residues/contaminants* Points earned for mass spectrometric identification minimum of 4 points needed *Taken from Directive 2002/657/EC
28 Multi-residue/multi-analyte methods Only single target analyte methods needed e.g. acrylamide, 3-MPCD. Some areas begin as single target then expand e.g. Sudan dye and melamine Some areas fall in between e.g. mycotoxins Some areas multi-analyte methods essential e.g. pesticides, drugs Challenge to develop methods for multiclass as compromises to be made e.g. recovery
29 Conclusions Every circumstance matrix, analyte, laboratory facilities.. different Many options available to food chemist many choices Skill and experience required in careful selection of method
30 Thank you very much for your attention FoodLife International Ltd.
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