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Method Development in LC-MS/MS–where do I start?

Method Development in LC-MS/MS–where do I start?. John Keegan Public Analyst’s Laboratory, Dublin john.keegan@hse.ie. Contents. Public Analyst’s Laboratories Where do I start? Resources Quality Control Case Study Examples: Semicarbazide (Food Contact Material)

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Method Development in LC-MS/MS–where do I start?

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  1. Method Development in LC-MS/MS–where do I start? John Keegan Public Analyst’s Laboratory, Dublin john.keegan@hse.ie

  2. Contents • Public Analyst’s Laboratories • Where do I start? • Resources • Quality Control • Case Study Examples: • Semicarbazide (Food Contact Material) • Melamine (Contaminant and Food Contact Material) • Citrinin (Mycotoxin) • Conclusions

  3. Public Analyst Laboratories • Department of Health–Health and Wellbeing • Public Analyst’s Laboratories (3)–Chemistry • Dublin • Cork • Galway • Public Health Laboratories (7)–Microbiology • Have responsibility for FOOD of NON-ANIMAL ORIGIN (FNAO)–EU definition

  4. Public Analyst’s Laboratory, Dublin • Responsible for Dublin Mid-Leinster and Dublin North-East Regions Population–1.5 M plus extensive National Testing

  5. Public Analyst’s Laboratory, Dublin • Staff numbers–~50 (split between the chemical and microbiological disciplines) • Provides an accredited multidisciplinary chemical and microbiological analytical service to the HSE Dublin Mid-Leinster and Dublin North-East Areas and a National Service in its wide area of specialised testing • Each PA laboratory is almost completely fully specialised (no overlap) in testing to maximise resources between the three laboratories, including: • Dublin–contaminants, mycotoxins, food contact materials • Cork–vitamins, metals • Galway–food compositional • Three PA laboratories have some common testing (e.g. water analysis, some chemical analysis)

  6. Public Analyst’s Laboratory, Dublin • Accredited to ISO 17025:2005 (INAB 99T) • National Reference Laboratory role • Process Contaminants (incl. PAHs) • Mycotoxins (incl. Plant Toxins) • Food Contact Materials • Wide variety of customers (FSAI, EHS, SFPA, DAFM, JRC, Other Agencies, Private Customers)

  7. Method Development in LC-MS/MS– where do I start? • General familiarity with Analytical Method Development • Most Laboratories accredited (INAB, FDA etc.) • Procedures in place for carrying out Method Development • Writing Validation Protocols • In-house SOP for Validation of Analytical Methods • In-house QC Procedures (additional requirements for MS/MS) • Build on expertise already gained from HPLC, GC etc. • Regulation 882 2004–Official Controls in Food And Feed Law under revision • Hierarchy of Analytical Methods

  8. Resources 1 • Chemical Literature (e.g. Science Direct: www.sciencedirect.com) • Royal Society of Chemistry (www.rsc.org) • American Chemical Society (www.acs.org) • ChemSpider (www.chemspider.com) • CEN Standards and ISO Standards (www.nsai.ie and http://shop.standards.ie/nsai)

  9. Resources 2 • J AOAC Int (http://aoac.publisher.ingentaconnect.com/content/aoac/jaoac) • Provider of Analytical Methods • EFSA Scientific Opinions • Provide a summary of Sample Preparation Methods • Provides references to Methods of Analysis and information on analytical methods of choice

  10. Resources 3 • Official Control Laboratories (OCLs) • Consult with your NRL or other OCLs • National Reference Laboratories (NRLs) • Consult with NRLs from other Member States • Consult with appropriate EURL • EU Reference Laboratories (EURL) • EU Regulation 10/2011 (Plastics in Food Contact Materials) • More than 900 substances • Maintains a repository of analytical methods • Contributions of analytical methods from Member States • Added to from time-to-time by contributions from NRLs and industry

  11. Resources 4 • CEN TC275 WG5 • Development of new Analytical Methods • Participation in interlaboratory studies • Provided with well-developed Analytical Methods • Successful participation in trials • Validation vs. Verification • Implementation within your own laboratory

  12. Resources 5 • Analytical Standards Providers • Analytical standards need to be available • Internal standards are desirable • Proficiency Test Providers • Relevant Analytes and Matrices • Requirement for Accreditation • Certified Reference Materials • Rarely available • Improvise (proficiency test samples, sample with known level of contaminant present)

  13. Quality Control 1 • Quality Control • HPLC Methods • Calibration Curve • Recovery • Precision • Peak symmetry • Resolution • Area counts • Plate Counts

  14. Quality Control 2 • Quality Control • LC-MS/MS • Ion ratio • Signal-to-noise ratio • Retention time window

  15. Quality Control 3 • Calibration Curves • Preparation of Calibration Curves–A Guide to Best Practice, September 2003, LGC/VAM/2003/032 • LOD and LOQ • Guidance Document on the Estimation of LOD and LOQ for Measurements in the Field of Contaminants in Feed and Food (https://ec.europa.eu/jrc/sites/jrcsh/files/eur22756en.pdf ) • Uncertainty of Measurement • EuraChem / CITAC Guide CG 4–Quantifying Uncertainty of Measurement 3rd Edition (2012) • COMMISSION DECISION of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (2002/657/EC) (Confirmatory Methods)

  16. Some Examples • Analytical method for the determination of Semicarbazide in food • Analytical Method for the determination of Melamine in Food • Analytical Method for the determination of citrinin in red yeast rice supplements

  17. Semicarbazide in Food (1) • In July 2003 European Food Safety Authority was informed by industry that Semicarbazide (SEM) had been found in food products from different manufacturers packed in glass jars with lids sealed with PVC gaskets • Nitrofuran antibiotics (prohibited in EU) • Nitrofurazone metabolised to SEM (marker) • Azodicarbonamide (ADC) is a flour improver (prohibited in EU) • ADC is a Plastics blowing agent • Carrageenan (chicken products, ice creams etc.) • Disinfection and bleaching (Hypochlorite) during food production

  18. Semicarbazide in Food (2) • Work carried out by Nestlé, Unilever, Crown Cork & Seal Technologies, Amcor White Cap Technical Centre, BAG (Switzerland) and CSL (UK) • The Analyst, 2004, 129, 276–281 • -basis of all future work • Method of Analysis • LC-MS/MS with o-NBA derivatisation

  19. Semicarbazide in Food (3) • Ad-hoc group of Experts convened – Oct. 2003 • EFSA informed that SEM found in other food products through Rapid Alert System (RASFF) • Toxicological impact not known (weak carcinogen) • Impact on Public Health (particularly babies and young children) • Precautionary principle applied

  20. Semicarbazide in Food (4) • Council Directive 89/109/EEC relates to materials and articles intended to come into contact with foodstuffs • Council Directive 2002/72/EC authorises the use of ADC as a blowing agent in plastic materials • Commission Directive 2004/1/EC prohibits the use of ADC from 2 August 2005 • Transition period of 18 months allowed to remove ADC from manufacture of gaskets

  21. Semicarbazide in Food (5) • In-house development of the analytical method • FAPAS organised a Proficiency Tests in different matrices • JRC organised two interlaboratory studies • Ginn et al., J. AOAC Int., 89(3), 2006 • De la Calle and Szilagyi, J. AOAC Int., 89(6) 2006 • In-house Analytical Method was accredited in 2006 • Samples analysed during these years • Method withdrawn from INAB Scope of accreditation • Life cycle of the Analytical Method was ~5 years

  22. Semicarbazide in Food (6) • 2 g Sample + 10.0 ml 0.2 M HCl + 100 l Int. Std. + 240 l o-NBA solution • Incubate for 16 h @ 40C • Hydrolysis with 10.0 ml phosphate buffer + 0.5 ml 2 M NaOH + centrifuge + clean-up the supernatant liquid • Phenomenex Strata™ SDB-L tubes, condition tubes with EtOAc, MeOH + UPW, 13 ml eluate, wash tube with UPW, elute with EtOAc • Evaporate eluate to dryness + reconstitute with MeOH:UPW (60:40), filter + analyse

  23. Semicarbazide in Food (7) • MS Conditions • Positive electrospray (ESI+) • Cone voltage – 36 • Source temperature – 120C • Desolvation temperature – 450C • Cone gas flow – 100 l/h • Desolvation gas flow – 700 l/h • Collision gas – Argon • Transitionsmonitored • 212→168 (internal standard – 13C, 15N2-SEM) • 209 →192 • 209 →166 • 209 →134

  24. Melamine in Food (1) • 2007–Dogs and cats becoming ill or dying from eating certain brands of pet food • Production of laminates, adhesives, resinsFCMs • Possible metabolite of cyromazine (pesticide) • Melamine found in wheat gluten and rice protein destined for use in pet food manufactured in China • Cause of illness and death attributed to melamine/cyanuric acid crystals in kidneys • Resulted in the recall of large quantities of pet food

  25. Melamine in Food (2) • 2008–An increased incidence of kidney stones and renal failure was reported in infants in China–consumption of contaminated infant formula • Addition of melamine to raw milk to boost the apparent ‘protein content’ as measured by the Kjeldahl method • Not relevant to the EU because infant formula not permitted to be exported to the EU • However, contaminated milk powder also used in composite products–biscuits, sweets, cakes, soy sauce and milk powder itself that are exported to the EU, therefore there is a need to control these products • Request to develop a method of analysis for melamine in these products

  26. Melamine in Food (3) • EU Commission published legislation imposing special conditions on the import of composite products form China • Develop an Analytical Method for the analysis of melamine in food • Basis was previous methods to analyse pet food • Initial results were poor–poor recoveries from spiking experiments in a number of the matrices to be analysed • Breakthrough–application note from an instrument supplier using a different extraction solvent

  27. Melamine in Food (4) • Another problem with the method–availability of labelled internal standards • Initially only 13C315N3-melamine available • Expensive • Reluctance to add internal standard at the extraction stage • Internal standard added only after sample preparation stage, only takes account of ion suppression/enhancement, gives no indication of losses due to sample preparation stage • Standards suppliers–another breakthrough • 13C3-melamine internal standard became available for use • Relatively cheap • Enabled internal standard to be added at the sample preparation stage • Matrix-matched standards could be prepared • Recovery and ion suppression/enhancement covered

  28. Melamine in Food (5) • Weigh 0.5 g sample, add 9.95 ml 0.2 M HClO4+50 l I.S. and shake (30 min) • Centrifuge and extract supernatant liquid using Oasis MCX Cartridges, wash with water and MeOH, elute with 0.5% Ammonia in MeOH • Evaporate to dryness and reconstitute in Mobile Phase

  29. Melamine in Food (6) • UPLC Conditions • Waters Acquity UPLC HILIC 1.7 m 2.1100 mm column with guard column • Column oven temperature: 355C • Sample temperature: 105C • Injection volume: 5 l • Run time: 4 min • Mobile phase: 10 mM Ammonium acetate in 90:10 ACN:UPW 

  30. Melamine in Food (7) • Mass Spec Conditions: ions monitored: • Problem now eliminated, legislation repealed in 2015 • Analysis of melamine transferred to the main contaminants legislation: EU Regulation 1881 2006

  31. Melamine–Food Contact Materials • Exploit previously gained knowledge in the analysis of the specific migration of melamine from Melamine Kitchenware (FCMs) • EU Regulation 10/2011 • Migration Limit of 2.5 mg/kg • Simulant is 3% aq. Acetic acid–no sample preparation, dilution if concentration exceeds top standard on the calibration curve, same mass spec conditions

  32. Citrinin (1) • EFSA published a Scientific Opinion in 2012 • Stated that HPLC methods with FLD was the method of choice for analysis • Legislation published in 2014–Regulation 1881/2006, as amended • Applies to Food supplements based on rice fermented with red yeast Monascus purpureus

  33. Citrinin (2) • CEN Working Group TC275 WG5–exploit involvement in this group • Analytical method for cereals, red rice, herbs and food supplements–Belgian NRL • Expedited in-house development of method of analysis, benefit from an analytical method sufficiently well developed to be subjected to interlaboratory trials • Method accredited in 2015

  34. Conclusions • LC-MS/MS Methods of Analysis are of increasing importance in Official Controls • LC-MS/MS analytical methods are replacing HPLC methods, provide quantification and confirmation • No set procedure for each analytical method • Life-cycle of Analytical Methods becoming shorter

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