Method Development in LCMS MS where do I start John

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

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

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

Public Analyst’s Laboratory, Dublin n Responsible for Dublin Mid-Leinster and Dublin North-East Regions Population– 1. 5 M plus extensive National Testing

Public Analyst’s Laboratory, Dublin n n 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: q Dublin–contaminants, mycotoxins, food contact materials q Cork–vitamins, metals q Galway–food compositional Three PA laboratories have some common testing (e. g. water analysis, some chemical analysis)

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

Method Development in LC-MS/MS– where do I start? n n n General familiarity with Analytical Method Development Most Laboratories accredited (INAB, FDA etc. ) Procedures in place for carrying out Method Development q q q n n 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 q Hierarchy of Analytical Methods

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

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

Resources 3 n Official Control Laboratories (OCLs) n n National Reference Laboratories (NRLs) n n n Consult with your NRL or other OCLs Consult with NRLs from other Member States Consult with appropriate EURL EU Reference Laboratories (EURL) n n n 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

Resources 4 n CEN TC 275 WG 5 n n n Development of new Analytical Methods Participation in interlaboratory studies Provided with well-developed Analytical Methods Successful participation in trials Validation vs. Verification n Implementation within your own laboratory

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

Quality Control 1 n Quality Control q HPLC Methods n n n n Calibration Curve Recovery Precision Peak symmetry Resolution Area counts Plate Counts

Quality Control 2 n Quality Control q LC-MS/MS n n n Ion ratio Signal-to-noise ratio Retention time window

Quality Control 3 n Calibration Curves q n LOD and LOQ q n 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/eur 22756 en. pdf ) Uncertainty of Measurement q n Preparation of Calibration Curves–A Guide to Best Practice, September 2003, LGC/VAM/2003/032 Eura. Chem / CITAC Guide CG 4–Quantifying Uncertainty of Measurement 3 rd 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)

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

Semicarbazide in Food (1) n n n 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) q 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

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

Semicarbazide in Food (3) n n n 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

Semicarbazide in Food (4) n n 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

Semicarbazide in Food (5) n n n In-house development of the analytical method FAPAS organised a Proficiency Tests in different matrices JRC organised two interlaboratory studies n n q q 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

Semicarbazide in Food (6) n n n 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 Na. OH + centrifuge + clean-up the supernatant liquid Phenomenex Strata™ SDB-L tubes, condition tubes with Et. OAc, Me. OH + UPW, 13 ml eluate, wash tube with UPW, elute with Et. OAc Evaporate eluate to dryness + reconstitute with Me. OH: UPW (60: 40), filter + analyse

Semicarbazide in Food (7) n MS Conditions q q q q n 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 Transitions monitored q q 212→ 168 (internal standard – 13 C, 15 N 2 -SEM) 209 → 192 209 → 166 209 → 134

Melamine in Food (1) n n n 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

Melamine in Food (2) n n n 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

Melamine in Food (3) n n n 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

Melamine in Food (4) n Another problem with the method–availability of labelled internal standards n n n Initially only 13 C 3 15 N 3 -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 n n n 13 C 3 -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

Melamine in Food (5) n n n Weigh 0. 5 g sample, add 9. 95 ml 0. 2 M HCl. O 4+50 l I. S. and shake (30 min) Centrifuge and extract supernatant liquid using Oasis MCX Cartridges, wash with water and Me. OH, elute with 0. 5% Ammonia in Me. OH Evaporate to dryness and reconstitute in Mobile Phase

Melamine in Food (6) n UPLC Conditions q q q 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 m. M Ammonium acetate in 90: 10 ACN: UPW

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

Melamine–Food Contact Materials n n 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

Citrinin (1) n n 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

Citrinin (2) n n CEN Working Group TC 275 WG 5–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

Conclusions n n 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