Multi-residue analysis of antimicrobial agents by LC-MS Prof. Ramon Companyó K. Praveen Kumar, PhD student Department of Analytical Chemistry Faculty of Chemistry University of Barcelona
Outline of the talk Antimicrobial agents. The large family Legislation. A look at a complex and strange world (for scientists) Materials to analyze Which method? Screening and confirmatory methods. Multiresidue and multiclass methods. Method validation Sample treatment (extraction and extract purification). The stumbling stone Measurement. LC/MS, the most powerful tool Non-targeted analysis. The last step 2
Antimicrobial agents. The large family Regulation (EC) 1831/2003 Antimicrobials: substances produced either synthetically or naturally, used to kill or inhibit the growth of micro-organisms, including bacteria, viruses or fungi, or of parasites, in particular protozoa. Antibiotic: antimicrobials produced by, or derived from, a micro-organism, which destroys or inhibits the growth of other micro-organisms. International Federation for Animal Health-Europe. 2012 Annual Report Sales of animal health products Product Value in % Vaccines 1,226 M 27.4 Antimicrobials 750 M 16.8 Parasiticides 1,298 M 29,1 Topical 315 M 7.1 Other products 878 M 19.6 Total 4,467 M 100 3
Legislation. A complex and strange world TOXICOLOGICAL Regulation (EC) No 470/2009. Classification of pharmacologically active substances With MRL Pursuant to an opinion of the European Medicines Agency With provisional MRL Established in cases where scientific data are incomplete. Defined period (5 + 2 years) MRL not necessary for the protection of human health Prohibited substances because the presence of the active substance or residues thereof in foods of animal origin may constitute a hazard to human health Regulation (EU) No 37/2010 Allowed substances (MRL) Prohibited substances Food ANALYTICAL Decision 2003/181/EC and Decision 2004/25/EC Minimum required performance limits (MRPL) for prohibited substances Decision 2002/657/EC Performance of analytical methods and the interpretation of results and method validation Guidelines for clarification and updating Codex Alimentarius Commission. Codex Committee on Residues of Veterinary Drugs in Foods. Proposed Draft Guidelines on Performance Characteristics for multiresidue Methods (May 2012) Codex Alimentarius Commission. Maximum Residue Limits for Veterinary Drugs in Foods (July 2012) 4
Legislation. A complex and strange world Food Family With EU MRL With Codex MRL Aminoglycosides 8 5 Amphenicols 2 Beta-lactams (penicillins and cephalosporins) 18 2 Lincosamides 2 2 Macrolides 6 4 Quinolones 7 3 Sulfonamides All 1 Tetracyclines 4 3 Polyether 2 Polypeptide 2 Other antimicfrobial 10 Coccidiostats 5 2 Avermectins 5 Benzimidazole derivatives 6 Other antihelmintic agents 7 Antimicrobial agents prohibited in the EU Chloramphenicol Dapsone Nitrofurans Nitroimidazoles (3) 5
Legislation. A complex and strange world Feed Presence of antimicrobial agents in feeds Veterinary prescription: therapeutic and prophylactic purposes As feed additives for growth promotion As a result of cross-contamination during production Regulation 1831/2003 The use of antimicrobials as feed additives is banned since 1st January 2006. There is a moratorium on the use of coccidiostats and histomonostats. Directive 2009/8 Recognise the unavoidable carry-over of coccidiostats or histomonostats in nontarget feed in spite of the application of good manufacturing practices Establishes maximum levels of these drugs in the cross-contaminated feeds, according to the sensitivity of the non-target species fed with these feeds. 6
Materials to analyze Animal origin Muscle Kidney Liver Fat Milk Eggs Honey Urine Hair Food products Feed and feed components Raw materials (cereals, tubers, leguminous, fruits, milk,...) Feed additives Functional feed ingredients Premixtures Compound feeds Environmental samples Surface water (freshwater, seawater) Groundwater Wastewater Soils Sediments 7
Screening and confirmatory methods. Multiresidue and multiclass methods. Method validation Screening methods (Decision 2002/657/EC. Codex Alimentarius Commission. 2012) Methods that are used to detect the presence of a substance or class of substances at the level of interest. Often they take advantage of a structural feature common to a group of substances These methods have the capability for a high sample throughput and are used to sift large numbers of samples for potential non-compliant results Applicable in laboratories but also in field analysis (farms, slaughterhouses, factories) Qualitative or semiquantitative Sample treatment must be simple Specifically designed to avoid false compliant results Positive results must be confirmed by a confirmatory method Guidelines for the validation of screening methods for residues of veterinary medicines. EU Community Reference Laboratories for Residues. 2010 8
Screening and confirmatory methods. Multiresidue and multiclass methods. Method validation Confirmatory method (Decision 2002/657/EC) Methods that provide full or complementary information enabling the substance to be unequivocally identified and if necessary quantified at the level of interest Confirmatory method (Codex Alimentarius Commission. 2012) Methods that provide full or complementary information enabling the analyte to be identified with an acceptable degree of certainty at the concentration of interest Zero tolerance (as low as possible), MRPL, MRL or agreedupon level They are mainly based on LC coupled to mass spectrometry Requirements on: Retention times Ion intensities Ion ratios Number of identification points 9
Screening and confirmatory methods. Multiresidue and multiclass methods. Method validation Multiresidue method (Codex Alimentarius Commission. 2012) Method which is suitable for the identification and quantification of a range of analytes, usually in a number of different matrices. For the purposes of this document, a multiresidue method is considered to be a method which includes three or more analytes in the same class or more than one class of veterinary drugs in its scope. Multiresidue methods: one group of veterinary drugs Multiclass methods: drugs from several groups 10
Screening and confirmatory methods. Multiresidue and multiclass methods. Method validation Multiclass methods Basic Amphoteric Big - Small sulfadiazine Highly polar Moderately polar Banned Authorised 11
Screening and confirmatory methods. Multiresidue and multiclass methods. Method validation (Codex Alimentarius Commission. 2012) Accuracy (trueness, bias) Recovery Precision Sensitivity LOD LOQ Measurement uncertainty (Decision 2002/657/EC) Trueness/recovery Precision Selectivity Ruggedness/robustness Stability of analytes Decision Limit (CCα) Detection capability (CCβ) 12
Sample treatment. The stumbling stone GENERIC EXTRACTION Farms Slaughterhouses Wholesale markets Retail markets Import consignments Land Proteins/lipids precipitation SPE d-spe Gel permeation chromatography ((GPC) SAMPLE OFFICIAL SAMPLE EXTRACTION SOLVENT EXCHANGE EXTRACT PURIFICATION EXTRACT DILUTION SOLVENT EXCHANGE Mechanical shaking Ultrasound assisted extraction Microwave assisted extraction (MAE) Pressurized liquid extraction (PLE) PREPARED EXTRACT 13
Sample treatment. The stumbling stone H.G. J. Mol et al. Toward a generic extraction method for simultaneous determination of pesticides, mycotoxins, plant toxins and veterinary drugs in feed and food matrixes. Anal. Chem. (2008) 80:9450-9459 Analytes: 86 veterinary drugs 136 pesticides 36 natural toxins Matrices: Honey Feed Maize Meat Egg Milk 2.5 g of sample + 5 ml of water Vortex End-over-end shaking (1 h) Centrifugation (10 min) + 15 ml ACN 1% formic acid 0.5 ml in an autosampler vial Concentration levels: 0.01 0.25 mg/kg LC-MS/MS 14
Sample treatment. The stumbling stone Generic extraction methods for multiresidue analysis H.G. J. Mol et al. Toward a generic extraction method for simultaneous determination of pesticides, mycotoxins, plant toxins and veterinary drugs in feed and food matrixes. Anal. Chem. (2008) 80:9450-9459 15
An example. A multiclass method for the analysis of antibiotic residues in egg Laboratory of the Public Health Agency of Barcelona V. Jiménez et al. Development and validation of a multiclass method for the analysis of antibiotic residues in eggs by liquid chromatography tandem mass spectrometry. Journal of Chromatography A, 1218 (2011) 1443 1451 Analytes: sulfonamides (14) Trimethoprim Penicillins (7) Quinolones (9) Tetracyclines (4) Macrolides (5) Lincomycin 1 g of whole egg + 1.5 g of diatomaceous earth containing EDTA PLE 1:1 mixture of ACN and succinic acid buffer ph 6.0 2 cycles (8 min, 70⁰C, 1500 psi) Make up to a final volume of 20mL Centrifuge Evaporate a 4mL aliquot near to dryness at 50 C Re-dissolved in 1mL of water LC-MS/MS (QqQ) 16
Response An example. A multiclass method for the analysis of antibiotic residues in egg 1000000 800000 600000 Sulfadimethoxine 250000 200000 150000 Trimethoprim MATRIX EFFECTS 400000 100000 200000 50000 0 0 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 80000 35000 Penicillin G 60000 28000 Sarafloxacin 21000 40000 14000 20000 7000 0 0 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 800000 140000 600000 400000 Oxolinic acid 105000 70000 Tetracycline CALIBRATION Method matrix matched standards Internal standards: [ 13 C 6 ]Phenylsulfamethazine Demeclocycline Roxitromycin [D 5 ]Norfloxacin Piperacillin sodium salt 200000 0 0 200 400 600 800 1000 1200 600000 Tylosin 450000 300000 150000 0 0 200 400 600 800 1000 1200 35000 0 0 200 400 600 800 1000 1200 500000 Lincomycin 400000 300000 200000 100000 0 0 200 400 600 800 1000 1200 Concentration (µg kg -1 ) 17
An example. A multiclass method for the analysis of antibiotic residues in egg BIAS < 17% < 10% (70%) CCα: 0.5-3.8 ng/g CCβ: 0.9-4.6 ng/g PRECISION RSD r : 5 17% RSD R : 9 26% The method is within the scope of accreditation (ISO 17025) and routinely applied in the Laboratory of the Public Health Agency of Barcelona 18
Measurement. LC/MS, the most powerful tool Mass accuracy Mass accuracy, Δm i = m a m e in Da = m a m e x 10 3 in mda = (m a m e )/ m e x 10 6 in ppm m a = monoisotopic accurate mass m e = monoisotopic exact mass Mass measurement accuracy with TOF and orbitrap 5 ppm 19
Measurement. LC/MS, the most powerful tool Resolution/Resolving power Resolving power = m/δm h Resolving power : 40,000 FWHM at 200m/z Resolution = 200.2031/(200.2031-200.1900) = 30549 FWHM FWHM 10% valley definition 50% 10% Δm m 20
Measurement. LC/MS, the most powerful tool Timeline for LC MS in residue analysis adapted from H.F.De Brabander et al. J. Chromatogr. A 1216 (2009) 7964-7976 Chromatographic resolution, speed and sensitivity HPLC < 10 µm particle size 3000 psi UHPLC < 2 µm particle size Up to 15000 psi 1970 1980 1990 2000 2010 LC MS/MS LC TOF LC Orbitrap Unit resolution Up to 60000 FWHM < 2ppm accuracy Up to 240000 FWHM < 2ppm accuracy Resolution and mass accuracy, analytes/matrix throughput 21
Confirmatory methods criteria from 2012 CAC Guidelines Measurement. LC/MS, the most powerful tool Requirements 1970 1980 1990 2000 2010 LC MS/MS LC TOF LC Orbitrap Up to 60000 FWHM < 2ppm accuracy Up to 240000 FWHM < 2ppm accuracy RT Two MRM product ions Ion ratio RT 1 precursor ion and 1 product ion at resolution > 20,000 FWHM Accuracy <5 ppm Ion ratio
Measurement. LC/MS, the most powerful tool How important is mass accuracy? 1.2 Da window 0.003 Da window 0.0012 Da window 1 μg/l spiked standard in blank urine sample
How important is the resolving power? An example. Analysis of ronidazole in meat False positive in ronidazole analysis in muscle LC-QqQ Chromatogram of a suspicious sample Relative retention time 201.0>140.0/204.1>143.0 Ion ratio 201.0>140.0/201.0>55.0 Suspected sample 1.002 (±2.5%) 4.635 (±25%) Matrix matched standard 1.004 4.456 Analysis of veterinary drugs in food, feed and environmental samples 24
How important is the resolving power? An example. Analysis of ronidazole in meat Ion chromatogram extracted at 1 Da mass window LC-HRMS (Q-Orbitrap) chromatogram of a suspicious sample Precursor ion Ion chromatogram extracted at 0.0008 Da mass window Product ion Precursor ion IS spiked sample Precursor ion Product ion Analysis of veterinary drugs in food, feed and environmental samples 25
How important is the resolving power? An example. Analysis of ronidazole in meat Matrix peaks - 201.10965 and 201.12297 Ronidazole 201.0618 False non compliant (positive) sample!
Non-targeted analysis. The last step The puzzle? LC-HRMS data Compound identification steps Accurate mass with < 2 ppm error Mol. weight Pseudo mol. Ion with acc. mass e.g. M-H- Adduct e.g. M+HCOO- Adducts Mol. formula A+1 Isotopic pattern A+2 A+3 Precursor isolated MS/MS Structural information Fragments e.g. M-CO2
Non-targeted analysis. The last step Non-targeted analysis by database approach Sample preparation (generic extraction) M.Krauss, Anal. Bioanal. Chem. (2010) 397:943 951 LC-HRMS Blank substraction Targeted analysis of different chemical families Known knowns Non target peaks detection and selection
Non targeted analysis. The last step S.Stein, Anal. Chem., 2012, 84, 7274 7282 Identification of molecular formula using isotopic pattern and fragments Search in the inhouse database No Search in HR spectral database e.g. MassBank No Insilico fragmentation and spectraless database search - Metfrag+Chemspider/Pubchem No Yes Yes Yes Identification and confirmation with Isotopic pattern match and fragmentation match Known unknowns Identification and confirmation with Isotopic pattern match and fragmentation match Known unknowns Identification and confirmation with Isotopic pattern match and fragmentation match Known unknowns Recurrent database and structural elucidation Unknown unknowns
Targeted vs. non-targeted analysis Outlook Targeted analysis - MS/MS still a workhorse in food safety with better features sensitivity and scan speed (more MRMs /scan more analytes) High resolution and accurate MS is necessary to avoid isobaric matrix interferences, and improve confidence in results Non-target analysis with HRMS- From Yes/No results to safe/unsafe samples 30
Multi-residue analysis of antimicrobial agents by LC-MS Thank you for your attention Prof. Ramon Companyó K. Praveen Kumar, PhD student Department of Analytical Chemistry Faculty of Chemistry University of Barcelona