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Sample Preparation General Procedure Next Steps ▶ Evaluate the extraction procedure in other relevant matrices ▶ Evaluate short-term and long-term effects of clean-up ▶ Determine analyte stability in standard mixtures and sample extracts References Robert C., Gillard N., Brasseur P. Y., Pierret G., Ralet N., Dubois M., and Delahaut Ph., “Rapid multi-residue and multi-class qualitative screening for veterinary drugs in foods of animal origin by UHPLC-MS/MS”, Food Additives and Contaminants, Part A 30(3):443-457 (2013). Kaufmann A., Butcher P., Maden K., Walker S., and Widmer M., “Multi-residue quantification of veterinary drugs in milk with a novel extraction and cleanup technique: Salting out supported liquid extraction (SOSLE)”, Analytical Chimica Acta 820:56-68 (2014). Introduction A modern multi-class, multi-residue method for detection, identification and quantitation of veterinary drugs using liquid chromatography- tandem mass spectrometry (LC-MS/MS) is a highly effective approach for residue monitoring with the ultimate goal of protecting consumers from potentially unsafe exposure to drug residues in foods of animal origin (milk, meat, poultry, seafood, fish, egg and honey). In this study, we developed and optimized a method for over 150 compounds belonging to a variety of veterinary drug classes, including amphenicols, anthelmintics, antibiotics (beta-lactams – penicillins and cephalosporins, lincosamides, macrolides, quinolones, sulfonamides, tetracyclines, and others), antimicrobial growth promoters, antiprotozoals, beta-agonists, coccidiostats, dyes, NSAIDs, and tranquilizers. Infant formula was chosen to establish a robust, efficient and reliable method for screening, identification and quantitation of the included veterinary drug residues. Modern Multi-Class, Multi-Residue Method Using LC-MS Benefits ▶ Cost-effective ▶ Time-effective ▶ Selective detection of individual analytes ▶ Improved sensitivity for low LODs/LOQs ▶ Identification/confirmation Challenges ▶ A large spectrum of drug classes ▶ Parent drugs and metabolites ▶ Different physical/chemical properties – Hydrophilic to hydrophobic – Acidic, neutral and basic – Stability – Interaction with matrix components ▶ Compromise between analyte scope and performance characteristics ▶ Matrix effects and potential interference from co-extractives Matrices of Interest for Our Method Development ▶ Infant formula and its relevant ingredients Veterinary Drugs of Interest for Our Method Development Total Numbers of Interest ▶ 161 veterinary drug analytes and 10 internal standards ▶ All the analytes included to cover for the needs of global regulation and different government monitoring programs Multi-Class ▶ 11 classes ▶ Anthelmintic (23), Antibiotic (102), Antimicrobial Growth Promoter (3), Antiprotozoal (1), Beta agonist (8), Coccidiostat (10), Dye (5), NSAID (1), Other-no class (1), Pesticide (2) and Tranquilizer (5) Antibiotic ▶ 102 analytes, 9 sub-classes ▶ Aminoglycoside (7, analyzed in separate method), Amphenicol (5), Beta Lactom-cephalosporin (12), Beta lactam-penicillin (8), Macrolides and lincosamides (12), Other (9), Quinolones (16), Sulfonamides (24), Tetracyclines (9) Structures of Representative Antibiotics and Other Drugs Development and Optimization of a Multi-Class, Multi-Residue Method for Veterinary Drug Analysis in Infant Formula Ingredients and Products Hui Zhao, John Zulkoski, Azeem Hasan and Katerina Mastovska Nutritional Chemistry and Food Safety, Covance Laboratories Inc., Madison, WI, USA [email protected], [email protected] Presented at RAFA 2015 Method Development Plan Optimization of MS/MS Conditions for Individual Compounds ▶ Product ion scans ▶ Selection of MRMs and collision energy optimization ▶ Up to 10 MRMs per analyte Analytes in Positive Mode (~150) Divided into 9 Groups ▶ Anthelmintic (Mix A-22) ▶ Beta lactam of cephalosporin and penicillin (antibiotic) (Mix B-20) ▶ Macrolides and lincosamides (antibiotic) (Mix C-13) ▶ Quinolones and others (antibiotic) (Mix D-23) ▶ Sulfonamides (antibiotic) (Mix E-24) ▶ Tetracyclines (antibiotic) (Mix F-9) ▶ Beta agonists, coccidiostat, and antimicrobial growth promoter (Mix G-22) ▶ Tranquilizers, Dyes and Pesticides (Mix H-12) ▶ Amphenicol, aminoglycoside, and other (antibiotic) (Mix I-4) Optimization of LC Conditions ▶ To improve the elution profile ▶ To reduce potential interferences between compounds ▶ To separate compounds, which share the same precursor and even product ions LC Conditions ▶ Column: Agilent ® C18 Zorbax ® Eclipse ® Plus, 2.1x100 mm, 1.8 μm ▶ Column temperature: 40 o C ▶ Flow rate: 0.5 mL/min ▶ Injection volume: 5 µL Elution Profile Comparison for All Analytes MRM (dMRM) Acquisition Method Optimization ▶ Mass spectrometer: Agilent ® Triple Quadrupole 6495A MS/MS ▶ Cell accelerator voltage (CAV) ▶ ESI source conditions ▶ Evaluate the MRMs for sensitivity and selectivity to choose the best two MRMs for the method ▶ Verify the selectivity for the final sample preparation procedure for multiple matrices Sample Preparation ▶ Extraction – Water and acetonitrile solvent mixture – Ratio between aqueous and organic solvent – Addition of acid – Addition of EDTA buffer ▶ Clean-up (optional) – Hexane defatting – Modified QuEChERS (dSPE) – Supported liquid extraction (SLE) – Enhanced Matrix Removal-Lipid (EMR) – SPE Ratio of Extraction Solvent Antibiotics Streptomycin (Aminoglycoside) MF C21H39N7O12 MW=581.57 Florfenicol amine (Amphenicol) MF C10H14FNO3S MW=247.291 Ceftiofur (Cephalosporin) MF C19H17N5O7S3 MW=523.56 Amoxicillin (Penicillin) MF C16H19N3O5S MW=365.4 Tylosin A (Macrolide) MF C46H77NO17 MW=915.52 Lincomycin (Lincosamide) MF C18H34N2O6S MW=406.21 Ciprofloxacin (Quinolone) MF C17H18FN3O3 MW=331.34 Sulfamethizole (Sulfonamide) MF C9H10N4O2S2 MW=270.02 Trimethoprim (other) MF C14H18N4O3 MW=262.22 Other Drugs Cambendazole (Anthelmintic) MF C14H14N4O2S MW=302.35 Ractopamine (β-agonist) MF C18H23NO3 MW=301.17 Ipronidazole (Coccidiostat-nitroimidazole) MF C7H11N3O2 MW=169.09 Decoquinate (Coccidiostat) MF C24H35NO5 MW=417.54 Malachite Green (Dye) MF C23H25ClN2 MW=364.91 Chlorpromazine (Tranquilizer) MF C17H19ClN2S MW=318.10 Virginiamycin (M1) (Antimicrobial Growth Promoter) MF C28H35N3O7 MW=525.59 Strychnine (Pesticide) MF C21H22N2O2 MW=334.41 Isometamidium (Antiprotozoal) MF C28H25N7 MW=460.55 Main Phases Optimization of MS/MS conditions for individual compounds Optimization of LC conditions Development of the final LC-MS/MS method Sample preparation procedure development and optimization Validation, data acceptance criteria and implementation in routine analysis Example: Sulfadoxin, m/z 311.1 6 x10 +ESI Product Ion:1 (rt: 3.765-3.877 min, 6 scans) Frag=380.0V CF=0.000 DF=0.000 (311.1000 -> **) Sulfadoxin_Optimizer_5uL_P2.d 311.1000 6 x10 +ESI Product Ion:2 (rt: 3.770-3.882 min, 6 scans) Frag=380.0V CF=0.000 DF=0.000 [email protected] (311.1000 -> **) Sulfadoxin_Optimizer_5uL_P2.d 156.1000 311.2000 107.9000 92.0000 140.2000 245.0000 6 x10 +ESI Product Ion:3 (rt: 3.774-3.886 min, 6 scans) Frag=380.0V CF=0.000 DF=0.000 [email protected] (311.1000 -> **) Sulfadoxin_Optimizer_5uL_P2.d 108.1000 92.0000 156.1000 140.1000 65.2000 5 x10 +ESI Product Ion:4 (rt: 3.779-3.891 min, 6 scans) Frag=380.0V CF=0.000 DF=0.000 [email protected] (311.1000 -> **) Sulfadoxin_Optimizer_5uL_P2.d 92.0000 108.2000 64.9000 154.1000 80.1000 5 x10 +ESI Product Ion:5 (rt: 3.783-3.895 min, 6 scans) Frag=380.0V CF=0.000 DF=0.000 [email protected] (311.1000 -> **) Sulfadoxin_Optimizer_5uL_P2.d 65.2000 80.2000 107.9000 53.1000 132.1000 154.2000 Counts vs. Mass-to-Charge (m/z) 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 CE = 0 CE = 45 CE = 60 CE = 15 CE = 30 Initial Optimized Aqueous to organic solvent ratio 1:3 water - ACN 1:1 water - ACN Spike recovery of 60-130% for the majority of analytes/classes Lower spike recoveries (<60%) for tetracycline and some quinolones Two steps: 1:1 water - ACN followed by 1:1 extract dilution with ACN Note: 0.1% formic acid added to ACN to assist with protein precipitation Aqueous EDTA buffer 0.025M EDTA 0.05M EDTA Majority of analytes/classes maintained spike recovery of 60-130% Tetracyclines improved spike recoveries from 0-15% to 60-85% Problematic quinolones improved spike recoveries from 50-60 % to 70-120% 0.1M EDTA 0.1M EDTA- 0.05M succinate 0.1M EDTA- McIlvaine LC-MS/MS analysis Reconstitute in 3:1 (v/v) water-acetonitrile Clean-up (optional) Centrifuge 5 minutes Add 10 mL 0.1% formic acid in acetonitrile, shake 5 minutes Add 10 mL 0.05M EDTA buffer in water, shake to homogeneous Add internal standard mix Weigh 2 g sample Initial Mobile Phases and Gradients Optimum Mobile Phases and Gradients Mobile Phase A 0.1% formic acid in water 0.1% formic acid in water Mobile Phase B 0.1% formic acid in acetonitrile 0.1% formic acid in methanol Gradient Min %A %B Min %A %B 0 98 2 0 98 2 0.5 98 2 0.75 98 2 9 10 90 7 60 40 11 10 90 11 0 100 11.1 98 2 13 0 100 17 98 2 13.1 98 2 17 98 2 Addition of EDTA Buffer
