Analysis of Veterinary Drugs in Meat with the Agilent 6495 Triple Quadrupole LC/MS Application Note Food Authors Abstract Tarun Anumol, Joan M. Stevens, and A method using an Agilent 1290 Infinity II LC coupled to an Agilent 6495 Triple Jerry Zweigenbaum Quadrupole LC/MS for the rapid and sensitive analysis of 120 veterinary drugs Agilent Technologies Inc. in bovine meat has been developed. The analytical run time is 12 minutes, while limits of detection and quantification range between 0.1–2 ng/mL and 0.1–5 ng/mL, respectively. Three optimized MRM transitions were selected for all but three veterinary drugs, ensuring selectivity and robustness. Quantification of real samples was possible with most compounds having R2 >0.99 when two sets of matrix-matched calibration curves were performed. The method is reproducible and repeatable as indicated by the results of intra- and interday variability tests that produce relative standard deviations of <15 % for more than 90 % of the compounds tested. Introduction Instrumentation Separation of analytes for this method was performed using The monitoring of veterinary drugs in food is critical due to an Agilent 1290 Infinity II LC with a 20 µL injection loop and contamination and the possibility of increased antimicrobial multiwash capability. An Agilent 6495 Triple Quadrupole resistance by pathogenic microorganisms [1]. Veterinary drug LC/MS with the iFunnel and Jet Stream technology was used administration in animals is important to treat diseases and as the detector. Analysis was performed in simultaneous promote growth. However, improper dosing or illegal practices positive and negative electrospray ionization mode. All can lead to contamination in meat for human consumption. data acquisition and processing was performed using As a result, veterinary drugs in food are regulated in several Agilent MassHunter software (Version 07.00). Tables 1 and 2 regions including the US, Europe, China, Australia, and others show the instrument conditions. [2-4]. Analysis of veterinary drugs is challenging due to their Table 1. Optimized LC Conditions many classes with diverse structures and varying chemical Parameter Value properties. To meet the needs of analytical labs, rapid and Instrument Agilent 1290 Infinity II with 20 µL flex loop and efficient techniques using multiclass, multiresidue methods multiwash analyzing >100 veterinary drugs in a single run are required. Column Agilent ZORBAX C-18 Eclipse Plus Additional goals are detection limits of low μg/kg, with 2.1 × 150 mm, 1.8 µm (p/n 959759-902) good reproducibility and high sample throughput. The use Guard column Agilent ZORBAX C-18 Eclipse Plus of ultrahigh performance liquid chromatography (UHPLC) 2.1 × 5 mm, 1.8 µm (p/n 821725-901) coupled to tandem mass spectrometers (MS/MS) is the gold Column temperature 30 °C standard for this analysis. This technique offers the requisite Injection volume 15 µL analytical sensitivity and robustness while allowing for time Mobile phase A) Water + 0.1 % formic acid and labor savings compared to other techniques for analysis B) Acetonitrile of veterinary drugs. Run time 12 minutes Equilibration time 2 minutes This application note describes the development of a rapid Flow rate 0.5 mL/min UHPLC/MS/MS method with the Agilent 1290 Infinity II Gradient Time (min) A (%) UHPLC and an Agilent 6495 Triple Quadrupole LC/MS for 0.0 98 the analysis of 120 veterinary drugs in animal meat. The 1.0 98 1.5 85 method used three transitions for each analyte (except three) 2.5 70 satisfying US and EU specifications for identification. The 6.0 55 sensitivity of the method was determined by calculating the 8.5 20 limits of detection and quantification in kidney and liver. Other 10.0 0 method validation protocols such as linearity, robustness, and 11.0 0 11.2 98 reproducibility were also evaluated in this study. Table 2. Optimized MS conditions Experimental Parameter Value Mass spectrometer Agilent 6495 Triple Quadrupole LC/MS Standards and reagents Gas temperature 150 °C All native veterinary drug standards were bought from Gas flow rate 18 L/min Sigma-Aldrich (St. Louis, MO), and prepared between 300 Sheath gas temperature 300 °C and 1,000 µg/mL in solvent (either acetonitrile, methanol, Sheath gas flow rate 11 L/min dimethyl sulfoxide, or water depending on solubility). The Nebulizer pressure 35 psi Capillary voltage 4,000 V (3,000 V) three internal standards used in this study (flunixin-d3, nafcillin-d5, and doxycycline-d3) were acquired from Toronto Nozzle voltage 500 V (1,500 V) Research Chemicals (Toronto, ON). LC/MS grade acetonitrile Ion funnel HPRF 200 v (90 V) and water were procured from Burdick and Jackson Ion funnel LPRF 100 V (60 V) (Muskegon, MI), while formic acid (>98 %, Suprapur) was Delta EMV 200 V obtained from EMD Millipore (Darmstadt, Germany). Time segments Time (min) Flow 0.0 Waste 0.7 MS 2 Sample preparation Results and Discussion The Agilent Enhanced Matrix Removal—Lipid (EMR—L) product was used for sample extraction of veterinary drugs Compound selection and optimization in this study. The EMR—L selectively removes lipids while The 120 veterinary drugs analyzed in this study were not trapping contaminants of interest, and has been shown selected based on a monitoring list used by the United to be effective in extracting several classes of compounds States Department of Agriculture’s Agricultural Research including pesticides, toxins, and PAHs in food [5,6]. Details of Service (USDA-ARS) [9]. The compound-specific parameters the procedure followed for veterinary drug extraction using including precursor ion, three most abundant unique product EMR—L, and product information can be found in previously ions, and collision energy were determined by running each published literature [7,8]. Briefly, 2 g samples of homogenized standard through the Agilent Optimizer software. Three bovine kidney and liver were weighed and placed into specific transitions were selected for each compound (except 50-mL polypropylene tubes. A 10 mL solution of acetonitrile thiouracil, metronidazole, and clindamycin) to satisfy both US with 5 % formic acid was added to the sample and mixed with and EU regulations for identification by mass spectrometry. an orbital shaker for 5 minutes, followed by centrifugation at Table 3 shows the optimized transitions, retention times, 4,000 rcf for 5 minutes. After this, 5 mL of the supernatant and other relevant parameters for each compound. The was added to the 1 g EMR—L tube, which had been activated tolerance levels for each veterinary drug were obtained from previously with 5 mL of 5 mM ammonium acetate solution. the USDA-ARS, and were used to prepare calibration curves, The tube was then vortexed and centrifuged at 4,000 rcf for and perform spike studies, described later. Care was taken 5 minutes. The 5 mL of supernatant from this solution was to select transitions that did not have matrix interferences. transferred to a 15-mL centrifuge tube to which 2 g of MgSO 4 Cimaterol had matrix interferants for the 220.1 → 202.1 and were added from the EMR—L pouch with vortexing and 220.1 → 160.1 transitions, therefore, extra transitions were centrifugation, as before. Finally, a 100 µL extract was obtained. The ion ratio intensities were helpful in identifying collected from the tube and diluted with 400 µL of ultrapure these issues (as opposed to reporting cimaterol as incurred). water in a 1-mL polypropylene vial, ready for LC/MS analysis. Figure 1 represents a chromatogram of cimaterol in standard and liver blank with the different MRM transitions that indicate the presence of two of the transitions in matrix but at different ion ratios than would be expected based on the standard. Table 3. Optimized Compound Parameters and Tolerance Levels with Retention Times for 120 Veterinary Drugs Compound Class Tolerance (ng/g) Precursor ion Product ion Collision energy RT (min) Delta RT Thiouracil Thyreostat 400 129 90.1 8 0.95 1.0 129 82.3 16 Florfenicol amine Phenicol 300 248.1 230.1 8 0.99 0.8 248.1 130.1 28 248.1 91.1 50 Florfenicol Phenicol 300 358 241 16 1 0.6 358 206 28 358 170 32 Sulfanilamide Sulfonamide 100 173 156 5 2 0.6 173 92 25 173 76 5 Methyl-thiouracil Thyreostat 400 143 126 20 2.5 0.6 143 86 20 143 84 20 Amoxicillin β-Lactam 10 367 349.1 4 2.56 0.6 367 208 8 367 114 56 3 Table 3. Optimized Compound Parameters and Tolerance Levels with Retention Times for 120 Veterinary Drugs (continued) Compound Class Tolerance (ng/g) Precursor ion Product ion Collision energy RT (min) Delta RT Salbutamol β-Agonist 10 240.2 222.2 4 2.6 0.6 240.2 166.1 4 240.2 148.1 16 Tildipirosin Macrolide 100 734.5 561.5 36 2.65 0.6 734.5 174 44 734.5 98.2 56 Cimaterol β-Agonist 10 220.1 202.1* 4 2.66 0.6 220.1 160.1* 12 220.1 143.1 14 220.1 116.1 20 Hydroxy- metronidazole Coccidiostat 10 188.1 126.1 16 2.7 0.6 188.1 123.1 8 188.1 68.0 22 Lincomycin Lincosamide 100 407.2 359.2 16 2.7 0.6 407.2 126.1 24 407.2 42.2 68 Hydroxy-dimetridazole Coccidiostat 50 158.1 140 8 2.8 0.6 158.1 55.2 20 158.1 42.2 40 Metronidazole Coccidiostat 10 172.1 128 12 2.83 0.6 172.1 82.1 24 Dipyrone metabolite Anti- inflammatory 200 218.1 187.1 18 2.85 0.6 218.1 125 16 218.1 97 14 Levamisole Anthelmintic 100 205.1 178.1 20 2.9 0.6 205.1 123 32 205.1 91.1 44 Albendazole-2- Anthelmintic 50 240.1 198 20 2.97 0.6 aminosulfone 240.1 133.1 20 240.1 105 40 Ampicillin β-Lactam 10 350 160 4 3 0.6 350 114 36 350 106 16 Dimetridazole Coccidiostat 10 142.1 96.1 16 3 0.6 142.1 81.1 28 142.1 54.1 36 Thiabendazole Anthelmintic 100 202 175 24 3 0.6 202 131 36 202 65 52 Ronidazole Coccidiostat 10 201.1 140.1 8 3.09 0.6 201.1 55.2 20 201.1 154.9 8 Desethylene Fluoroquinolone 100 306.1 288.2 20 3.1 0.6 Ciprofloxacin 306.1 268.1 28 306.1 217 44 * Potential matrix interferants in liver extract.