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Determination of Veterinary Drug Residues in Milk Using Polymeric SPE and

UHPLC-MS/MS Analysis

UCT Part Numbers

ECHLD126-P EnviroClean® HL DVB 200 mg/6 mL SPE cartridge ______

VMF0 24GL 24 position glass block manifold ______

Summary: SLDA100ID21 -18UM ® This application note outlines a multi-class, multi-reside method for the Selectra DA UHPLC column (100 × 2.1 mm, 1.8 µm) determination of 49 representative veterinary drugs in milk using a simple, solid- phase extraction (SPE) procedure and analysis by UHPLC-MS/MS. To achieve fast and ______simultaneous extraction of the various drug residues, a generic liquid extraction

procedure using EDTA/acetic acid buffer is conducted prior to extraction on a SLDAGDC20 -18UM polymeric SPE cartridge. UHPLC separation is carried out with a Selectra® DA column, ® Selectra DA guard cartridge which exhibits alternative selectivity to a C18 phase and is capable of enhanced

(10 × 2.0 mm, 1.8 µm) retention for the more polar drugs. The method was evaluated for each compound ______at three varying concentrations (1, 10 and 100 μg/kg). For most compounds, recoveries were between 70% and 120% and reproducibility was <20%. In addition,

SLGRDHLDR the majority of compounds could be accurately detected at a concentration of 1 Guard cartridge holder μg/kg, demonstrating that the presented method is sufficient to monitor a wide range of veterinary drugs in milk. The drugs investigated belonged to several different

classes, including β-agonists, macrolides, amphenicols, sulfonamides, tetracyclines and quinolones.

Introduction: Veterinary drugs are frequently administered to food-producing animals, including dairy cows, to treat and prevent disease and/or increase growth rates. The inappropriate or illegal use of these drugs can result in the presence of their residues in food of animal origin which could pose a potential threat to human health. Milk is an important food commodity that is consumed by a large portion of the population, including infants. To ensure food safety and prevent the unnecessary exposure of consumers to veterinary drugs, it is vital to test milk for drug residues. The United States, European Union (EU), CODEX and other international organizations have established maximum residue limits (MRLs) for veterinary drugs in a variety of biological matrices, including milk [1-3]. The MRLs for milk are typically lower than those set for other biological matrices (muscle, liver and kidney) and span a wide concentration range (low µg/kg to >1000 µg/kg). In addition, a number of drugs are prohibited for use in food producing animals or are unauthorized for use in lactating animals and require very low detection limits (≤2 μg/kg).

Milk is a complex matrix containing dissolved fats, carbohydrates, proteins and minerals (including calcium), which can complicate the development of a fast, easy and reliable analytical method for the identification and quantification of veterinary drug residues. Development of a multi-class, multi-residue (MMR) method can be challenging not only due to the inclusion of a large number of drugs with diverse physicochemical properties, but also on account of the complex sample matrix and the instability of certain drug classes (e.g. β-lactams, tetracyclines and macrolides). A MMR method should ideally be capable of extracting a wide range of drugs, reduce major matrix interferences, obtain good analyte recovery, be reproducible and achieve adequate limits of detection (LOD’s). The use of a generic sample preparation procedure, such as SPE using a polymeric sorbent, is a suitable approach for achieving these goals. Ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) is the detection system of choice for veterinary drugs as it allows rapid detection of trace-level residues in complex matrices. However, the diverse physicochemical properties of the veterinary drugs still pose challenges and analytical conditions must be optimized to obtain adequate sensitivity of all the compounds as well as good retention and peak shape of problematic compounds. Sample Preparation Procedure:

1. Sample extraction a) Weigh 5 g of milk into a 15 mL polypropylene centrifuge tube. b) Add 5 mL of 0.1M disodium EDTA + 2% acetic acid. c) Vortex for 5 minutes to deproteinize the milk. d) Centrifuge at ≥3500 × g for 5 minutes.

Note: A larger volume of extraction solvent or a second extraction of the milk sample (5mL buffer) can

be carried out if deemed necessary.

2. SPE extraction

a) Condition SPE cartridge with: 1. 1 × 3 mL methanol 2. 1 × 3 mL ultrapure water b) Apply the supernatant to the SPE cartridge, taking care to avoid any transfer of the lipid layer. If required, use a low vacuum to draw the sample through (≤5 mL/min).

3. Wash cartridge

a) 1 × 3 mL ultrapure water.

b) 1 × 3 mL 10% methanol. c) Dry cartridge under vacuum (≥10 inHg) for 5-10 minutes to remove residual water. d) 1 × 3 mL hexane. e) Dry cartridge under vacuum (≥10 inHg) for 2 minute to remove residual hexane.

4. Elution a) Elute with 3 mL acetone.

b) Evaporate the sample to dryness at 35-40°C under a gentle stream of nitrogen. c) Reconstitute in 1 mL of methanol:water (50:50, v/v). d) Filter extract with a 0.22 µm nylon (or other suitable membrane) syringe filter into an autosampler

vial.

LC-MS/MS Parameters:

HPLC Conditions HPLC system Thermo ScientificTM DionexTM UltimateTM 3000 UHPLC MS system Thermo ScientificTM TSQ VantageTM (MS/MS) HPLC column UCT Selectra® DA, 100 × 2.1 mm, 1.8 µm (p/n: SLDA100ID21-18UM) Guard column UCT Selectra® DA, 10 × 2.0 mm, 1.8 µm (p/n: SLDAGDC20-18UM) Guard column holder p/n: SLDGRDHLDR Column temperature 60°C Flow rate 400 µL/min Injection volume 5 µL

Mobile Phase Gradient Mobile Phase A Mobile Phase B Time (min) Water + 0.1% formic acid Methanol + 0.1% formic acid 0.0 95 5 0.5 70 30 4.0 70 30 5.0 40 60 8.0 40 60 8.5 0 100 12 0 100 12.1 95 5 16.5 95 5

MS Conditions Instrumentation Thermo ScientificTM TSQ VantageTM Ionization mode ESI+ & ESI- Spray voltage 4000 V Vaporizer temperature 450°C Capillary temperature 350°C Sheath gas pressure 55 arbitrary units Auxiliary gas pressure 45 arbitrary units Ion sweep gas 0 arbitrary units Declustering potential 0 V Collision gas Argon (1.7 mTorr) Cycle time 0.5 sec Software XcaliburTM version 2.2

MRM Transitions Compound Polarity tR (min) Precursor ion Product ion 1 Product ion 2 + 2.14 156.0 92.1 108.1 Albuterol + 2.55 240.1 121.1 148.1 Albuterol-D3 (IS) + 2.55 243.1 124.2 151.2 Lincomycin + 3.13 407.2 126.1 359.2 Ampicillin + 3.82 350.1 106.1 192.0 + 3.90 291.1 123.1 230.1 13 Trimethoprim- C3 (IS) + 3.90 294.1 233.2 264.2 Thiamphenicol - 4.10 353.9 121.1 185.0 + 4.15 251.0 92.1 156.0 + 4.30 256.0 92.1 156.0 + 4.35 320.1 233.1 276.1 Ormetoprim + 4.53 275.1 123.1 259.1 Thiabendazole + 4.57 202.0 131.1 175.1 Thiabendazole-D6 (IS) + 4.57 208.0 137.1 181.1 Oxytetracycline + 4.60 461.1 337.1 426.1 Cefalexin + 4.85 348.0 158.0 174.0 + 5.06 362.1 261.1 318.2 + 5.25 332.1 231.1 288.2 15 13 Ciprofloxacin- N- C3 (IS) + 5.25 336.1 235.1 291.2 Tetracycline + 5.30 445.1 154.0 410.2 + 5.38 254.1 92.1 148.1 13 Sulfamethoxazole- C6 (IS) + 5.38 260.0 98.2 162.1 + 5.41 265.0 92.1 156.0 + 6.04 352.1 265.1 308.2 + 6.12 271.0 92.1 156.0 Chloramphenicol - 6.43 320.9 121.0 152.0 Cefotaxime + 6.50 456.0 125.0 167.0 + 6.51 360.1 245.1 316.2 Demeclocycline + 6.55 465.0 430.1 448.1 Sulfachloropyridazine + 6.76 285.0 92.1 156.0 Sulfamethazine + 6.80 279.1 124.1 186.0 13 Sulfamethazine- C6 (IS) + 6.80 285.1 124.2 186.1 Azithromycin + 6.85 749.1 116.0 591.5 + 6.88 386.1 299.1 342.2 Clindamycin + 6.96 425.1 126.1 377.2 Chlortetracycline + 7.05 479.0 154.0 444.1 Cefazolin + 7.15 455.0 111.9 156.0 Doxycycline + 7.24 445.1 321.1 428.2 Diphenhydramine + 7.34 256.1 115.1 165.1 Carbadox + 7.40 263.0 129.1 231.1 + 7.60 311.0 108.1 156.0 Erythromycin + 7.91 734.4 158.0 576.4 13 Erythromycin- C2 (IS) + 7.91 736.4 160.1 578.4 Cephalothin + 8.10 419.0 204.0 359.1 Penicillin G + 8.15 367.1 114.0 160.0 Anhydroeythromycin + 8.23 716.4 158.0 558.4 Clarithromycin + 8.53 748.4 158.0 590.4 Ceftiofur + 8.73 524.0 124.9 241.0 Penicillin V + 8.83 383.1 114.0 160.0 Tylosin + 8.97 916.4 173.9 772.5 Roxithromycin + 9.30 837.4 158.0 679.5 + 9.39 262.0 160.1 216.0 Oxacillin + 9.52 434.1 144.0 160.0 Cloxacillin + 10.10 468.0 160.0 178.0 + 10.40 262.0 126.1 202.0 Virginiamycin + 10.80 526.2 337.1 355.1

Results and Discussion:

Chromatographic separation The unique chemistry of the Selectra® DA column, which contains a polyaromatic stationary phase, provides orthogonal selectivity to a traditional C18 column and offers a high degree of retention and selectivity for aromatic compounds. The stationary phase is capable of retaining analytes through hydrophobic (dispersive) interactions as well as through pi-pi (π- π) interactions which exhibit a substantial increase in retention for dipolar, unsaturated or conjugated analytes. The Selectra® DA column is ideally suited for the analysis of veterinary drug residues, as most compounds (and metabolites) possess aromatic functionality.

In the final UHPLC-MS/MS method, methanol was chosen as the organic mobile phase solvent, as it was found to give better overall peak shape than acetonitrile, particularly for the tetracycline and fluoroquinolone . A hold was included in the gradient to improve chromatographic separation and all compounds were successfully eluted in <12 min. The enhanced retention of the Selectra® DA column ensured that the most polar compound included in the method, sulfanilamide, didn’t elute until >2 minutes (30% methanol). Although it was possible to start the gradient at a higher percentage of organic solvent (20%) and reduce the overall run time, this required the use of a smaller injection volume (2 μL) which negatively affected the method sensitivity. Ultimately, the best sensitivity was obtained by starting the gradient at 5% methanol and using a 5 μL injection volume.

RT: 0.00 - 11.99 SM: 5G 4.50 NL: 100 1.64E6 TIC MS 95 10ppb_sa mple_6

90

85

80

75

70

65 10.38

60 6.94

55 3.89

50

45

RelativeAbundance 8.98

40

7.32 35

5.03 30 3.11 25 2.54

5.23 6.01 20 8.50 6.50 10.09

15 7.58 8.20 9.50 10 9.24 7.89 11.21 5 10.78

1.58 2.05 2.86 3.51 11.79 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 Time (min) Figure 1. TIC chromatogram of an extracted milk sample (10 μg/kg) containing the 49 veterinary drugs and 7 internal standards.

Sample preparation procedure: Prior to instrumental analysis, a sample pre-treatment step is required to concentrate the analytes of interest and eliminate non-desirable matrix components. This is particularly important for the analysis of veterinary drugs in milk because of their low regulatory limits and the larger sample size required to obtain necessary method sensitivity. One of the biggest difficulties in milk analysis is the high fat, protein, and calcium content that can often interfere with instrumental analysis. The instability of certain drug classes, namely β-lactams, tetracyclines and macrolides, causes additional complications by limiting the conditions that can be used for sample extraction and cleanup. Therefore, the sample preparation procedure was optimized to remove as much co-extracted matrix components as possible while minimizing any loss of the veterinary drug residues.

A simple deproteinization procedure using an EDTA/acetic acid buffer (sample pH should be 4-4.5) followed by centrifugation to separate the proteins and lipids was carried out prior to SPE extraction and cleanup. The inclusion of EDTA in the extraction buffer prevents the complexation of drugs with metal ions (e.g. calcium), particularly the tetracyclines and fluoroquinolones. After application of the sample supernatant to the SPE cartridge, the sorbent was washed with 10% methanol to remove polar matrix components and hexane to remove lipophilic compounds. Acetone was used as the SPE elution solvent as it was found to be more effective than methanol, particularly for hydrophobic compounds that contain multiple aromatic functional groups and are strongly retained on the DVB sorbent. Furthermore, acetone is a volatile organic solvent that is readily removed by evaporation under mild conditions (35-40°C). Filtration of the sample extract prior to LC-MS/MS analysis and the use of isotopically labeled internal standards and matrix- matched calibration curves are recommended in order to obtain the best possible results.

For most compounds, the recovery was between 70% and 120% and the reproducibility <20%. Only a small number of compounds gave results outside of the acceptable limits, which was due to analyte instability (cefalexin and ceftiofur) or inadequate sensitivity at the lowest concentration level (sulfanilamide and thiamphenicol). In addition, all compounds could be accurately detected at a concentration of 10 μg/kg and the vast majority of compounds at 1 μg/kg, demonstrating that the presented method is suitable for monitoring a wide range of veterinary drug residues in milk.

Accuracy, Precision and Method Performance Data for Whole Milk 1 µg/kg (n=12) 10 µg/kg (n=12) 100 µg/kg (n=12) Recovery RSD Recovery RSD Recovery RSD LCL Linearity Analyte (%) (%) (%) (%) (%) (%) (µg/kg) (R2) Albuterol 100.1 6.5 106.6 4.4 105.9 1.7 0.5 0.9996 Ampicillin 109.8 10.4 92.3 7.8 96.3 3.6 0.5 0.9983 Anhydroeythromycin 112.7 9.8 107.8 6.7 100.2 4.4 0.5 0.9971 Azithromycin 71.0 7.7 83.4 8.0 82.8 6.2 0.5 0.9981 Carbadox 82.3 11.4 95.3 12.2 95.9 6.5 0.5 0.9962 Cefalexin 69.3a 7.8 78.4 25.9 82.1 21.6 1.0 0.9981 Cefazolin 96.1 1.9 97.3 7.2 101.7 3.8 1.0 0.9980 Cefotaxime 96.6 11.3 90.7 6.2 92.1 3.2 0.5 0.9979 Ceftiofur 57.1 7.7 67.8 20.3 66.2 25.9 0.5 0.9982 Cephalothin 105.4 5.4 96.5 11.1 99.8 6.0 0.5 0.9997 Chloramphenicol 98.0 12.2 110.3 7.4 101.5 4.1 0.5 0.9959 Chlortetracycline 108.1 4.7 89.7 8.6 92.2 5.4 0.5 0.9999 Ciprofloxacin 95.0 3.5 99.4 4.2 100.9 1.8 0.5 0.9988 Clarithromycin 104.5 7.1 99.3 7.3 100.8 4.9 0.5 0.9986 Clindamycin 82.4 19.2 81.0 12.3 86.2 19 0.5 0.9978 Cloxacillin 87.6 11.5 79.0 6.7 84.2 3.5 0.5 0.9982 Demeclocycline 100.3 11.1 95.2 5.8 95.9 7.3 0.5 0.9991 Diphenhydramine 92.3 8.2 96.6 6.0 97.8 8.0 0.5 0.9999 Doxycycline 102.9 4.3 87.2 6.1 92.7 5.3 0.5 0.9988 Enrofloxacin 96.4 4.2 84.4 5.6 99.5 4.8 0.5 0.9985 Erythromycin 97.1 8.0 102.6 3.8 100.1 1.4 0.5 0.9970 Flumequine 80.8 13.9 93.0 6.8 91.8 10.5 0.5 0.9992 Lincomycin 109.3 5.8 87.4 9.1 94.0 4.6 0.5 0.9974 Lomefloxacin 107.1 4.4 111.0 4.6 108.5 1.9 0.5 0.9989 Norfloxacin 96.4 4.7 98.0 5.5 98.1 3.3 0.5 0.9992 Ofloxacin 101.2a 4.9 83.7 9.1 100.1 7.3 0.5 0.9988 Ormetoprim 95.3 6.1 101.6 10.1 98.0 4.2 0.5 0.9974 Oxacillin 92.8 10.5 83.4 5.8 88.6 6.6 0.5 0.9990 Oxolinic acid 101.0 5.4 97.5 7.9 99.5 3.9 0.5 0.9986 Oxytetracycline 98.8 11.6 90.9 13.8 96.2 4.2 0.5 0.9984 Penicillin G 103.2 5.4 98.0 7.1 99.9 2.5 0.5 0.9995 Penicillin V 101.7 10.1 88.9 6.2 96.7 5.9 0.5 0.9990 Roxithromycin 97.2 9.1 92.4 10.5 95.6 4.2 0.5 0.9986 Sarafloxacin 79.0 16.2 102.4 11.5 98.5 6.1 0.5 0.9994 Sulfachloropyridazine 85.6 9.0 82.7 15.1 88.7 14.3 0.5 0.9994 Sulfadiazine 88.7 10.1 89.7 7.8 86.7 6.1 0.5 0.9985 Sulfadimethoxine 74.4 14 74.6 7.4 81.4 5.5 0.5 0.9991 Sulfamerazine 87.7 10.7 88.9 10.8 100.1 12.4 0.5 0.9975 Sulfamethazine 97.7 9.1 99.4 4.3 99.7 2.4 0.5 0.9996 Sulfamethizole 91.0 13.5 95.2 12.2 98.8 5.0 0.5 0.9990 Sulfamethoxazole 89.0 9.2 101.6 3.6 101.7 2.5 0.5 0.9994 Sulfanilamide 87.1a 1.6 82.6 12.2 75.2 8.2 0.5 0.9993 Sulfathiazole 90.0 8.9 92.4 12.8 98.4 4.4 0.5 0.9990 Tetracycline 108.8 10.0 99.0 5.2 99.4 7.7 0.5 0.9993 Thiabendazole 96.5 5.7 102.3 4.5 101.6 1.5 0.5 0.9997 Thiamphenicol 81.3 23.3 103.3 7.7 80.2 17.2 0.5 0.9962 Trimethoprim 94.8 5.6 105.6 3.6 103.1 1.8 0.5 0.9992 Tylosin 82.5 7.4 71.4 5.2 79.4 6.1 0.5 0.9995 Virginiamycin 89.3 16.1 91.4 9.3 92.7 10.0 0.5 0.9979 an=6.

Figure 2. Example of an eight-point matrix-matched calibration curve (0.5-200 µg/kg, equivalent to 2.5- 1000 ng/mL in final extract).

6102-05-02

UCT, LLC • 2731 Bartram Road • Bristol, PA 19007 800.385.3153 • 215.781.9255 www.unitedchem.com Email: [email protected] ©UCT, LLC 2016 • All rights reserved Streamlined Method for the Determination of More Than 100 Veterinary Drugs in Animal Tissue Using Dispersive-SPE Clean-up and LC-MS/MS Detection

UCT Part Number: ECC1850CT (500 mg endcapped C18)

August 2012

More than 100 veterinary drugs can be extracted and analyzed using this fast and easy multi- class, multi-residue method.

Procedure: 1) Extraction a) Weigh 2 g of homogenized tissue sample into a 50 mL centrifuge tube b) Add 10 mL of acetonitrile/water (4/1, v/v) c) Shake or vortex for 5 min d) Centrifuge for 5 min at >3700 rcf

2) dSPE Clean-up a) Transfer the supernatant into product ECC1850CT b) Add 10 mL hexane that has been pre-saturated with acetonitrile c) Shake or vortex for 30 sec d) Centrifuge for 5 min at >3700 rcf e) Aspirate hexane to waste f) Evaporate 5 mL of the extract under nitrogen at 45ºC to <0.7 mL g) Add water with 0.1% formic acid to reach a final volume of 1 mL (1 g/mL sample equivalent) h) Transfer sample to a HPLC vial (filter with 0.2µm PVDF membrane if desired) i) Sample is ready for LC-MS/MS analysis

3) LC-MS/MS analysis

MS: Waters TQD HPLC: Waters Acquity UHPLC system

LC Parameters

LC column Waters Acquity HSS T3 (C18), 1.8 μm, 2.1 x 100 mm Guard column Agilent Eclipse Plus C18, 5 μm, 4.6 x 12.5 mm Flow rate 0.5 ml/min Injection volume 20 μl Oven temperature 40 ˚C Autosampler temperature 4 ˚C Equilibration time 3.3 min

Mobile Phase A: 0.1 % formic acid in water/MeCN (95/5, v/v) B: 0.1 % formic in MeCN

Time %B 0.0 0.2 0.1 0.2 8.0 99.8 9.5 99.8 9.6 0.2 13.0 0.2

For analysis of late eluting compounds, 50 μL/min of 27 mM ammonium formate in MeOH:MeCN (75:25) is infused from 5.05 to 9.45 min using the instrument’s infusion syringe to enhance the + signal of the late-eluting anthelmintics ([M+NH4] ion formation).

MS Instrument Settings Capillary voltage 3000 V Extractor voltage 3 V Desolvation temperature 450°C Source temperature 150°C Dwell time 5 msec

Analyte Drug class RT Precursor Cone Product Collision Product Collision (min) ion V 1 energy (V) 2 energy (V) Desacetyl Cephapirin β-Lactam 0.69 382.1 32 152 28 124.2 48 Florfenicol Amine Phenicol 0.68 248.1 25 230.2 10 130.1 35 Sulfanilamide 1.19 173 40 92.9 20 75.9 36 Amoxicillin β-Lactam 1.47 366.1 20 114 22 349.3 10 Salbutamol β -Agonist 1.46 240.2 20 148.2 20 222.3 10 Zilpaterol β -Agonist 1.46 262.3 27 244.3 12 185.2 30 Cimaterol β -Agonist 1.51 220 16 143 24 115.9 34 DCCD β -Lactam 1.72 549.1 40 183 30 241.1 20 Lincomycin Lincosamide 1.87 407.3 20 126.1 30 359.2 20 Sulfadiazine Sulfonamide 2.00 251.1 30 156.1 15 108 20 Ampicillin β -Lactam 2.01 350.1 26 106.1 24 114 30 Desethylene Ciprofloxacin Fluoroquinolone 2.06 306.2 35 288.2 20 245.2 20 Sulfathiazole Sulfonamide 2.10 256.1 25 156.1 15 108 25 Sulfonamide 2.18 250.1 32 156.1 18 108.1 28 Norfloxacin Fluoroquinolone 2.16 320.2 36 276.2 18 233.1 26 Tulathromycin Macrolide 2.17 806.8 38 72 56 577.5 24 Oxytetracline Tetracycline 2.21 461.2 25 426.4 20 443.4 15 Ciprofloxacin Fluoroquinolone 2.22 332.2 35 245.2 25 288.4 20 Ractopamine β -Agonist 2.27 302.2 26 164 16 107 32 Sulfamerazine Sulfonamide 2.30 265.1 28 91.9 28 155.9 16 Fluoroquinolone 2.31 358.1 28 96 26 314.2 18 Tetracyline Tetracycline 2.35 445.2 30 154.1 30 410.2 20 Enrofloxacin Fluoroquinolone 2.38 360.2 35 316.4 20 245.3 25 2-Quinoxalinecarboxylic Acid Other 2.43 175 22 129 16 131 16 Sulfamethizole Sulfonamide 2.55 271.1 28 156.1 16 92 30 Sulfamethazine Sulfonamide 2.54 279.1 35 186.1 20 156.1 20 Sulfamethazine-13C6 (IS) - 2.54 285.2 32 186.1 18 124.1 26 Cefazolin Cephalosporin 2.56 455.1 20 156 16 323.2 12 Sulfonamide 2.58 281.1 30 156.1 20 126.2 20 β -Lactam 2.62 400.3 35 356.4 20 299.2 30 Sarafloxacin Fluoroquinolone 2.58 386.1 20 342.2 20 299.2 30 Clenbuterol β -Agonist 2.56 277.2 25 259.2 10 132.1 30 Pirlimycin Lincosamide 2.74 411.3 30 112.2 40 363.3 20 Chlortetracycline Tetracycline 2.84 479.2 30 154.1 30 444.3 20 Clindamycin Lincosamide 2.89 425.3 45 126.2 40 377.4 20 Gamithromycin Macrolide 2.91 777.8 62 83 54 116 50 Sulfachloropyridazine Sulfonamide 2.95 285 28 156.1 16 108 26 Tilmicosin Macrolide 3.06 869.8 45 174.2 35 696.6 35 Sulfonamide 3.10 311.2 35 156.1 20 108.1 30 Sulfamethoxazole Sulfonamide 3.11 254 26 92.1 30 156 18 Sulfaethoxypyridazine Sulfonamide 3.14 295.1 30 156.1 20 140.2 20 Florfenicol Phenicol 3.15 358.1 24 241 18 206 28 Chloramphenicol Phenicol 3.36 323.1 16 275 16 165 26 Erythromycin Macrolide 3.49 734.8 30 158.2 36 115.9 54 Sulfadimethoxine Sulfonamide 3.57 311.1 35 156.1 20 108 30 Sulfaquinoxaline Sulfonamide 3.59 301.1 34 156.1 18 108 28 Prednisone Corticosteroid 3.67 359.2 22 341.1 10 146.9 26 Tylosin Macrolide 3.66 916.8 45 174.2 35 101.1 35 Penicillin G-d7 (IS) - 3.86 342.1 46 183.1 26 160.1 24 Penicillin G β -Lactam 3.86 335.1 18 176 16 160.1 18 Beta/Dexa-methasone Corticosteroid 4.11 393.2 20 373.2 10 147.1 28 Sulfanitran Sulfonamide 4.16 336.2 26 156 12 134.1 28 Sulfabromomethazine Sulfonamide 4.21 357.1 35 92 30 156.1 25 Zeranol (α-Zearalanol) Other 4.37 323.2 16 305.2 10 189.1 24 Oxacillin β -Lactam 4.39 402.1 22 160 20 243.1 18 Atrazine (QC) - 4.49 216.1 34 174 18 103.9 30 Nafcillin β -Lactam 4.79 415.2 20 199.1 14 171.1 38 Oxyphenylbutazone NSAID 4.83 325.2 26 120.1 24 148.2 30 Flunixin NSAID 4.86 297.1 42 279.1 22 109 50 Flunixin-d3 (IS) 4.82 300.1 40 282.1 24 112 54 Dicloxacillin β -Lactam 5.03 470.2 22 160.1 14 311.1 16 Phenylbutazone NSAID 5.93 309.1 28 120 20 91.8 30 Melengesterol Acetate Other 6.30 397.4 30 279.3 20 337.5 15 2-thiouracil Thyreostat 0.85 128.9 32 111.9 12 69.9 18 2-mercapto-1-methylimidazole Thyreostat 1.14 114.9 40 87.9 16 73.9 16 6-methyl-2-thiouracil Thyreostat 1.22 142.9 32 83.9 18 125.9 14 -OH 1.42 188 22 123 14 126 18 Dipyrone Tranquilizer 1.60 218.1 24 96.9 12 187 10 Dimetridazole-OH Nitroimidazole 1.63 158 22 140 12 93.9 22 Metronidazole Nitroimidazole 1.63 172 26 127.9 14 81.9 24 5-hydroxythiabendazole Anthelmintic 1.70 218 50 190.9 26 147 32 Albendazole 2-amino-sulfone Anthelmintic 1.85 240 36 133 28 198 20 Ronidazole Nitroimidazole 1.85 201 18 139.9 10 54.8 20 Levamisole Anthelmintic 1.86 205 40 178 22 90.9 34 Dimetridazole Nitroimidazole 1.86 142 32 95.9 16 80.9 24 Thiabendazole Anthelmintic 1.94 202 44 174.9 26 130.9 32 6-propyl-2-thiouracil Thyreostat 2.15 171 38 154 18 112 20 2-mercaptobenzimidazole Thyreostat 2.30 150.9 42 92.8 20 118 22 Azaperone Tranquilizer 2.34 328.3 34 165 20 122.9 36 Fluoroquinolone 2.39 396.2 36 352.2 18 295.1 24 Albendazole sulfoxide Anthelmintic 2.44 282.1 28 240 14 207.3 24 Xylazine Tranquilizer 2.48 221.1 42 164 26 147 24 Ipronidazole-OH Nitroimidazole 2.54 186.1 22 168 14 121.8 20 Morantel Anthelmintic 2.60 221.1 50 122.9 36 163.9 28 2-amino-Mebendazole Anthelmintic 2.63 238.1 50 104.9 26 132.9 36 6-phenyl-2-thiouracil Thyreostat 2.73 205 38 103 26 187.9 18 2-amino-Flubendazole Anthelmintic 2.77 256 50 122.9 28 94.9 38 Cambendazole Anthelmintic 2.83 303.1 34 261.1 18 217 28 Bacitracin Other 2.87 475.3 26 85.9 24 199.1 30 Carazolol Tranquilizer 2.90 299.3 34 116 20 97.9 22 Doxycycline Tetracycline 2.91 445.3 28 428.2 20 97.9 46 Oxibendazole Anthelmintic 2.95 250.1 34 218.1 18 175.9 28 Oxfendazole Anthelmintic 3.01 316.1 40 158.9 32 191 22 Albendazole sulfone Anthelmintic 3.02 298.1 38 266 20 159 36 Ipronidazole Nitroimidazole 3.20 170.1 34 124 18 109 24 Clorsulon Flukicide 3.39 377.7 24 341.8 12 241.9 20 Haloperidol Tranquilizer 3.53 376.2 40 165 24 122.9 42 Acetopromazine Tranquilizer 3.55 327.2 32 86 20 254 22 Promethazine Tranquilizer 3.58 285.2 24 85.9 16 198 20 Fenbendazole sulfone Anthelmintic 3.65 332.1 40 300 22 158.9 38 Albendazole Anthelmintic 3.65 266.1 34 234 20 191.1 32 Mebendazole Anthelmintic 3.70 296.1 36 264.1 20 104.9 36 Flubendazole Anthelmintic 3.90 314.1 38 282 22 94.9 50 Propionylpromazine Tranquilizer 3.91 341.2 32 85.9 22 268.1 24 Chlorpromazine Tranquilizer 4.04 319.2 32 86 20 246 22 Triflupromazine Tranquilizer 4.26 353.2 34 85.9 22 280 28 Fenbendazole Anthelmintic 4.33 300.1 38 268 20 158.9 36 Oleandomycin triacetate Macrolid 4.37 814.7 38 200.1 30 98 48 Nitroxynil Flukicide 4.41 288.8 40 126.8 20 115.9 34 Virginiamycin M1 Other 4.49 526.4 26 508.3 12 108.9 44 Ketoprofen Tranquilizer 4.71 255.1 28 104.9 24 209 14 Haloxon Anthelmintic 5.28 415.1 44 272.9 34 210.9 36 Triclabendazole sulfoxide Flukicide 5.37 372.8 36 357.8 18 212.9 30 Emamectin benzoate Anthelmintic 5.49 886.8 52 158 40 126 46 Diclofenac Tranquilizer 5.55 296 20 214.9 20 250 12 Triclabendazole Flukicide 5.99 359 52 343.9 26 274 38 Other 6.05 613.5 30 189 28 132.9 64 Oxyclozanide Flukicide 6.08 399.6 38 363.8 14 175.9 24 Niclosamide Flukicide 6.20 325 36 170.9 30 289 16 Tolfenamic acid Tranquilizer 6.23 262.1 22 244 14 180 40 Bithionol Flukicide 6.76 352.9 36 160.8 24 191.2 28 Eprinomectin Anthelmintic 7.44 914.8 18 186.1 20 154 40 Abamectin Anthelmintic 7.94 890.8 16 305.3 28 145 42 Closantel Flukicide 8.07 660.9 70 126.8 54 344.8 32 Doramectin Anthelmintic 8.30 916.9 22 331.3 26 113 56 Moxidectin Anthelmintic 8.32 640.5 16 528.3 8 498.2 10 Rafoxanide Flukicide 8.50 623.9 62 126.1 48 344.8 30 Selamectin Anthelmintic 8.62 770.7 36 145 30 112.9 40 Ivermectin [M+Na]+ Anthelmintic 8.77 897.8 82 183 58 329.2 56

Accuracy and Precision A multi-day, multi-analyst validation demonstrated that the final method is suitable for screening of 113 analytes, identifying 98 and quantifying 87 out of the 127 tested drugs at or below US regulatory tolerance levels in bovine muscle. Overall, the method demonstrated reasonably good quantitative performance with recoveries ranging between 70–120% for 87 out of 127 analytes, and recovery of < 50% for only 20 analytes. 85 analytes gave RSDs ≤ 20% and 100 analytes gave RSDs ≤ 25%.

Adapted from: L. Geis-Asteggiante, S.J. Lehotay, A.R. Lightfield, T. Dutko, C. Ng, L. Bluhm, Ruggedness testing and validation of a practical analytical method for >100 veterinary drug residues in bovine muscle by ultrahigh performance liquid chromatographyndashtandem mass spectrometry, Journal of Chromatography A 1258 (2012) 43-54.

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