Determination of Beta-Agonists, Including Ractopamine, in Animal Tissues and Urine by Liquid Chromatography-Tandem Quadrupole Mass Spectrometry

DETERMINATION OF BETA-AGONISTS, INCLUDING RACTOPAMINE, IN ANIMAL TISSUES AND URINE BY LIQUID CHROMATOGRAPHY-TANDEM QUADRUPOLE MASS SPECTROMETRY

Simon Hird1 and James Jowett2 1 Waters Corporation, Wilmslow, UK 2 Fera Science Ltd, York, UK

Compound Animal muscle Animal offal Poultry offal Urine  The method was shown to be suitable for checking compliance INTRODUCTION Terbutaline (TER) 5.0 5.0 5.0 1.5 Cimaterol (CIM) 0.25 0.25 0.25 0.25 with the Russian action level for imported meat of 0.1 µg/kg Beta agonists (β-agonists) are synthetic compounds that Salbutamol (SAL) 2.5 2.5 2.5 0.5 ractopamine as shown in Figure 4 Zilpaterol (ZIL) 2.5 2.5 2.5 0.5 mimic some of the effects of naturally-occurring compounds Cimbuterol (CIMB) 0.25 0.25 0.25 0.25 RAC m/z 302>164 RAC m/z 302>164 by binding to beta-receptors on the surface of cells within Hydroxymethyl clenbuterol (HMC) 0.05 0.1 0.1 0.1 Clenproperol (CLE) 0.25 0.25 0.25 0.25 the muscle, fat and other tissues of animals. Some are used Ractopamine (RAC) 0.5 0.5 0.5 0.5

in human medicine but have also been used in livestock RAC m/z 302>107 RAC m/z 302>107 Table 1. STC values (µg/kg) for β-agonists in different samples production to enhance growth and alter body composition1. The two MRM transitions that showed the best selectivity were Salbutamol Zilpaterol Clenbuterol Mabuterol used for each of the β-agonists and one for each stable isotope H3C RAC-d6 m/z 308>168 RAC-d6 m/z 308>168 CH3 CH OH H N Cl HO 3 2 F analogue. The optimum dwell time was set automatically using the HN NH Cl NH CH CH3 3 CH HO CH3 F 3 CH3 autodwell function based upon 4s wide peaks and 12 data points CH 3 F NH CH3 H2N per peak. Figure 4. Chromatograms showing ractopamine and stable N CH3 Cl NH HO isotope analogue from analysis of matrix-extracted standard of HO O Compound RT (mins) MRM CE (ev) Compound RT (mins) MRM CE (eV) Cimaterol Turbutaline pork meat spiked at 0.05 µg/kg and associated blank OH Terbutaline 1.57 226.1 > 107.0 30 Ractopamine-d6 2.30 308.2 > 168.0 15

H2N OH 226.1 > 125.0 25 Clenbuterol 2.49 277.1 > 203.1 15 CH3  Measurement precision, from the peak areas of replicate

HO CH3 Terbutaline-d9 1.57 235.1 > 153.0 15 279.1 > 205.1 15 Salmeterol NH CH3 (n=25) injections of β-agonists matrix extracted standards (1x N NH CH3 OH Cimaterol 1.57 202.1 > 116.0 30 Clenbuterol-d9 2.48 286.1 > 204.0 15 CH3 STC), was good for all but two of the compounds but improved HO O 220.1 > 143.0 25 Tulobuterol 2.48 228.1 > 154.0 15 Isoxsuprine HO when internal standards used (all analytes RSD < 4%) Cimaterol-d7 1.57 227.1 > 161.0 20 228.1 > 172.0 10 Clenproperol Cimbuterol HO NH Salbutamol 1.59 240.2 > 121.0 30 Tulobuterol-d9 2.47 237.2 > 155.0 15  Ion ratios were within tolerance with the exception of

H H H C N O 3 CH 240.2 > 148.0 20 Brombuterol 2.72 367.1 > 214.0 30 clenproperol in liver samples; an isobaric interference was HO 3 H Cl Cl N Salbutamol-d3 1.59 243.2 > 151.0 20 367.1 > 293.1 15 observed on the second transition, so an alternative MRM Ractopamine CH3 HN CH3 HO OH Zilpaterol 1.59 262.2 > 185.1 25 Isoxsuprine 2.81 302.2 > 107.0 30 transition would be needed for confirmatory analyses OH HO CH3 262.2 > 202.1 20 302.2 > 150.0 20 NH OH  Retention times of the analytes from these replicate injections Zilpaterol-d7 1.59 269.1 > 251.1 10 Mabuterol 2.84 311.1 > 217.1 25 CH3 H3C N were very stable showing a maximum shift of ±0.01 minutes HO H N Cimbuterol 1.79 234.2 > 160.0 15 311.1 > 237.1 15 N CH H H 3 OH 234.2 > 143.0 25 Mabuterol-d9 2.83 320.1 > 238.1 15 Performance of the method for confirmatory purposes Figure 1. The structures of some of the key β-agonists Cimbuterol-d9 1.77 243.2 > 161.0 15 Clenpenterol 2.83 291.1 > 203.0 15  The linearity of response for two of the β-agonists in bovine Hydroxymethyl 2.15 293.1 > 203.0 15 293.1 > 205.0 15 The administration of β-agonists as growth-promoting Clenbuterol liver, using bracketed calibration over a suitable concentration agents in food-producing animals is banned in many 295.1 > 205.0 15 Mapenterol 3.18 325.1 > 217.1 25 range, is shown in Figure 5 Clenproperol 2.21 263.1 > 132.0 25 325.1 > 237.1 15 2 countries2. However, ractopamine and zilpaterol are  Coefficients of determination (r ) were > 0.99 and the residuals 265.1 > 132.0 25 Mapenterol-d11 3.16 336.1 > 238.1 15 <4% authorized for production of some animals in a limited Clenproperol-d7 2.21 270.1 > 169.0 25 Salmeterol 4.22 416.3 > 232.2 20 number of countries (e.g. USA, Canada and Brazil). Ractopamine 2.30 302.2 > 107.0 30 416.3 > 380.3 20 Maximum residue limits (MRLs) for ractopamine in cattle and 302.2 > 164.0 15 Salmeterol-d3 4.23 419.4 > 383.3 20 pig muscle (both 10 µg/kg) have been adopted by the Codex Table 2. Retention times and MRM parameters for β-agonists and Alimentarius Commission and have been implemented in stable isotope analogues (quantitative transitions in bold) some countries (e.g. Canada). Others have set alternative limits for ractopamine (e.g. USA - 30 and 50 µg/kg in cattle RESULTS AND DISCUSSION and pig muscle). However, many countries have banned The objective of this work was to evaluate the performance of the meat products containing β-agonists. In the EU, β-agonists Xevo TQ-S micro instrument for determination of 16 β-agonists in other than clenbuterol (for cattle and horse) are prohibited appropriate matrices; batches of various animal target tissues (e.g. for use in food producing animals. As no minimum required liver), edible products (e.g. muscle) and urine collected from performance limits (MRPLs)3 were set for these banned β- animals on the farm. Performance has been evaluated based upon agonists, the EU relies on recommended concentrations sensitivity for analytes in context with international regulations, the (RCs) to indicate required performance of methods. absence of isobaric interference, precision of UPLC-MS/MS measurements, accuracy and precision through analysis of spiked Figure 5. Calibration graphs for β-agonists in bovine liver Importing countries can set even lower limits for testing samples and proficiency test materials and compliance with  The mean measured concentrations of ractopamine and based upon trading decisions to provide better warranties to identification and quantification criteria. clenbuterol compared well with the assigned values in three their customers and to gain commercial advantage. Russia Chromatography bovine liver proficiency test materials (BVL, Germany) and established an action level of 0.1 µg/kg for ractopamine in The UPLC conditions were modified from those reported in an precision was < 6% RSD imported beef consignments, 100x times lower than Codex 4 earlier Waters application note8. The CORTECS C18+ column is a  Requirements given in Commission Decision 2002/65/7/EC MRL, driving down further the analytical limits needed. charged surface, high efficiency C18 column, based on a solid- for identification were met Most countries have established strict surveillance programs core particle, which delivers excellent peak shape for these basic  Ion ratios for ractopamine (0.95 to 1.00) and clenbuterol for official control purposes. Monitoring compliance with analytes at low pH, whilst providing sufficient retention of the most (0.62 to 0.64) were well within tolerance of ±20 % of the regulatory limits requires the use of highly sensitive and polar β-agonists, both at a lower operating pressure than a column reference values selective methods based upon liquid chromatography- packed with fully porous sub 2µm particles. The gradient was  The retention times for ractopamine (2.30 to 2.31 min) tandem mass spectrometry. As the frequency of detection of adjusted to provide sufficient chromatographic resolution of all and clenbuterol (2.49 min) showed little variation and analytes from isobaric interference on both transitions. residues is typically low, samples tend to be screened using were well within acceptance tolerance of ±2.5% a reduced but adequate level of quality control. Any suspect Performance of the method for screening purposes  Mean measured recovery and precision from analysis of two positives can be re-analyzed with the same method but with  No interfering compounds were detected at the retention times of samples of bovine liver spiked with ractopamine (1.25 µg/kg) addition analytical quality control suitable for quantification the β-agonists or stable isotope analogues in all the tested blank and clenbuterol (0.2 µg/kg) was 100% (1.9% RSD) and 101% (2.1% RSD), respectively (e.g. multi-level matrix-extracted calibration) and validated as samples, with the exception of the second transition for clenproperol in liver samples suitable for confirmation4 with values for Decision Limit (CC ) as low as reasonably achievable.  Excellent sensitivity and selectivity was demonstrated by the CONCLUSION response for each analyte peaks detected from the analysis of matrix-extracted standards prepared at 1 x STC  UPLC-MS/MS using the Xevo TQ-S micro provides METHODS excellent sensitivity for detection, identification and ZIL m/z 262>185 RAC m/z 302>164 Extracts were generated by Fera using methods based upon quantification of β-agonists in a range of samples 5,6,7 those of the EURL . Tissue samples were extracted by SAL m/z 240>148 CLE m/z 365>132  The method can be used for both screening and blending with an aqueous buffer/methanol solution and subjected CIM m/z 220>103 HMC m/z 293>203 confirmation for official control purposes with a high to enzyme hydrolysis to cleave any conjugated β-agonists. Solid- degree of confidence but also to meet the challenging phase extraction (SPE) was carried out on a mixed mode-type TER m/z 226>107 CIMB m/z 243>160 requirements of pre-export testing, which often demands cartridge. Samples of urine were extracted by SPE after enzyme lower limits of quantification hydrolysis. Stable isotope analogues were added to all samples ISO m/z 302>107 SALM m/z 416>380 prior to extraction. Matrix-extracted standards were prepared at  Scientists must validate the method in their own BRO m/z 367>293 MAP m/z 325>237 the screening target concentration (STC) when screening and at laboratories and demonstrate that their calculated multiple levels for any confirmation. Typically, STC values are set TUL m/z 228>154 MAB m/z 311>237 Detection Capability (CC ) and Decision Limit (CC ) at half the maximum limit, which for β-agonists varies (Table 1). values are fit for purpose (i.e. as low as reasonably Sample extracts were filtered prior to UPLC-MS/MS. CLEN m/z 277>203 CLENP m/z 291>203 achievable)

Instrument Conditions: Figure 2. Chromatograms showing β-agonists from analysis of References matrix-extracted standards of bovine liver at the STC 1. Centner TJ, Alvey JC, Stelzleni AM. Beta agonists in livestock feed: status, health concerns, UPLC system: ACQUITY UPLC I-Class with FTN autosampler MS Instrument Xevo TQ-S micro and international trade. J Animal Sci. 2014 92(9):4234-40 Column: CORTECS UPLC C18+ column (1.6µm, 2.1 100 mm) Source: electrospray ZIL m/z 262>185 RAC m/z 302>164 2. Nino AMM, Granja RH, Wanschel AC, Salerno AG. The challenges of ractopamine use in Mobile phase A: 0.1 % formic acid (aq.) Polarity: positive ion mode meat production for export to European Union and Russia. Food Control 2017 72:289-292 Mobile phase B: 0.1 % formic acid in acetonitrile Capillary voltage: 1.0 kV SAL m/z 240>148 CLE m/z 365>132 3. European Commission. Commission Decision 2003/181/EC of 13 March 2003 amending Flow rate: 0.6 mL/min Desolvation temperature: 600 C Decision 2002/657/EC as regards the setting of minimum required performance limits Injection volume: 5 µL Desolvation gas flow: 1000 L/Hr CIM m/z 220>103 HMC m/z 293>203 (MRPLs) for certain residues in food of animal origin. Official Journal of the European Column Temp: 40 C Source temperature: 140 C Communities 2003 L71:17-18. Sample temp: 10 C Cone gas flow: 0 L/Hr TER m/z 226>107 CIMB m/z 243>160 4. European Commission. Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods Run-time: 11 minutes Cone voltage: 40 V (75 V for cimaterol 202>116) and the interpretation of results. Official Journal of the European Communities 2002 L221:8- SALM m/z 416>380 ISO m/z 302>107 36. Time (min) % A % B Curve 5. Radeck W and Gowik P. Validation of a multi-residue method for confirmation and 0.0 100 0 - MAP m/z 325>237 BRO m/z 367>293 0.2 100 0 6 quantification of ß agonists in liver in accordance with CD 2002/657EC. First International Symposium on Recent Advances in Food Analysis, Prague 2003. 5.0 50 50 6 MAB m/z 311>237 TUL m/z 228>154 5.1 0 100 6 6. Radeck W and Gowik P. Validation of a multi-residue method for confirmation and quantification of 22 beta-agonists in urine. Fifth International Symposium on Hormone and 8.0 0 100 6 CLENP m/z 291>203 8.1 100 0 6 CLEN m/z 277>203 Veterinary Drug Residue Analysis, Antwerp 2006. 7. Radeck W. Confirmatory method for the determination of 30 ß-agonists in muscle using LC- The data were acquired using MS/MS, 2008, unpublished results MassLynx 4.1 software and processed Figure 3. Chromatograms showing β-agonists from analysis of matrix-extracted standards of bovine urine at the STC 8. Ren Y and Yang J. Determination of Common Beta-Agonist Residues in Meat Products by using TargetLynx XS Application UPLC-MS/MS. Waters Application Note 720004388EN. 2012