Development and validation of LC-MS/MS method for simultaneous quantitation of testosterone, trenbolone, salbutamol and taleranol in chicken muscle

Indian Journal of Chemistry Vol. 3B, September 4, pp 2-27 Development and validation of LC-MS/MS method for simultaneous quantitation of testosterone, trenbolone, salbutamol and taleranol in chicken muscle Meenakshi Dahiya, Nidhi Dubey* & G N Singh Indian Pharmacopoeia Commission Sector 23, Rajnagar, Ghaziabad 002, India E-mail: drnidhiipc@gmail.com Received 22 October 3; accepted (revised) 8 June 4 A comparatively simple, sensitive and rapid analytical method has been developed and validated to determine the residues of testosterone, trenbolone, salbutamol and taleranol in chicken muscle using LC-MS/MS in positive ionization mode. Two main parameters that are focused for the identification of target compound is retention time and ion ratio. m/z at 289 > 97, 270 > 98, 240 > 48 and 322 > 304 have been taken as quantifier ion and m/z at 289 >9, 270 >226, 240 > 66 and 322 > 286 have been taken as qualifier ion for testosterone, trenbolone, salbutamol and taleranol, respectively. The LOD and LOQ are found to be 0.2 ng/kg and 0.4 ng/kg for testosterone and 0.µg/kg and 0.2 µg/kg trenbolone, salbutamol, taleranol, respectively which is below the MRL value fixed by Export Inspection Council, Ministry of Commerce and Industry, Govt. of India. Considering the risk of misuse of veterinary drugs and importance of their monitoring, the present method has advantages, such as simple and robust extraction procedure, lesser consumption of organic solvent, shorter run time of.0 min, high throughput analysis, precise, the ability to quantify residues at low detection limits in complex matrix and screening of multiple drug residues in a single run with high selectivity and accuracy. Keywords: LC-MS/MS, method development, validation, testosterone, trenbolone, salbutamol, taleranol, chicken muscle Veterinary drugs are commonly used on animals as therapeutic agents to increase feed efficiency and to prevent outbreak of disease. The use of unauthorized drugs or the failure to follow label directions of approved drug may result in unsafe residues in these food products and endanger human health. Therefore, it is necessary to develop and validate analytical methods to monitor drug residues in animal derived food for human consumption. Earlier, products of animal origin harvested by hunting were considered as noble food contributing to strength, health, longevity and the well being of man. However, with advances in science, many xenobiotic and natural compounds (steroids, hormones and β-agonists) have been used and sometime misused to improve the growth of cattle, sheep and other livestock animals 2. Testosterone is one such steroid hormone from the androgen group (C-9 steroids) and is found in mammals, reptiles 3, birds 4 and other vertebrates. Androgens stimulate or control the development and maintenance of masculine characteristics and most often used as anabolic steroids. Steroid hormones are legally used in veterinary medicine under veterinary prescription. However, besides their use under regulated conditions, their use for growth promotion is forbidden. Salbutamol also known as albuterol, is a β-adrenergic receptor agonist, primarily used in the treatment of bronchial asthma and other forms of allergic airways disease. The drug is also used in obstetrics for the prevention of premature labour and as a nasal decongestant 6,7. Trenbolone and taleranol are the other anabolic steroids. The use of these drugs may cause accumulation of their residues in the animal tissues which ultimately find their way into food products derived from animal origin. Chicken muscle contaminated with residues of veterinary drugs in concentration above the MRL is considered adulterated and inappropriate for consumption representing risk to public health and commercial risk to the drug industry. The MRL value is.0 µg/kg for all four drug residues as per Export Inspection Council, Ministry of Commerce and Industry, Govt. of India 8. The occurrence of drug residues in food and food products originating from veterinary treatments has become increasingly noticeable. Residues of these drugs are responsible for several health hazards. It therefore becomes essential that the residues be strictly regulated from food safety point of view. Various methods are available for the simultaneous determination of drug residues 9-. However, extraction procedure in several methods is complicated and lengthy 2-6. The present paper describes an analytical method developed for simultaneous determination of residues of testosterone, trenbolone, salbutamol and taleranol in chicken muscle using LC-MS/MS with ESI positive ionization mode. Liquid chromatography-electrospray ionization tandem mass spectrometry is a promising technique for residual analysis because of its high selectivity, specificity and sensitivity and no need for derivatization steps. This technique reduces the analysis time, eliminates predictable sources of error

22 INDIAN J. CHEM., SEC B, SEPTEMBER 4 and decreases the use of hazardous and expensive reagents. In this study, rapid and convenient multiresidual methods were established in order to ensure public health and monitor food safety. The goal was achieved by combining mass spectrometric method with a simple preparation procedure. LC- MS/MS gives good selectivity and sensitivity. The two stage mass selection enables the detection, identification and quantification of target compounds at very low level in complex matrix. Materials and Methods Reagents and chemicals: Reference standards of testosterone, trenbolone and salbutamol with purity of 98.0%, 99.32% and 99.% were purchased from Sigma Aldrich and taleranol with purity of 98.92% was received as a gift from IVRI India. Acetonitrile, water and methanol (liquid chromatographic grade) were purchased from Merck Specialties Private Limited and formic acid (analytical reagent grade) was purchased from S.D. Fine Chem Limited. LC-MS/MS Instrumentation and Chromatographic conditions LC-MS/MS system: Waters 269 series Alliance quaternary liquid chromatographic system (Waters, USA) with a Triple Quadrupole Mass Spectrometer, Quattro micro API (Micro mass, UK) equipped with an electro spray interface and masslynx 4. software (Micro mass) for data acquisition and processing was used. The instrument was provided with a -vial capacity management system. Balance: Balance with readability of 0.0 mg and capacity of 2 g (Mettler Toledo XP-), was used. Vortex: Model-Spinix (Tarsons Products Pvt Ltd). Syringe filter: Syringe filters were of pore size 0.22 µm and 0.4 µm, with diameter of 2 mm (Advanced Micro Devices Private Limited). Nitrogen evaporator: Rapid Vap (Labconco Corporation). Centrifuge: The extracts were centrifuged by using a high-speed refrigerated centrifuge, The rotor head was suitable for eight tubes of 0 ml size (Remi Sales and Engineering Ltd). Centrifuge tubes: Disposable 0 ml conical centrifuge tubes with screw caps (Tarsons Products Pvt Ltd). Preparation of standard solution: Approximately.0 mg testosterone and mg of trenbolone, salbutamol and taleranol reference standards were accurately weighed into individual 0 ml volumetric flask and dissolved and made to volume using methanol. This gave a stock solution of 0 µg/ml for testosterone and 0 µg/ml for trenbolone, salbutamol and taleranol each. From all the four stock solutions ml of aliquot was taken in a 0 ml volumetric flask and made to volume using methanol so as to give a std mix solution having a concentration of 0. µg/ml for testosterone and.0µg/ml for trenbolone, salbutamol and taleranol. The solutions were stored at 2 C to 8 C Preparation of calibration standard solutions: From the standard mix solution having concentration of 0. µg/ml for testosterone and.0 µg/ml for trenbolone, salbutamol and taleranol, appropriate aliquots were taken and further diluted with methanol so as to give a series of calibration standard solutions having concentration ranging from 0.2 ng/ml to ng/ml for testosterone and.0 ng/ml to 0 ng/ml for trenbolone, salbutamol and taleranol. All solutions were stored at 2 C to 8 C. Preparation of matrix-matched calibration standard solutions: For the preparation of matrixmatched calibration solutions the standards were spiked into the blank muscle s after extraction at the same concentration level as the solution of calibration standards. Preparation of mobile phase: The mobile phase was prepared by mixing two solutions i.e., A and B in the ratio of :80 (A : B) and filtered through 0.4- micron filter membrane using the Millipore filtration unit. Solution A: 0.% formic acid in water prepared by adding 0. ml of formic acid to 00 ml of water and solution B: 0.% formic acid in methanol prepared by adding 0. ml of formic acid in 00 ml of methanol. Preparation of : Muscle s were obtained from local market and were stored at C. Approximately.0±0. g of the muscle equilibrated at RT, was taken in the centrifuge tube and homogenised with ml of 0:0 mixture of acetonitrile and methanol for min at RT. The solution was then centrifuged at ambient temperature for min at 7000 rpm followed again by centrifugation at 4 C at 7000 rpm for further min. The supernatant layer was collected in a dry separating funnel. The residue was extracted using the same process twice. The combined organic solvent from all the three extractions was passed through anhydrous sodium sulphate and washed with n-hexane saturated with acetonitrile. This solvent was then evaporated to dryness under a stream of nitrogen and the dried extract was re-dissolved in mobile phase before injecting into LC-MS/MS.

NOTES 23 LC-MS-MS conditions Column: The separation was carried out using X Terra MS C-8 column (2. mm 0 mm; µm) and mobile phase comprising of A: 0.% formic acid in water; B: 0.% formic acid in methanol; (A:B- :80 in the isocratic mode). The LC column was set at 30 C. ESI Interface: Optimal parameters of the ESI interface were optimized by infusing ng/ml standard solution of testosterone, trenbolone, salbutamol and taleranol one by one in the mobile phase using a Harvard syringe pump. LC-MS/MS determination was performed by operating the mass spectrometer in positive ionisation mode. Typical MS settings: Capillary voltage (kv): 3.; cone voltage (V): 30; source temperature ( C): 0; dessolvation temperature ( C): 40. Results and Discussion Liquid chromatographic separation: A comparatively simple, sensitive and accurate method has been developed for the determination of testosterone, trenbolone, salbutamol and taleranol in muscle using LC-MS/MS with positive ESI mode. Optimum separation was achieved using 0.% formic acid in water (A) and 0.% formic acid in methanol (B) in the ratio :80(v/v) as mobile phase; well resolved peaks for testosterone, trenbolone, salbutamol, and taleranol were obtained within min of injection. Extraction procedure: For the extraction of testosterone, trenbolone, salbutamol and taleranol in muscle, a simplified extraction procedure has been developed. Since all the four analytes are soluble in solvents like methanol and acetonitrile, a combination of methanol and acetonitrile has been taken for extracting the residues from meat s. Any fat components which might have been coextracted along with the residues were washed off with n-hexane saturated with acetonitrile. The extract was dried off under nitrogen and the dried extract was dissolved in methanol and injected into LC-MS/MS. Compd Mass spectrometery: For the purpose of evaluating the fragment ions and the intensity of the signals, the reference standard solutions of all the four, testosterone, trenbolone, salbutamol, and taleranol were infused one by one using both positive and negative ESI mode of the mass spectrometer detector. The result shows intense signal for testosterone and trenbolone in positive ionization mode whereas no significant response was observed in the negative ionization mode for the two compounds, in case of salbutamol and taleranol intense signals were observed in both positive and negative ionization mode. Even when the conditions were optimized using liquid chromatography, all the ions were distinctly observed in both the modes and signal response was also comparable in both the modes indicating that either of the modes could be used for the purpose of development of the method for salbutamol and teleranol. Since all the four compounds showed intense signals in positive ionization mode, the method was develop by using water and methanol as mobile phase in positive ionization mode to detect the residue of all four drugs at very low concentration. This saves run time and also the running cost of equipment. For the purpose of developing and validating the method, the most distinct ions used are tabulated in Table I. Method performance characteristics: The method was validated as per the International Union of Pure and Applied Chemistry (IUPAC) and Eurachem guidelines 6. Specificity and Selectivity: The chromatographic interferences from the muscle were investigated by comparing the chromatograms of blank and the spiked s. For this purpose the chromatographic conditions were optimized to get good peak shape and sensitivity of the analyte. No significant interfering peaks from the endogenous compound were observed at the retention time of Table I MRM setting for positive ion MS/MS analysis of testosterone, trenbolone, salbutamol and taleranol Retention time (min) ESI mode Parent ion (m/z) Testosterone 2.0 + Ve 289.00 Trenbolone.86 + Ve 270.98 Salbutamol.00 + Ve 240.2 Taleranol.64 + Ve 322.99 Product ion (m/z) 97.00 9.00 98.94 226.92 48.00 66.00 304.8 286.8 Capillary voltage(kv) Cone voltage (V) Collision energy (ev) Dwell time (milli second) 3. 2 2 0 3. 30 30 0 3. 0 3. 0

24 INDIAN J. CHEM., SEC B, SEPTEMBER 4 testosterone, trenbolone, salbutamol and taleranol, thus proving reliability of the method. Total chromatographic run time was min. The shorter run time makes the method more productive. shows the chromatograms of muscle spiked with testosterone, trenbolone, salbutamol and taleranol, respectively. Precision and Accuracy: The precision and accuracy studies were carried out for both inter-day and intra-day repeatability and reproducibility by analyzing six replicates of three spiked s of muscle at five different concentrations. The intra-day and inter-day precision (% RSD) ranged from 0.876-3.447 and.700-2.809, respectively for the spiked s of testosterone (Table II), from 0.7-2.98 and.30-2., respectively for trenbolone (Table III), from 0.660-3.44 and.04-2.74, respectively for salbutamol (Table IV) and from 0.02-3.246 and.048-2.9, respectively for taleranol (Table V). The accuracy and precision (intra- and inter-day) were determined by analyzing six replicates of spiked s at five concentrations on three different days. Linearity: Six calibration standards evenly spread over the concentration range of interest were analyzed. The calibration standards were run in triplicate. The average r 2 values are 0.9997, 0.9988, 0.999 and 0.998 for testosterone, trenbolone, salbutamol and taleranol, respectively. The matrix matched calibration curves were found to be linear (r 2 > 0.99) over the concentration range of 0.2 ng/ml to ng/ml for testosterone and.0 ng/ml to 0 ng/ml for trenbolone, salbutamol and taleranol. The matrix effect was investigated by comparing standards in solvent with matrix matched standards at different concentration levels. The matrix effect was found to be small for all the four analytes, but it was observed that the matrix suppressed the signal response to some extent for all the four analytes. For the calculation purpose the MMC (matrix matched calibration) was used as this corrects the matrix effects. The % accuracy observed for the mean of back-calculated concentration for three linearities was within 99.82-.0, 97.9-.46, 98.-0.38 and 98.33-.78 for testosterone, trenbolone, salbutamol and taleranol, respectively. The corresponding precision (% RSD) values ranged from 0.664-2.86, Figure Typical MRM chromatogram of muscle spiked with drug residues analyzed by LC-MS/MS

NOTES 2 Table II Intra-day and Inter-day precision and accuracy data of testosterone in muscle 0.2 2. Inter-day precision and accuracy (n=8) Mean SD % RSD % Accuracy Mean SD % RSD % Accuracy 0.249 0.009 3.447 99.73 2 0.2 0.006 2.337 0.40 0.249 0.007 2.809 99.69 3 0.247 0.007 2.867 98.93.006 0.07.76 0.62 2.08 0.0.994.77.009 0.07.700 0.88 3.003 0.02.9 0.27 2.47 0.064 2.30.89 2 2.498 0.049.98 99.93 2.03 0.06 2.42 0.3 3 2.464 0.42.690 98.6 4.98 0.43 2.900 98.36 2 4.984 0.044 0.876 99.68 4.988 0.22 2.443 99.76 3.062 0.26 2.479.24 9.976 0.24.247 99.76 2 9.876 0.6.07 98.76 9.942 0.32.323 99.42 3 9.974 0.6.6 99.74 Table III Intra-day and Inter-day precision and accuracy data of trenbolone in muscle Sample Inter-day precision and accuracy (n=8) Mean SD % RSD % Accuracy Mean SD % RSD % Accuracy 0.008 0.0.48 0.7 2.02 0.06.3.23 3.008 0.04.379 0.83 4.98 0.072.44 99.6 2.027 0.074.467 0.3 3.09 0.060.97 0.37.03 0.9.82 0.3 2 9.998 0.22.22 99.98 3.034 0.6.7 0.34.296 0.29.279.48 2.46 0.60 2.98 0.73 3 9.989 0.39.976 99.94 0.8 0.36 0.7 0.22 2 0.39 0.926.838 0.78 3 0.287 0.394 0.784 0.7.009 0.04.402 0.94.009 0.068.38 0.7.022 0.3.30 0.22.43 0.434 2. 0.72 0.262 0.92.78 0.2 Table IV Intra-day and Inter-day precision and accuracy data of salbutamol in muscle Inter-day precision and accuracy (n=8) Mean SD % RSD % Accuracy Mean SD % RSD % Accuracy 0.004 0.04.444 0.42 2.002 0.0.09 0.7 3.00 0.008 0.827 0.3.076 0.09.864.2 2.2 0.40 2.737 2.4 3.34 0.4 2.023 2.68.097 0.6 2.043 0.97 2.029 0.9 2.079 0.29 3.04 0.22 2.234 0.4 9.926 0.26.287 99.63 2 9.9 0.249.248 99.76 3 9.889 0.3 0.660 99.4 0.483.029 2.039 0.97 2 0.242 0.46 0.828 0.48 3 0.948.602 3.44.90.004 0.0.090 0.37. 0. 2.64 2.2.060 0.2 2.02 0.60 9.922 0.8.04 99.6 0.7.099 2.74.

26 2 INDIAN J. CHEM., SEC B, SEPTEMBER 4 Table V Intra-day and Inter-day precision and accuracy data of taleranol in muscle Inter-day precision and accuracy (n=8) Mean SD % RSD % Accuracy Mean SD % RSD % Accuracy 0.00 0.026 2.66 0.3 2.002 0.009 0.87 0.8 3 0.998 0.0 0.967 99.7.4 0.3 2.70 2.08 2.7 0.66 3.246 2.34 3.8 0.087.688 3.62 9.940 0.63.638 99.40 2 9.999 0.00 0.02 99.99 3 9.963 0.092 0.922 99.63 9.977 0. 0. 99.88 2 9.862 0.30.8 99.3 3 9.88 0.82 0.99 99.09 49.96 0.963.928 99.9 2 0.72.67 3.089.44 3 0.0.274 2.48 0.02.002 0.06.6 0.6.34 0.29 2. 2.68 9.967 0.4.048 99.67 9.88 0.22.064 99.43 0.229.26 2.9 0.46 0.2 2. Table VI Recovery data of testosterone in muscle (n = 6) Measured concentration of testosterone 0.238 0.007 3.607 9.27 2 0.236 0.006 2.374 94.3 3 0.237 0.007 2.88 94.87 0.98 0.036 3.908 9.7 2 0.942 0.02 2.274 94. 3 0.884 0.06.76 88.37 2.437 0.023 0.943 97.48 2 2.424 0.046.892 96.96 3 2.407 0.040.64 96.30 4.723 0.23 2.639 94.4 2 4.3 0.0 2.3 9.0 3 4.37 0.084.80 90.74 9.608 0.30.3 96.08 2 9.624 0.76.827 96.24 3 9.468 0.74.803 96.48 0.-3.82,.0-4.483 and 0.09-3.72% for testosterone, trenbolone, salbutamol and taleranol, respectively. No single calibration point was dropped during validation. Results indicated that the method was accurate and precise within the analytical range. Recovery and matrix effect: The extraction recovery studies of the analytes were carried out at five different concentrations. For this, five different portions of pre-analysed muscle were spiked with µg/kg,.0 µg/kg, µg/kg, µg/kg and 0 µg/kg, respectively in triplicate on three different days and then extracted and determined by the same method as mentioned earlier. The recoveries were within the range of 88.37-97.48% for testosterone (Table VI), 88.8-99.3% for trenbolone (Table VII), 88.38-99.83% for salbutamol (Table VIII) and 89.23-99.30% 0 Table VII Recovery data of trenbolone in muscle (n = 6) Measured concentration of trenbolone 0.908 0.024 2.89 90.80 2 0.889 0.03.443 88.8 3 0.9 0.03 3.448 9. 4.736 0.8 2.290 94.7 2 4.87 0.4 2.43 97.4 3 4.794 0.3 2.827 9.87 9.84 0.70.737 98.4 2 9.74 0.03 0.47 97.4 3 9.77 0.24 2.9 97.7 9.3 0. 0.99 9.66 2 9.340 0.39.89 96.70 3 9.906 0.308.04 99.3 48.687 0.349 0.77 97.37 2 48.78 0.999 2.048 97.2 3 49.284.022 2.074 98.7 for taleranol (Table IX). The extraction recoveries were found to be satisfactory as the values were consistent, precise and reproducible. Limit of detection (LOD) and limit of quantitation (LOQ): LOD was determined by considering signal to noise (S/N) ratio of 3: for the strongest mass transition with respect to the background noise obtained from the blank, whereas, LOQ was determined similarly by considering signal to noise ratio (S/N) :. In order to establish the LOD and LOQ the matrix meat was spiked with all the four analytes (taking into account the weight and the dilution factor) the LOD and LOQ were found to be 0.2 ng/kg and 0.4 ng/kg for testosterone and 0. µg/kg and 0.2 µg/kg trenbolone, salbutamol, taleranol, respectively.

27 INDIAN J. CHEM., SEC B, SEPTEMBER 4 2 0 0 Table VIII Recovery data of salbutamol in muscle (n = 6) Measured concentration of salbutamol 0.884 0.02.38 88.38 2 0.89 0.04.626 89.08 3 0.894 0.06.780 89.43 4. 0. 2.463 90.2 2 4.38 0.6 2.62 90.76 3 4.60 0.044 0.96 9. 9.007 0.094.043 90.07 2 8.942 0.63.824 89.42 3 8.929 0..734 89.29 7.90 0.40 0.784 89.3 2 9.686 0.384.9 98.43 3 9.024 0.69 2.990 9.2 49.97 0.63.308 99.83 2 46.046.076 2.338 92.09 3 49.497.093 2.9 98.99 Table IX Recovery data of taleranol in muscle (n = 6) Measured concentration of taleranol 0.899 0.036 4.08 89.8 2 0.949 0.036 3.772 94.87 3 0.892 0.029 3.264 89.23 4.4 0.79 3.938 9.09 2 4.878 0.099 2.037 97.7 3 4.796 0.028 0.76 9.92 9.226 0.36 3.86 92.26 2 9.436 0.340 3.603 94.36 3 9.4 0.83.937 94. 9.93 0.364.89 9.97 2 9.048 0.3.064 9.24 3 9.44 0.238.244 9.72 49.648 0.842.708 99.30 2 48.228 0.60 0.33 96.46 3 48.322 0.2 0.29 96.64 Robustness: Robustness of the method was determined by analyzing the same set of spiked s (i.e. s spiked at concentration levels of.0 µg/kg,.0 µg/kg and µg/kg) under different parameters, such as, same column chemistry from different manufacturers and different analysts. The method was found to be robust even with small changes in analytical conditions like change in flow rate (± 0.0 ml/min), a change in column temperature (± C), use of same column from different manufacturers (Waters C8 column and Varian C-8) and showed no dramatic change in retention time, peak area and symmetry of peaks. References Aumaitre A, Livestock Production Science, 9, 999, 3. 2 Saeed T, Naimi I, Ahmad N & Sawaya W N, Food,, 999, 69. 3 Cox R M & John-Alder H B, J Expt Biol, 8 (Pt 24), 0, 4679. 4 Reed W L, Clark M E, Parker P G, Raouf S A, Arguedas N, Monk D S, Snajdr E, Nolan V & Ketterson E D, Am Nat, 67, 06, 667. The use of gas chromatography tandem mass spectrometry (GC MS/MS) for optimization of targeted research on residues in matrices of animal origin, (Ph.D. Thesis, Ghent University, Ghent, Belgium), 02. 6 The Extra Pharmacopoeia, 30th Edn. (The Pharmaceutical Press, London), 993. 7 Goodman and Gilman's the Pharmacological Basis of Therapeuticus, 7th Edn. (MacMillan Publishing Company, New York), 98. 8 India-Fresh Poultry meat and poultry meat products: Residue monitoring plan (RMP) for Export to European Union for year 3-4, (Export Inspection Council, Ministry of Commerce and Industry, Govt. of India), 3. 9 Howells L & Sauer M J, Analyst, 26, 0,. Roudaut B, Analyst, 23, 998, 24. Sekench F & Logman L, J AOAC Int, 82, 999, 340. 2 Howells L & Sauer M J, Analyst, 26, 0,. 3 Sheridan R & Desjardins L, J AOAC Int, 89, 06, 88. 4 Durden D A, J Chromatography B, 80, 07, 34. Thompson M, Ellision S I R & Wood R, Pure Appl Chem, 74, 02, 83. 6 The Fitness for Purpose of Analytical Methods: A Laboratory Guide to Method Validation and Related Topics 998 (EURACHEM Working Group LGC, Queens Rd, Teddington, Middlesex, TW 0LY, United Kingdom), 998.