Residues of Salbutamol and Identification of Its Metabolites in Beef

Article pubs.acs.org/JAFC

Residues of Salbutamol and Identification of Its Metabolites in Beef Cattle Kai Zhang,†,‡ Chaohua Tang,†,‡ Qingshi Meng,† Wei Du,§ Tao Bo,§ Qingyu Zhao,† Xiaowei Liang,† Shengsheng Liu,† Zhixu Zhang,§ and Junmin Zhang*,† †

Institute of Animal Science, Chinese Academy of Agricultural Sciences, Scientific Observing and Experiment Station of Animal Genetic Resources and Nutrition in North China, Ministry of Agriculture, Beijing 100193, China § Agilent Technologies (China) Co., Ltd., Beijing 100102, China ABSTRACT: Salbutamol, a selective β2-agonist, endangers the safety of animal products because of its illegal use in food animals. In this work, residues of salbutamol and its metabolites were investigated to select appropriate targets and marker residues for monitoring the illegal use of salbutamol. Ten metabolites of salbutamol were identified from plasma, urine, liver, and kidney samples; of these, six were newly identified. There were significant differences (P < 0.01) between the parent (nonconjugated) and total (conjugated + nonconjugated) salbutamol concentrations in plasma, urine, liver, and kidney tissues. Salbutamol residues in urine were relatively higher than those in plasma and other internal tissues during the dosing period and were rapidly eliminated from plasma, heart, spleen, and kidney tissues during the withdrawal time. Total salbutamol was identified as more preferable than parent salbutamol as a marker residue, and urine and eye tissues were found to be more suitable as targets for preslaughter and postslaughter monitoring of the illegal use of salbutamol in beef cattle. KEYWORDS: salbutamol, cattle, residues, metabolites, monitoring

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INTRODUCTION Salbutamol, a phenol-β2-agonist, is widely used for the treatment of human respiratory diseases and asthma.1 In addition, it also acts as a nutrient repartitioning agent and has been used to improve the carcass leanness and production performances of livestock.2,3 Unfortunately, the residues of β2-agonists in edible tissues of animals accumulate in the human body through the food chain and can have adverse effects on human health.4−6 Many countries, including China and those of the European Union, prohibit the use of salbutamol as a growth promoter in food animals.7,8 However, salbutamol is still illegally used in animal production because it can be easily obtained as an overthe-counter drug. The potential risk of β2-agonists to human health is of great concern; thus, the abuse of salbutamol in food animals must be monitored. Food safety regulators commonly use the parent drug as a marker residue during screening, but salbutamol is rapidly cleared from the body and is mainly present in animals as its conjugate metabolites. Consequently, the selection of appropriate target tissues and marker residues for monitoring the abuse of salbutamol in food animals is important. Recently, several analytical methods were developed to analyze the residues of salbutamol in different tissues, and these methods are effective tools for monitoring the salbutamol residues in animal products because they focus directly on sensitivity and rapid detection.9−11 Some studies have reported the depletion of salbutamol in serum, urine, and internal tissues of monogastric animals such as swine and chickens.12,13 Numerous articles have reported the metabolites of salbutamol in plasma and urine samples of rats, swine, and humans.14−17 However, the elimination and metabolism of salbutamol in ruminant animals (e.g., beef cattle) after administration with anabolic dosages remains unclear. © XXXX American Chemical Society

Therefore, the present study identified the primary metabolites of salbutamol in plasma, urine, liver, and kidney tissues of cattle by ultrahigh-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOFMS/MS) and then investigated the parent (nonconjugated) and total (conjugated + nonconjugated) salbutamol residues in plasma, urine, and internal tissues during treatment and after exposure using UHPLC-MS/MS. The obtained results can help in the selection of appropriate target tissues and marker residues in different biological samples to facilitate monitoring the illegal use of salbutamol in beef cattle.

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MATERIALS AND METHODS

Chemicals and Reagents. Salbutamol hydrochloride (95% purity) used for feeding animals was provided by the Institute of Quality Standards and Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences (Beijing, China). The salbutamol sulfate standard (99.5% purity) and salbutamol-d3 internal standard (98.4% purity) were purchased from Dr. Ehrenstorfer (Augsburg, Germany) and C/D/N Isotopes, Inc. (Quebec, Canada), respectively. β-Glucuronidase/arylsulfatase (Helix pomatia, 20 U/mL) was purchased from Merck KGaA (Darmstadt, Germany). Oasis MCX solidphase extraction (SPE) columns (60 mg, 3 mL) were purchased from Waters Corporation (Milford, MA). All chemicals used were of analytical grade or higher. Experimental Animals. In this experiment, nine healthy male Chinese Simmental beef cattle weighing 281.11 ± 29.36 kg were fed 0.15 mg of salbutamol (kg of body weight)−1 day−1 for 21 consecutive days (the given dose of this study was calculated based on a previous report2). Received: Revised: Accepted: Published: A

January 13, 2017 March 15, 2017 March 21, 2017 March 21, 2017 DOI: 10.1021/acs.jafc.7b00189 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Table 1. Recoveries and Precision for the Determination of Salbutamol in Plasma, Urine, Liver, Kidney, Muscle, Heart, Lung, Fat, Eye, Spleen, Small Intestine, Large Intestine, and Rumen Wall Tissues of Cattle (n = 5) sample

spiked levels (ng/mL or ng/g)

mean recovery (%)

intraday RSD (%)

interday RSD (%)

plasma urine liver kidney muscle heart lung fat eye spleen small intestine large intestine rumen wall

0.2, 5, 50 0.2, 5, 50 0.5, 5, 50 0.5, 5, 50 0.5, 5, 50 0.5, 5, 50 0.5, 5, 50 0.5, 5, 50 0.5, 5, 50 0.5, 5, 50 0.5, 5, 50 0.5, 5, 50 0.5, 5, 50

84.5, 109.9, 100.2 82.7, 103.8, 88.5 74.9, 85.1, 79.5 72.6, 82.7, 73.2 80.3, 78.2, 86.1 73.4, 70.9, 79.4 74.2, 73.8, 84.8 74.4, 72.7, 80.5 76.6, 70.9, 77.7 71.6, 74.0, 71.5 75.9, 80.5, 71.1 74.4, 79.0, 76.9 70.1, 74.5, 79.3

6.8, 5.2, 3.5 10.2, 5.7, 8.1 5.4, 7.6, 3.8 7.9, 3.7, 10.6 4.6, 9.5, 6.1 6.6, 3.8, 5.4 5.0, 7.8, 9.7 6.1, 8.4, 10.0 4.8, 5.4, 4.3 11.0, 10.1, 4.5 4.8, 7.9, 12.4 12.5, 11.3, 3.0 9.9, 8.7, 7.9

9.3, 6.2, 8.4 8.5, 9.8, 4.1 3.1, 9.7, 4.3 3.8, 11.3, 14.8 11.2, 9.3, 9.8 6.5, 5.0, 7.6 14.9, 9.9, 11.1 4.5, 7.8, 4.8 12.0, 4.6, 6.0 14.1, 7.3, 6.1 7.5, 4.3, 8.5 11.5, 7.6, 8.1 10.4, 8.2, 8.6

into a 10 mL centrifuge tube, and 50 μL of 100 ng/mL salbutamol-d3 internal standard, 4 mL of ammonium acetate buffer (pH 5.2), and 500 μL of 10% perchloric acid were added. (To determine the total residues of salbutamol, another 70 μL of β-glucuronidase/arylsulfatase was added, and the mixture was vortexed and incubated at 37 °C for 16 h.) After centrifugation for 5 min at 7741g, the supernatants were loaded onto an SPE column (Oasis MXC 60 mg, Waters Corp.), which was activated with 3 mL of methanol and 3 mL of 0.1% formic acid, washed with 3 mL of 0.1% formic acid and 3 mL of methanol, and finally eluted with 3 mL of 5% ammonium methanol. The eluant was evaporated to dryness under nitrogen gas at 40 °C, reconstituted in 1 mL of a mixture of acetonitrile and 0.1% formic acid (1:9, v/v), filtered through a 0.22μm filter (Waters Corp.), and analyzed by UHPLC-MS/MS. Tissue samples were prepared based on the Chinese national standard (SN/T 1924−2011). Briefly, tissue samples (2.00 g) were placed in 50 mL centrifuge tubes with 5 mL of ammonium acetate buffer (pH 5.2) and 50 μL of 100 ng/mL salbutamol-d3 internal standard, and then the mixture was homogenized for 1 min using a T18 Ultra-Turrax digital homogenizer (IKA Works GmbH & Co. KG, Staufen, Germany). Subsequently, another 5 mL of ammonium acetate buffer was added. (For hydrolyzed samples, 70 μL of β-glucuronidase/arylsulfatase was added, and the mixture was vortexed and incubated at 37 °C for 16 h.) After centrifugation for 10 min at 7741g, the supernatants were collected, mixed with 10 mL of n-hexane, and centrifuged for 10 min at 14636g. The upper n-hexane layer was discarded, and the lower aqueous layer was filtered and then transferred to an SPE column. All subsequent procedures were the same as described above for the plasma and urine samples. UHPLC-MS/MS Analysis. The determination of salbutamol was performed using a Waters Acquity UHPLC system (Waters Corp.) equipped with an Acquity UHPLC BEH C18 column (2.1 × 50 mm; 1.7-μm particle size). The column oven was set to 40 °C. The mobile phase consisted of 5% acetonitrile and 0.1% formic acid (eluent A), and 95% acetonitrile and 0.1% formic acid (eluent B). The gradient conditions were as follows: 0 min, 99% A; 1 min, 99% A; 5 min, 10% A, 5.5 min, 10% A; 6 min, 99% A. The outlet of the UHPLC system was coupled to a Xevo TQ-S triple-quadrupole mass spectrometer (Waters Corp.) equipped with an electrospray interface source operated in the positive-ion mode. The target analyte was monitored in multiple reaction monitoring (MRM) mode. The operating conditions were as follows: capillary voltage, 3 kV; cone voltage, 24 V; ion source temperature, 150 °C; desolvation temperature, 350 °C; desolvation gas flow rate, 500 L/h. In MRM mode, one parent ion (m/z 240.0) and two product ions (m/z 148.0 and 120.8) were detected for salbutamol, and one parent ion (m/z 243.1) and two product ions (m/z 151.0 and 123.8) were detected for salbutamol-d3. Method Validation. In consideration of the matrix effect issue when MS/MS is used for the quantitation of salbutamol residues in various tissues, matrix-matched calibration curves were established using fortified blank samples with salbutamol standards at six levels (i.e., 0.1,

Blood and urine were collected from three cattle on treatment days 1, 7, 14, and 21 and on withdrawal days 0, 3, 7, 14, and 28 before morning feeding. Blood samples were collected from the jugular vein into heparinized tubes and centrifuged at 1482g for 10 min for plasma separation. Three cattle were slaughtered on withdrawal days 0 and 14. Liver, kidney, heart, spleen, lung, muscle, fat, small intestine, large intestine, rumen wall, and eye tissues were collected at each slaughter time point and stored at −80 °C before being assayed. All procedures in this study were performed in accordance with the guidelines for Animal Experiments and Standards of the Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China. Identification of Salbutamol and Its Metabolites. Sample Preparation. Sample pretreatments were developed as previously described,18 and to maximize the detection of salbutamol metabolites, samples were extracted with acetonitrile only and received no other treatment. Briefly, plasma or urine samples (1 mL) were mixed with 4 mL of acetonitrile and then centrifuged at 3024g for 5 min. The supernatants were collected, and the preparation steps were repeated one additional time. The supernatants were then evaporated to dryness under a stream of nitrogen gas at 40 °C, dissolved in 1 mL of distilled water, centrifuged at 17418g for 10 min, and filtered through 0.22-μm filters (Waters Corp.) before UHPLC-QTOF-MS/MS analysis. Liver or kidney samples (2.00 g) were mixed with 8 mL of acetonitrile, vortexed for 1 min, ultrasonically extracted for 15 min, and then centrifuged at 3024g for 10 min. The supernatants were collected, and the preparation steps were repeated one additional time. The supernatants were subsequently evaporated to dryness under a stream of nitrogen gas at 40 °C, dissolved in 2 mL of distilled water, and then centrifuged at 17418g for 10 min before UHPLC-QTOF-MS/MS analysis. UHPLC-QTOF-MS/MS Analysis. The separation was performed using a UHPLC system (Agilent 1290 series, Agilent Technologies, Santa Clara, CA) equipped with an Agilent Eclipse Plus C18 column of 2.1 × 100 mm and 1.8-μm particle size (Agilent Technologies). The column temperature was set to 30 °C. The mobile phase consisted of 0.2% acetic acid (A) and acetonitrile (B). The flow rate was 0.3 mL/min. The gradient conditions were as follows: 0 min, 97% A; 1 min, 97% A; 12 min, 2% A; 15 min, 2% A. The identification of salbutamol and its metabolites was carried out on an Agilent 6550 QTOF-MS/MS system in positive-ion mode, using the following operation parameters: capillary voltage, 3500 V; nebulizer pressure, 35 psi; drying gas flow rate, 7 L/ min; gas temperature, 275 °C. All data were acquired and processed using MassHunter Metabolite ID software (Agilent Technologies), which was set to automatically identify the possible metabolites by comparing the samples with the control and using the molecular mass of salbutamol and metabolism pathways (mass spectrum error of 0.99, curves data was not shown). The LOD and LOQ of salbutamol were found to be 0.1 and 0.2 ng/mL, respectively, for plasma; 0.1 and 0.3 ng/mL, respectively, for urine; and 0.3 and 0.5 ng/g, respectively, for internal tissues. Recovery and RSD data for salbutamol spiked in the matrixes are reported in Table 1. Mean recoveries ranged from 90.5% to 109.9% for plasma, from 88.5% to 103.8% for urine, and from 70.1% to 86.1% for internal tissues of cattle. The RSDs were below 15% for all samples. These

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RESULTS Validation of UHPLC-MS/MS. The matrix effects of β2agonists in biological fluids and animal tissues when the MS/MS method is used for quantification have been widely studied. Previous reports showed that the matrix suppression was observed for the determination of salbutamol in urine, muscle, kidney, and liver tissues.20,21 Therefore, in the present study, matrix-fortified standard curves using various blank matrixes D

DOI: 10.1021/acs.jafc.7b00189 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. Tentative structures of the identified salbutamol metabolites.

the N-oxide of salbutamol. M6 can be formed by the dehydration of M4, with a mass shift of 17.9742 Da (reduction of H2O). Two sequential desaturations of salbutamol yielded metabolite M7, with a mass shift of 4.0309 Da (reduction of H4). M8 was formed by oxide reduction and hydrogenation, with a mass shift of 13.9791 Da (addition of H2 and reduction of oxygen atom). The depropylation, dehydrogenation, and N-oxide of salbutamol generated metabolite M9, with a mass shift of 28.068 Da (reduction of C3H8 and addition of oxygen atom). M10 was formed by the debutylation and hydrogenation of salbutamol, with a mass shift of 54.0466 Da (reduction of C4H6). Residues of Salbutamol in Plasma, Urine, and Tissues. The proportions of parent and total salbutamol in plasma, urine, liver, and kidney tissues during the entire experimental period are shown in Figure 4. Parent salbutamol in cattle plasma, urine, liver, and kidney accounted for only 2%, 36.8%, 9.9%, and 35.5%, respectively, of the total salbutamol. Statistically significant differences between the concentrations of parent and total salbutamol were observed in plasma, urine, liver, and kidney tissues (P < 0.01). The mean parent and total concentrations of salbutamol in plasma and urine are shown in Figures 5 and 6, respectively. Parent salbutamol concentrations in the plasma were below the LOQ (0.2 ng/mL) except on treatment day 14, and the total salbutamol concentrations ranged from 12.20 to 15.72 ng/mL during the treatment period and were below the LOQ (0.2 ng/mL) on withdrawal day 7. No accumulation of salbutamol or its conjugates was found in plasma following 21 days of treatment. Compared with the concentrations of salbutamol residues in plasma, those in urine were much higher and changed rapidly. Residues of salbutamol in urine increased from 59.95 ng/mL (parent) and 865.41 ng/mL (total) on day 1

results indicate that the method employed is reliable for detecting the residues of salbutamol in various biological samples. The MRM chromatograms of salbutamol and salbutamol-d3 in urine samples are shown in Figure 1. Identification of Salbutamol and Its Metabolites. After the last dose, salbutamol and its metabolites were identified using QTOF-MS/MS measurements, and a total of 10 metabolites were detected in the cattle’s plasma, urine, liver, and kidney tissues (as summarized in Table 2). The extracted ion chromatograms with the corresponding MS spectra are shown in Figure 2. None of these compounds were found in blank samples. The parent drug of salbutamol was identified at m/z 240.1595, with three typical fragment ions at m/z 222.1483, 166.0859, and 148.0754, which is consistent with the previously reported results.14,15 Four metabolites (M1, M4, M5, and M6) were observed at m/z 416.1908, 256.1183, 254.1388, and 238.1441, respectively, in accordance with previous research.14,15 Six metabolites (M2, M3, M7, M8, M9, and M10) were newly identified at m/z 286.1654, 258.1704, 236.1286, 226.1804, 212.0915, and 186.1129, respectively. The tentative structures of these metabolites are shown in Figure 3. M1 was identified as conjugated glucuronic acid. This biotransformation has an associated mass shift of 176.0313 Da (addition of C6H8O6). The addition of a methoxyl group to salbutamol and the N-oxide yielded M2, corresponding to a mass shift of 46.0059 Da (addition of CH2O2). M3 was formed by the oxidation and hydrogenation of salbutamol, with a mass shift of 18.0109 Da (addition of H2O). The addition of a hydroxyl group led to metabolite M4, associated with a mass shift of 15.9588 Da (addition of oxygen atom). M5 was 13.9793 Da higher than the protonated parent drug and was formed by dehydrogenation and E


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Figure 4. Proportions of the parent salbutamol and its conjugates in different tissues during the entire experimental period.

Figure 6. Parent and total salbutamol concentrations in cattle urine after the cattle had been dosed with 0.15 mg of salbutamol (kg of body weight)−1 day−1 for 21 consecutive days. T X and W X denote treatment day X and withdrawal day X, respectively. Parent represents nonconjugated salbutamol; total represents conjugated + nonconjugated salbutamol.

Figure 5. Parent and total salbutamol concentrations in cattle plasma after the cattle had been dosed with 0.15 mg of salbutamol (kg of body weight)−1 day−1 for 21 consecutive days. T X and W X denote treatment day X and withdrawal day X, respectively. Parent represents nonconjugated salbutamol; total represents conjugated + nonconjugated salbutamol.

of treatment to 1654.40 ng/mL (parent) and 2657.26 ng/mL (total) on day 14 of treatment and then decreased to 0.45 ng/mL (parent) and 0.58 ng/mL (total) on withdrawal day 28. The residues of parent salbutamol in internal tissues are shown in Figure 7. The liver and kidney samples were analyzed before and after treatment with β-glucuronidase/arylsulfatase, and the concentrations of total salbutamol were 547.52 and 82.11 ng/g, respectively, on withdrawal day 0 and 27.59 and 8.60 ng/g, respectively, on withdrawal day 14. On withdrawal day 0, the highest concentrations of parent salbutamol were found in liver (53.69 ng/g), followed by kidney (32.20 ng/g), fat (31.16 ng/g), eye (13.92 ng/g), small intestine (7.48 ng/g), spleen (7.14 ng/ g), rumen wall (6.75 ng/g), large intestine (6.14 ng/g), lung (5.27 ng/g), heart (4.99 ng/g), and muscle (4.57 ng/g). On withdrawal day 14, the concentrations were the highest in the eye tissues (77.52 ng/g), followed by fat (5.17 ng/g), large intestine (4.47 ng/g), rumen wall (4.01 ng/g), small intestine (3.80 ng/g), liver (3.27 ng/g), and lung (2.60 ng/g), but the concentrations in the kidney, heart, spleen, and muscle tissues were below the LOQ (0.5 ng/g).

Figure 7. Parent salbutamol concentrations in various tissues of cattle after treatment with 0.15 mg (kg of body weight)−1 day−1 salbutamol for 21 days. W 0 d and W 14 d denote samples collected on on withdrawal day 0 and withdrawal day 14, respectively.

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DISCUSSION Salbutamol Metabolites. The primary metabolic pathways of salbutamol in the human body involve its combination with sulfate. The main forms of salbutamol in human urine after oral administration are the parent drug and its sulfate conjugates. However, compared with the parent drug, the proportion of conjugated glucuronic acid (M1) is less than 3%.16 Recent studies detected a total of seven metabolites of salbutamol in urine from rats, swine, and calves, including conjugated glucuronic acid (M1), hydroxylated salbutamol (M4), hydroxylation and the N-oxide of salbutamol (M5), dehydrated M4 (M6), and conjugated sulfate.14,15,22 In this study, a total of 10 metabolites were identified in plasma, urine, liver, and kidney tissues from cattle after the final administration of salbutamol, F


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a previous study reporting that, in heifer plasma samples, the highest concentration of parent salbutamol was 0.3 ng/mL 12 h after a single dose.19 No accumulation of salbutamol or its conjugates was observed in plasma, which is consistent with research reported by Pou et al. for calves that had been given a dose of 10 mg of salbutamol per day for 10 days.25 This might be because salbutamol is rapidly cleared from the animal’s plasma before the next dose. Hung et al. reported that salbutamol was undetectable in swine serum at a withdrawal time of 18 h after the animals had been given feed containing 3 mg of salbutamol/kg for 14 days, which also indicated rapid the clearance of salbutamol from the animals’ plasma.12 According to the available literature, the levels of salbutamol were undetectable on withdrawal days 2, 3, and 4 in heifer, chicken, and calf plasma samples, respectively.13,19,25 Taken together, the results from these studies indicate that plasma salbutamol concentrations are very low during the treatment period and quickly become undetectable after administration. Hence, when plasma is used as the target for monitoring the illegal use of salbutamol in cattle production, total salbutamol might be the appropriate marker residue. Urine is the major route of salbutamol excretion from animals. In this study, parent salbutamol concentrations were highest on treatment day 21 (1654.4 ng/mL) and were still detectable on withdrawal day 28 (0.45 ng/mL), and the total salbutamol concentrations were highest on treatment day 14 (2657.26 ng/ mL) and were still detectable on withdrawal day 28 (0.58 ng/ mL). According to Hung et al.’s report, salbutamol residues in swine urine changed from 145.12 ng/mL on withdrawal day 0 to 1.16 ng/mL on withdrawal day 30 after the animals had been given feed containing 3 mg of salbutamol/kg for 14 consecutive days.12 Zhang et al. found that the parent salbutamol concentrations were highest on withdrawal day 0 (50.2 ng/ mL) and were below the LOQ (0.2 ng/mL) on withdrawal day 5 and that the total salbutamol concentrations were highest (3582.1 ng/mL) on withdrawal day 1 and were still detectable on withdrawal day 7 (0.7 ng/mL) after a single dose.19 Together, these findings suggest that salbutamol residues in urine samples are relatively high during the dosing period and are eliminated slowly after the withdrawal time. Therefore, urine samples are more appropriate targets for monitoring the abuse of salbutamol, and total salbutamol has an advantage over parent salbutamol as a marker residue. Reports of β2-agonist intoxication in Spain and China suggested that the consumption of contaminated animal products was a main cause of poisoning.26,27 Consequently, the residues of salbutamol in internal tissues are of greater concern regarding human health than the residues in urine and plasma samples. In this study, the highest concentrations were found in liver (53.69 ng/g), followed by kidney tissues (32.2 ng/g) on withdrawal day 0. Previous studies also found similar results, as high salbutamol concentrations were also observed in liver and kidney tissues of swine (70.42 and 31.88 ng/g, respectively), calf (3920 and 130 ng/g, respectively), chicken (333.8 and 109.5 ng/ g, respectively), and guinea pigs (34.89 and 18.61 ng/g, respectively) on withdrawal day 0.12,13,22,28 This is not surprising because liver and kidney tissues are the main organs involved in the metabolism and excretion of salbutamol. Although salbutamol is widely used for the treatment of asthma, the concentrations of salbutamol observed in lung tisssues were relatively low (5.27 ng/g), likely because salbutamol is a shortacting β2-agonist. However, in the present study, the highest concentrations of salbutamol were detected in eye tissues (77.52

with six of the metabolites (M2−M3 and M7−M10) being newly identified. M1, M4, and M5 were identified in previous studies on urine samples from rats and swine, whereas M6 was only observed in urine samples from swine after administration of salbutamol.14,15 No conjugations of sulfate were detected in this study, which might be due to the complicated digestive characteristics of rumen microorganisms. The present study indicated that the primary metabolic pathways of salbutamol in beef cattle were oxidative metabolism, conjugation of glucuronic acid, and reduction of hydrocarbon. The metabolism of salbutamol in beef cattle is significantly different from that in humans, rats, and swine. Although the metabolites of salbutamol were identified in this study and a better understanding of the metabolic pathway of salbutamol in beef cattle was achieved, future studies are needed to determine the exact structures of the salbutamol metabolites. Residues of Salbutamol in Plasma, Urine, and Tissues. Proportions of Salbutamol and Its Conjugates. Previous studies have shown that salbutamol is primarily excreted in urine as a mixture of the parent drug and its conjugates, mainly sulfate conjugations.1,16,23 In the present study, plasma, urine, liver, and kidney tissues were hydrolyzed with β-glucuronidase/arylsulfatase to determine the proportions of parent salbutamol and its conjugates. Zhang et al. reported that the salbutamol concentrations in plasma and urine samples of heifers were significantly different (P < 0.001) before and after hydrolysis with β-glucuronidase/arylsulfatase and that the main forms of salbutamol in plasma and urine were its conjugates.19 Here, statistically significant differences (P < 0.01) were also observed between the concentrations of salbutamol with hydrolyzed and nonhydrolyzed plasma, urine, liver, and kidney samples. Based on the metabolites identified in this study, the primary conjugate metabolites of salbutamol in beef cattle were conjugated glucuronic acid (M1). No sulfate conjugation of salbutamol was identified. Therefore, the main form of salbutamol in beef cattle was metabolite M1, and the proportions were as high as 98%, 63.2%, 90.1%, and 64.5% in plasma, urine, liver, and kidney tissues, respectively. Metabolic studies in humans and animals indicated that approximately 83% of the drug in human plasma is in the form of its sulfate conjugates; 70%−90% of the administration dose was parent drug in dog urine; and 90% and 40% was its conjugates (mainly M1) in rabbit and rat urine, respectively.23,24 Montrade et al. reported that the major forms of salbutamol in calf urine were parent drug, as well as sulfate and glucuronide conjugates, with the latter accounting for only 9.7% (treatment period) and 4.9% (withdrawal time), respectively.22 Taken together, the main forms of salbutamol and their proportions differ among species, and many factors can influence this difference, such as the dosage and duration of administration, as well as the weight and age of the animals. The present study indicated that salbutamol is primarily in the form of conjugated glucuronic acid (M1) in beef cattle after treatment with an anabolic dose [0.15 mg (kg of body weight)−1 day−1] of salbutamol for 21 consecutive days. Residues of Salbutamol in Beef Cattle. Regulations commonly regard plasma, urine, and internal tissue samples as targets for the preslaughter and postslaughter monitoring of the illegal use of β2-agonists in food animals. Despite the widespread human use of salbutamol for the treatment of asthma, there is a lack of published pharmacological data on salbutamol, primarily because of the low plasma concentrations of salbutamol. In the present study, the highest concentration of parent salbutamol was 0.21 ng/mL on treatment day 14, which was consistent with G


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carcass measures, and health of finishing pigs. J. Anim. Sci. 2012, 90, 4081−4089. (3) Mersmann, H. J. Overview of the effects of beta-adrenergic receptor agonists on animal growth including mechanisms of action. J. Anim. Sci. 1998, 76, 160−172. (4) Brambilla, G.; Cenci, T.; Franconi, F.; Galarini, R.; Macrì, A.; Rondoni, F.; Strozzi, M.; Loizzo, A. Clinical and pharmacological profile in a clenbuterol epidemic poisoning of contaminated beef meat in Italy. Toxicol. Lett. 2000, 114, 47−53. (5) Pulce, C.; Lamaison, D.; Keck, G.; Bostvironnois, C.; Nicolas, J.; Descotes, J. Collective human food poisonings by clenbuterol residues in veal liver. Vet. Hum. Toxicol. 1991, 33, 480−481. (6) Martinez-Navarro, J. F. Food poisoning related to consumption of illicit β-agonist in liver. Lancet 1990, 336, 1311. (7) Regulation No. 176. Ministry of Agriculture, P. R. China. http:// www.moa.gov.cn/zwllm/tzgg/gg/201104/t20110422_1976307.htm (accessed April 22, 2011). (8) Directive 2003/74/EC of the European Parliament and of the Council of 22 September 2003 amending Council Directive 96/22/EC concerning the prohibition on the use in stockfarming of certain substances having a hormonal or thyrostatic action and of beta-agonists. Off. J. Eur. Communities: Legis. 2003, L262, 17. (9) Tang, Y.; Lan, J.; Gao, X.; Liu, X.; Zhang, D.; Wei, L.; Gao, Z.; Li, J. Determination of clenbuterol in pork and potable water samples by molecularly imprinted polymer through the use of covalent imprinting method. Food Chem. 2016, 190, 952−959. (10) Wang, P.; Liu, X.; Su, X.; Zhu, R. Sensitive detection of β-agonists in pork tissue with novel molecularly imprinted polymer extraction followed liquid chromatography coupled tandem mass spectrometry detection. Food Chem. 2015, 184, 72−79. (11) Sheu, S. Y.; Lei, Y. C.; Tai, Y. T.; Chang, T. H.; Kuo, T. F. Screening of salbutamol residues in swine meat and animal feed by an enzyme immunoassay in Taiwan. Anal. Chim. Acta 2009, 654, 148−153. (12) Hung, M. J.; Huang, H. H.; Chen, C. L.; Wu, Y. J.; Dixon, K. M.; Mao, C. L. Salbutamol residues in swine tissues and body fluids after feeding. Thai J. Vet. Med. 2010, 40, 399−404. (13) Malucelli, A.; Ellendorff, F.; Meyer, H. H. D. Tissue distribution and residues of clenbuterol, salbutamol, and terbutaline in tissues of treated broiler chickens. J. Anim. Sci. 1994, 72, 1555−1560. (14) Gu, X.; Liu, Y. M.; Yao, T.; Shi, H. L.; Li, J.; Zhao, Z.; Qin, Y. C. Identification of major metabolites of salbutamol in swine urine and plasma using ultra-high performance liquid chromatography-electrospray time of flight mass spectrometry. Chin. J. Anal. Chem. 2014, 42, 1692−1696. (15) Domínguez-Romero, J. C.; García-Reyes, J. F.; Martínez-Romero, R.; Martínez-Lara, E.; Del Moral-Leal, M. L.; Molina-Díaz, A. Detection of main urinary metabolites of β2-agonists clenbuterol, salbutamol and terbutaline by liquid chromatography high resolution mass spectrometry. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2013, 923−924, 128−135. (16) Mareck, U.; Guddat, S.; Schwenke, A.; Beuck, S.; Geyer, H.; Flenker, U.; Elers, J.; Backer, V.; Thevis, M.; Schän zer, W. Determination of salbutamol and salbutamol glucuronide in human urine by means of liquid chromatography-tandem mass spectrometry. Drug Test. Anal. 2011, 3, 820−827. (17) Joyce, K. B.; Jones, A. E.; Scott, R. J.; Biddlecombe, R. A.; Pleasance, S. Determination of the enantiomers of salbutamol and its 4O-sulphate metabolites in biological matrices by chiral liquid chromatography tandem mass spectrometry. Rapid Commun. Mass Spectrom. 1998, 12, 1899−1910. (18) Tang, C.; Liang, X.; Zhang, K.; Zhao, Q.; Meng, Q.; Zhang, J. Residues of ractopamine and identification of its glucuronide metabolites in plasma, urine, and tissues of cattle. J. Anal. Toxicol. 2016, 40, 738−743. (19) Zhang, K.; Liang, X.; Su, C.; Tang, C.; Zhao, Q.; Zhang, J.; Meng, Q. Salbutamol residues in plasma, urine and hair of heifers after a single dose and throughout. J. Anal. Toxicol. 2016, 40, 454−459. (20) Wang, L. Q.; Zeng, Z. L.; Su, Y. J.; Zhang, G. K.; Zhong, X. L.; Liang, Z. P.; He, L. M. Matrix effects in analysis of β-agonists with LC-

ng/g) on withdrawal day 14, which is consistent with the results of a study by Malucelli et al.13 on broiler chickens fed a diet containing 10 mg of salbutamol/kg for 14 days. It is worth noting that the concentrations of salbutamol in eye tissues increased from 13.92 ng/g on withdrawal day 0 to 77.52 ng/g on withdrawal day 14, which might be because salbutamol was absorbed by melanin in the eye tissues and continued to accumulate after administration. In vitro studies showed that salbutamol could bind to melanin, but compared with clenbuterol, salbutamol has a weak affinity to melanin.29,30 Research by Vulić et al. showed that the concentrations of salbutamol and clenbuterol in black hair were higher than those in white hair, which could be due to lower levels of in melanin in white hair than in black hair.31 Zhang et al. reported significantly higher salbutamol residues in red hair than in white hair, probably due to the affinity of salbutamol to melanin.32 Residues of salbutamol were rapidly depleted from kidney, heart, spleen, and muscle tissues and were below the LOQ (0.5 ng/g) on withdrawal day 14. In comparison, rapid elimination of salbutamol was also reported in kidney, spleen, heart, and muscle tissues of swine, and salbutamol was undetectable on withdrawal day 3.12 Altogether, these findings suggest that the residues and elimination of salbutamol differ according to the tissues and that eye tissues might be more suitable as postslaughter targets for monitoring the illegal use of salbutamol in beef cattle. In summary, we identified the metabolites of salbutamol and investigated the parent and total salbutamol residues in plasma, urine, and internal tissues of cattle fed salbutamol under anabolic dosage. Our findings indicate that total salbutamol is preferable to parent salbutamol as a marker residue for detecting the levels of salbutamol and that urine and eye tissues are more suitable as targets for preslaughter and postslaughter monitoring of the illegal use of salbutamol in beef cattle.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 86 10 6281 5852. Fax: 86 10 6281 5537. E-mail: [email protected]. ORCID

Junmin Zhang: 0000-0002-8405-0536 Author Contributions ‡

K.Z. and C.T. contributed equally to this study.

Funding

This work was supported by the Special Fund for Agro-Scientific Research in the Public Interest (Grant 201203088). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED UHPLC-QTOF-MS/MS, ultrahigh-performance liquid chromatography quadrupole time-of-flight-tandem mass spectrometry; SPE, solid-phase extraction; LOD, limit of detection; LOQ, limit of quantification; RSD, relative standard deviation; MRM, multiple reaction monitoring

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REFERENCES

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Residues of Salbutamol and Identification of Its Metabolites in Beef

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