Abstract
We aimed to evaluate whether living tissues such as urine, plasma and hair were suitable for monitoring clenbuterol (CL) abuse after its subchronic administration of a growth-promoting dose to the Chinese Simmental beef cattle. Eight male, white and red pied Chinese Simmental beef cattle were involved in the experiment, and the CL dose was 16 µg/kg BW/day. Liquid chromatography tandem mass spectrometry (LC–MS-MS) was used to determine CL residues in different tissues, and the addition of D9-clenbuterol internal standard was applied to increase determination accuracy. The recovery of plasma, urine, hair and in vivo tissues was 88.5–114.2, 83.9–114.3, 88.6–116.9 and 85.3–121.7%, respectively. The results showed that CL residue concentrations in the plasma, on Days 14 after withdrawal and later, were lower than the limit of detection (LOD) (0.06 ng/mL) and CL residue in urine was lower than LOD (0.16 ng/mL) 42 days after treatment. CL significantly accumulated in the white and red hair and maintained more than 7.19 ± 2.19 pg/mg within the early withdrawal period of 70 days. A large number of CL were determined in all tested biological tissues, in which residues were higher than the maximum residue limits (MRLs) after dietary administration of CL for 21 days and pre-slaughter withdrawal period of ∼6 h. A particular concern is the slow depletion of residues of CL in some tissues like gluteus and liver still exceeding theirs MRLs, respectively, on Days 14 or 28 days after withdrawal. Our study indicated that plasma and urine could be available for monitoring CL abuse only within a short period of time. However, hair (including light-pigmented) as a target matrix can be selected to perform the long-period monitor of CL.
Introduction
Clenbuterol (CL), a phenethanolamine β2-adrenergic agonist, has been used illegally to generate beef cattle of higher muscle mass and simultaneous lower fat accumulation (1, 2). The consumption of contaminated tissues has resulted in a worldwide food poisoning issue. Reports of CL intoxication in Spain (3, 4), France (5) and China (6) have shown the ingestion of liver-containing CL residues was a main cause of poisoning (7). Many reports have shown that CL accumulates in the liver to a greater degree than other edible tissues, and levels are maintained for a longer period of time. The living organisms' urine, plasma and hair are all possible convenient and effective matrices for monitoring CL abuse. However, when CL in urine is negatively detected, the concentrations which remain in other tissues are still possibly higher than the maximum residue limits (MRLs) in the later withdrawal period (8–10). Plasma is probably available for a shorter time. Recent studies have been focused on hair for its potential ability to monitor drug abuse (11–13) and for analytical methods (11, 13–16). However, traceable CL concentrations in hair appear to be correlated with hair color (17–19). In a previous study, CL residues accumulated in red hair in a dose-dependent manner (20). Further research is required to confirm the potential of CL increase in white hair, and it is unknown whether CL will accumulate in light-pigmented hair. Therefore, it is questionable as to whether or not hair is an effective matrix for monitoring CL abuse in food production animals covered with only light-colored hair.
The MRLs of CL were strictly confined by CODEX and the FDA (21) are 0.6 pg/mg in liver and kidney, while in muscle and fat, 0.2 pg/mg was observed. It is unclear whether CL in all edible tissues was lower than MRLs when these three tissues test negative for CL. Therefore, this study evaluated whether urine, plasma and hair are suitable for monitoring CL abuse in male Chinese Simmental beef cattle. The potential of CL accumulation in white hair was also clarified.
Material and methods
Chemicals and apparatus
The chemicals and apparatus were the same as the previous study (20). The CL purchased from Linen Technology Co., Ltd was of 96.5% in purity determined by Liquid chromatography–mass spectrometry (LC–MS). Except methanol, formic acid and acetonitrile were of chromatographic grade; other chemicals used throughout this study were of analytical grade.
Animal treatment and sampling procedure
Experimental animals and treatment
Eight male red and white pied Chinese Simmental beef cattle were housed and tie-down fed in separate pens, weighing a body mass of 220 ± 20 kg. All cattle were administered CL orally at a growth-promoting dose of 16 μg/kg BW/day. Then, 1.04 g CL was mixed in 250 g crush bran and corn powder and stepwise-diluted with commercially concentrated cattle feed. The initial weight of each steer was used to calculate the CL doses to be administered. About 200 g of mediated meal fed to each cattle before the total mixed ration (TMR) for 21 days in the medication period. Animal care and procedures were approved and conducted under established standards of the Institute of Animal Science, Chinese Academy of Agricultural Sciences.
Sampling procedure
Blank urine, plasma, and both white and red hair samples were collected from three steers on Day 0 (before treatment). On Days 1, 7 and 14 during treatment and Days 0, 3, 7, 14, 21, 28 and 42 after exposure, ∼9 mL of blood was collected from the jugular vein using disposable blood collecting needle and vacuum blood tube with lithium heparin, and centrifuged at 3,000 rpm for 5 min for plasma preparation. Urine was collected by an apparatus similar to that designed by Veenhuizen et al. (22). All urine and plasma samples were stored at −20°C. Hair samples were collected every 14 days after administration, and 2–3 g of white and red hair were collected from an unshaved site of each hide, sealed in zip lock bags and kept at 4°C for subsequent analysis. On Days 0, 3, 7, 14 and 28 after CL administration, one CL-treated beef cattle were sacrificed, and eye, muscles around eye, liver, lung, kidney, spleen, rib eye muscle and gluteus samples were collected, rinsed of blood and stored at −20°C.
Preparation and extraction procedure of samples
Preparation of hair samples
Hair samples were washed and soaked in 0.1 mol/L sodium dodecyl sulfate aqueous (SDS) for 30 min before ultrasound irradiation. The hair was dried at 40°C overnight after a thorough washing with distilled water. The dried hair samples were homogenized as suggested by Gleixner et al. (23) using a ball mill (MM400; Retsch Corp., USA), and the resulting hair powder was dried at room temperature overnight and stored at 4°C in closed plastic containers until subsequent analysis.
Extraction procedure
The fortified blank samples were used for determination of validation parameters. The samples of the urine, plasma and hair extraction pretreatment method, which was based on previous studies, were improved and simplified. The details were as follows: ∼1 mL of each urine sample (samples with high concentrations of CL residues were diluted to 2–10 ng/mL with ultrapure water) was transferred to a 10-mL pipette, and 0.5 mL of 30% perchloric acid and 50 μL 100 ng/mL of D9-CL internal standard (IS) were added and thoroughly mixed with the samples. The procedure for the extraction of plasma was similar to that of urine, but 30 μL glucose aldehyde glycosides enzyme were also added to samples and set in a 60°C shaking bath for 4 h. The D9-CL IS and glucose aldehyde glycosides enzyme was diluted with ammonium acetate buffer (pH 5.2) to avoid any destructive enzyme activity. As for hair, the procedure was referenced to Zhao et al. (24) and given in detail by the previous study (20). The supernatant was collected after centrifugation at 8,000 rpm for 10 min at 4°C (25). In vivo tissues were determined by Supervision & Testing Center of Milk and Dairy Product Quality, Ministry of Agriculture (Beijing) as Chinese national standard (SN/T 1924–2011 (26)). Lens, aqueous humor and the remaining segments of eye were separated for analysis.
The supernatant transferred to an solid phase extraction column activated with 3 mL methanol and 3 mL 0.1% formic acid. About 3 mL 0.1% formic acid and 3 mL methanol were used to wash the column. Furthermore, 3 mL 5% ammonium methanol was employed to elute the product. The nitrogen flush was performed to dry the eluate at 60°C, and the dried pellet was reconstituted in a solution of 0.1% formic acid and acetonitrile (9:1 by volume). CL standard samples were mixed with 5 ng/mL of D9-CL IS and diluted with a mixture of acetonitrile and 0.1% formic acid (1:9, v/v).
Analysis of CL
LC–MS-MS was applied for CL determination in this study. Acquity UPLC BEH C18, 50 mm × 2.1 mm, 1.7 μm particles (Waters, USA) and a Xevo TQ-S triple quadrupole mass spectrometer (Waters, USA) were all used. The UPLC parameters and the mass spectrometry parameters were referenced to SN/T 1924–2011 (26) and our previous study (20). Positive electrospray ionization (ESI+) was used, and the multiple reactions monitoring (MRM) mode was employed for the MS. Two product diagnostic ions (m/z 203.0 and m/z 168.1.) and one precursor product diagnostic ion (m/z 277.0 and m/z 204.0) were employed for CL and D9-CL IS, respectively.
Statistics
Statistics were performed in the SAS and the GLM procedure with significant level at P = 0.05. The residual data were normalized by square root (residues), and the Excel was employed to generate the box plot.
Results
Validation of LC–MS-MS
Figure 1 shows the total ion chromatograms (m/z) of CL. The accuracy of method is presented in Table I. The limit of detection (signal/noise = 3, LOD) and the limit of quantitation (signal/noise = 10, LOQ), which were 0.06 and 0.10 ng/mL for plasma, 0.16 and 0.20 ng/mL for urine, 0.32 and 0.36 pg/mg for hair, and 0.10–0.18 and 0.14–0.22 pg/mg for other tissues. The mean recoveries vary from 88.5 to 114.2% for urine, 83.0 to 114.3% for plasma and 88.6 to 116.9% for hair by validation of LC–MS-MS. The inter-day and intra-day variation of urine were from 3.8 to 10.0% and 4.4 to 11.0%, respectively. These values were 5.5–11.2% and 3.8–10.9% for plasma, and 1.0–8.3% and 3.6–8.0% for hair. The data obtained from this study proved efficient methods for preparation and subsequent determination.
Evaluation of Validation Procedure for Urine and Plasma Samples Fortified With CL
| Matrix | Urine | Plasma | Haira | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CLfortified (ng/mL) | 1.0 | 2.5 | 5.0 | 10.0 | 0.5 | 1.0 | 2.5 | 5.0 | 10.0 | 1.0 | 2.5 | 10.0 | 20.0 | 50.0 |
| Lower/% | 111.6 | 99.3 | 94.6 | 88.5 | 97.0 | 83.0 | 91.1 | 88.4 | 83.9 | 96.7 | 90.4 | 88.8 | 88.6 | 99.2 |
| Upper/% | 114.2 | 103.0 | 99.3 | 103.3 | 104.5 | 103.8 | 110.8 | 114.3 | 113.2 | 103.8 | 116.9 | 115.2 | 115.1 | 109.3 |
| RSD% | ||||||||||||||
| Inter-day | 9.5 | 3.8 | 7.2 | 10.0 | 9.1 | 11.2 | 5.5 | 10.4 | 8.0 | 8.3 | 5.1 | 2.8 | 3.5 | 1.0 |
| Intra-day | 6.8 | 4.4 | 7.7 | 11.0 | 10.1 | 6.7 | 3.8 | 10.9 | 8.0 | 7.7 | 8.0 | 7.2 | 6.0 | 3.6 |
| Matrix | Urine | Plasma | Haira | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CLfortified (ng/mL) | 1.0 | 2.5 | 5.0 | 10.0 | 0.5 | 1.0 | 2.5 | 5.0 | 10.0 | 1.0 | 2.5 | 10.0 | 20.0 | 50.0 |
| Lower/% | 111.6 | 99.3 | 94.6 | 88.5 | 97.0 | 83.0 | 91.1 | 88.4 | 83.9 | 96.7 | 90.4 | 88.8 | 88.6 | 99.2 |
| Upper/% | 114.2 | 103.0 | 99.3 | 103.3 | 104.5 | 103.8 | 110.8 | 114.3 | 113.2 | 103.8 | 116.9 | 115.2 | 115.1 | 109.3 |
| RSD% | ||||||||||||||
| Inter-day | 9.5 | 3.8 | 7.2 | 10.0 | 9.1 | 11.2 | 5.5 | 10.4 | 8.0 | 8.3 | 5.1 | 2.8 | 3.5 | 1.0 |
| Intra-day | 6.8 | 4.4 | 7.7 | 11.0 | 10.1 | 6.7 | 3.8 | 10.9 | 8.0 | 7.7 | 8.0 | 7.2 | 6.0 | 3.6 |
The results of LC–MS-MS expressed as recovery of urine, plasma and hair samples (n = 8).
aHair data were the same to the previous data (20).
Evaluation of Validation Procedure for Urine and Plasma Samples Fortified With CL
| Matrix | Urine | Plasma | Haira | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CLfortified (ng/mL) | 1.0 | 2.5 | 5.0 | 10.0 | 0.5 | 1.0 | 2.5 | 5.0 | 10.0 | 1.0 | 2.5 | 10.0 | 20.0 | 50.0 |
| Lower/% | 111.6 | 99.3 | 94.6 | 88.5 | 97.0 | 83.0 | 91.1 | 88.4 | 83.9 | 96.7 | 90.4 | 88.8 | 88.6 | 99.2 |
| Upper/% | 114.2 | 103.0 | 99.3 | 103.3 | 104.5 | 103.8 | 110.8 | 114.3 | 113.2 | 103.8 | 116.9 | 115.2 | 115.1 | 109.3 |
| RSD% | ||||||||||||||
| Inter-day | 9.5 | 3.8 | 7.2 | 10.0 | 9.1 | 11.2 | 5.5 | 10.4 | 8.0 | 8.3 | 5.1 | 2.8 | 3.5 | 1.0 |
| Intra-day | 6.8 | 4.4 | 7.7 | 11.0 | 10.1 | 6.7 | 3.8 | 10.9 | 8.0 | 7.7 | 8.0 | 7.2 | 6.0 | 3.6 |
| Matrix | Urine | Plasma | Haira | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CLfortified (ng/mL) | 1.0 | 2.5 | 5.0 | 10.0 | 0.5 | 1.0 | 2.5 | 5.0 | 10.0 | 1.0 | 2.5 | 10.0 | 20.0 | 50.0 |
| Lower/% | 111.6 | 99.3 | 94.6 | 88.5 | 97.0 | 83.0 | 91.1 | 88.4 | 83.9 | 96.7 | 90.4 | 88.8 | 88.6 | 99.2 |
| Upper/% | 114.2 | 103.0 | 99.3 | 103.3 | 104.5 | 103.8 | 110.8 | 114.3 | 113.2 | 103.8 | 116.9 | 115.2 | 115.1 | 109.3 |
| RSD% | ||||||||||||||
| Inter-day | 9.5 | 3.8 | 7.2 | 10.0 | 9.1 | 11.2 | 5.5 | 10.4 | 8.0 | 8.3 | 5.1 | 2.8 | 3.5 | 1.0 |
| Intra-day | 6.8 | 4.4 | 7.7 | 11.0 | 10.1 | 6.7 | 3.8 | 10.9 | 8.0 | 7.7 | 8.0 | 7.2 | 6.0 | 3.6 |
The results of LC–MS-MS expressed as recovery of urine, plasma and hair samples (n = 8).
aHair data were the same to the previous data (20).
Total ion chromatograms of an authentic sample extract 6–8 h after withdrawal (a) plasma; (b) urine; (c) hair; (d) muscle. The retention time was ∼1.87 min for CL.
Urine and plasma CL concentrations during and after withdrawal
The mean CL concentrations in urine and plasma on Days 1, 7 and 14 during treatment and on Days 0, 3, 7, 14, 21, 28 and 42 after discontinuation of CL (n = 3) are shown in Figure 2. On medication, CL concentrations in the urine and plasma continued to increase, and the concentrations in urine were much higher than that of plasma. The mean CL concentrations (6–8 h after the last treatment) in cattle urine treated with a promoting dose of CL on withdrawal Day 0 were 352.37 ± 37.40 ng/mL. The value decreased to 7.98 ± 2.56 ng/mL on the third day post-treatment, which was less than one-eighth of the level on withdrawal Day 0. The residual concentrations in the urine were 4.34 ± 0.61, 1.19 ± 0.35, 0.57 ± 0.15 and 0.41 ± 0.06 ng/mL on withdrawal Days 7, 14, 21 and 28, respectively. The value on Day 42 in the urine was lower than LOQ. Treatment with CL resulted in a mean CL concentration of 2.70 ± 0.54 ng/mL in cattle plasma on the day of withdrawal (6–8 h after the last treatment), which was dropped to 0.33 ± 0.06 ng/mL at Day 3 post-treatment withdrawal. As the withdrawal time continued, the CL concentration of plasma decreased relatively slowly. The CL residue concentrations in plasma on Days 14 and later were lower than the LOQ.
Urine and plasma CL concentration during and after treatment. M1, M7, M14 and M21, days on medication; W0, W3, W7, W14, W21, W28 and W42, days after withdrawal.
Hair CL concentrations after withdrawal
The mean CL concentrations in hair samples during and after treatment (n = 3) are shown in Figure 3. There was an increase in concentration of CL in hair during the medication period and did not decrease immediately after discontinuation of medication. The mean CL concentrations were 8.92 ± 0.42 pg/mg and 13.52 ± 8.69 pg/mg in white and red hair, respectively, on the day of withdrawal 0 (6–8 h after the last treatment), and CL concentration were increased within the first 14 days after withdrawal. Prior to a slow decrease on Day 28, 42 and 70 post-withdrawal, the residual concentrations of white hair peaked at 21.40 ± 5.14 pg/mg on withdrawal Day 14. However, there was no difference, in terms of residual concentration, observed in the red hair on Day 14, 28 and 42 and peaked at 27.55 ± 5.54 pg/mg in the red hair on Day 70 after withdrawal.
Hair CL concentrations during and after treatment. M7, M14 and M21, days on medication; W14, W28, W42 and W70, days after withdrawal.
CL residues of in vivo biological tissues after withdrawal
The CL concentrations in biological tissues on Days 0, 3, 7, 14 and 28 after discontinuation are shown in Figure 4. On withdrawal period of zero, the order of tissues with CL residues from the highest to the lowest are liver, eye, bile, kidney, lung, aqueous liquid, spleen, muscles around eye, heart, rib eye muscle and gluteus. A slow depletion of residues of CL was found in later withdrawal days, and the CL residues in some edible tissues were more than the MRLs published by the codex alimentarius commission. CL residue in gluteus on Day 14 after withdrawal exceeded 0.2 pg/mg, and CL residues in liver and spleen on Day 28 after withdrawal were more than 0.6 pg/mg. Eye and aqueous liquid had accumulated great concentrations of more than 2.0 pg/mg on Day 28 after withdrawal.
CL concentrations of in vivo biological tissues after treatment.
Discussion
CL residues in the urine and plasma tissues
Compared with plasma samples, CL concentrations measured in urine remained significantly higher after the treatment discontinuation, suggesting that CL is primarily eliminated through urine. Because it was in the winter, cattle produced less urine, which may partly explain why the urine data were much higher than other studies with a similar dose administrated to animals (27) in this study. The previous studies demonstrated that CL in the plasma and urine of cattle was eliminated more slowly than in the previous studies of broilers and pigs (9, 10, 27). Although CL residues were negatively detected in urine of pigs on Day 7 after treatment discontinuation, 4.34 ± 0.61 ng/mL of CL was measured in cattle urine. This effect may also be caused by different species and a lower metabolic rate on cold days. In this study, urine proved to be a more appropriate matrix for monitoring CL abuse in beef cattle as CL residues in urine lasts for a longer period of time (>14 days) than plasma.
CL residues in the white and red hair
Unlike other tissues, CL in the hair of treated cattle continued to increase during early withdrawal periods. This can be explained because there is an unique way for hair to accumulate CL. Henderson (28) suggested there are generally two possible ways for drug accumulation in hair. One is an endogenous pathway by which CL may be diffused from the bloodstream. While CL accumulated from sweat, sebum glands and external contamination account for alternative exogenous pathway. Almost all studies about the accumulation of CL in the hair indicate that residues continue to increase during the early withdrawal period. Dürsch et al. (29) also demonstrated that the concentration of CL accumulated in new hair was demonstrated to be higher than that in old hair, which might be resulted from the time gap between the incorporation of CL into the follicle and the release of the corresponding hair at the skin surface (30). The seasons are also related to changes in the hair growth rate (31) and may also be responsible for the residues accumulating in the hair. It is well known that melanin attributed to the accumulation of CL in tissues (23, 32, 33). In comparison to black hair, brown and blond hair was also shown to be in a much lower level of accumulated concentration. This was reinforced by the present study in which residues in a much lower level were determined in white hair samples (maximum at 21.40 ± 5.14 pg/mg) from cattle compared with other studies, and the peak value was lower in white hair than in that of red hair. The low residues of CL may be explained by a lack of melanin, which was reflected by the white hair color in this study. However, after 14 days of withdrawal, the concentrations were decreasing with withdrawal time. The CL residue tendency in the white hair was not the same as that in the red hair, and the latter maintained CL levels over a relatively long period of time after withdrawal. There are two pathways for CL accumulating in hair. Diffusion from bloodstream and accumulation from sweat, sebum glands and external contamination are two pathways for drug accumulation (11). In the melanin-lacked hair, one part of CL was probably accumulated by the exogenous pathway, and these combinations could easily be affected by external, environmental factors such as sunlight or washing, leading to a decrease in the accumulation concentrations of CL. Another factor is that length of hair was unknown and not taken into account, and CL is possibly diluted by a negative part of hair (16). Despite all of these possibilities, the white hair still proved to be an efficient matrix for monitoring the illegal use of CL.
CL residues in vivo biological tissues
Melanin was responsible for the accumulation of CL in tissues, and many studies have shown that CL was mainly enriched in retinal tissues (33, 34). In this study, CL concentrations in the eye and aqueous humor were much greater than that in other tissues, and previously, (35) it had been reported that melanin-rich ocular tissues accumulate more CL than other melanin-lacking tissues. A relatively slow metabolism rate partly resulted in CL residues staying in the eyes for a long time. However, in terms of all edible tissues, it was proposed that liver, kidney and lung maintained the highest CL residues (8, 36, 37). These findings can be explained because these are the most active tissues for drug metabolism (38, 39). The residues were decreasing over withdrawal time, although some adverse results existed because of individual difference. In fact, if one cattle tests positive for CL, one consuming tissues of the cattle at this time will probably get poisoned. In this study, more than 0.6 pg/mg of CL was measured in the liver and 0.2 pg/mg of CL was measured in the gluteus on Day 28 after withdrawal, proving it to be a risk for ingestion of these tissues. The fact that there were few differences in CL residues between gluteus and rib eye muscle corresponded with the results of other studies (40, 41), which also showed relatively lower CL residues in muscles than other tissues. In this study, more CL residues that detected in muscles around eyes than other part of the cattle were probably caused by great accumulation of CL in eye tissue.
Suitability of living tissues as matrices for monitoring CL abuse
It is well known that there are great variations of residue concentrations and elimination rates for drugs by different tissues. Previous studies have shown that the greatest concentration of CL was in retina tissues, and levels in liver and kidney were nearly as high. Elliott et al. (42) found that even 56 days after treatment, CL in liver could be detected at a relatively high concentration of 0.3–0.4 pg/mg. Meyer and Rinke (40) suggested that the consumption of liver or fat on withdrawal Day 14 would be ill advised. In this study, residues in the liver and gluteus of cattle on withdrawal 28 days exceeded the MRLs. This indicated that CL residues accumulated in some tissues and maintained high concentrations for a long period of time. Under these circumstances, plasma and urine, as two general matrices, would be poor targets for detecting CL abuse because of their short withdrawal periods, specifically, plasma, in which CL residues were lower than LOD 14 days after withdrawal. However, levels of CL detected in white hair on withdrawal days provide proof that white hair may also be used as a target matrix for CL abuse.
Conclusion
The results of this study indicate that CL in plasma could be determined in less than 14 days, during which, CL in liver and kidney far exceeded its MRLs. Urine maintains CL residues for a longer period of time, but a negative determination of urine does not ensure the safety of cattle tissue for ingestion. Compared with other living tissues, hair is the most suitable matrix to monitor CL levels in the cattle as CL is detectable for the longest period. As a whole, hair is necessary to be detected before slaughter though urine or plasma tests negative for CL.
Acknowledgments
We thank the Special Fund for Agro-scientific Research in the Public Interest (201203088-03).
