Liu Pengyan1, Wang Yingfeng2
(1College of Chemistry and Environmental Science, Hebei University, Baoding 071002; 2 Department of Chemistry, Capital Normal University, Beijing, 100037)
Abstract The residue analysis of clenbuterol in swine tissue, such as meat, liver, kidney, and lung was developed. Extracting conditions, selection of solid-phase extraction column and derivatization conditions were studied on. Perchloric acid extraction followed by two clean-up steps, such as extraction with isopropanol-ethyl acetate and solid-phase extraction with strong cation-exchange (SCX) resins. The analyte is determined by GC-SIMMS as the mono-trimethylsilyl derivative prepared using N,O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA) +Trimethylchlorosilane (TMCS) ( 99+1,v/v). The limit of detection spiked at 1mg/kg in tissue samples was less than 0.4mg/kg under the signal-to-noise ratio of 3:1, and the average recoveries (%) were more than 73.4 %. The RSD was less than 10% (n = 3). Under the selecting conditions, the analyte was clean-up with only one SCX resins column, so that the cost was decreased, the time was saved, and the procedure was simple. Moreover, the recovery and sensitivity were satisfied the determination requirements. The method could be used for routine ananlysis of clenbutrol residue confirmatory in animal tissues.
Clenbuterol (4-amino-3, 5-dichloro-a-tert-butylaminomethylbenzyl alcohol hydrochloride) is a beta-adrenergic drug used as a bronchial dilating agent for the treatment of pulmonary diseases in humans and animals. It is especially used in the case of chronic illness . In the 80’s, it was happened to discover that it can promote muscle growth and reduce body fat and increase muscle mass. After that it has been used as growth promoters in animal feed  and it is also extensively misused in farm animals where high doses give rise to a preferential muscle to fat ratio resulting in financial gain for the farmer. Therefore, residue of clenbuterol in edible tissues is happened sometimes. Consequently, it is essential to have a sensitive, rapid, low cost and reliable technique to detect the presence of these residues. Different analytical methods, such as GC-MS [3-9], HPLC-MS [10, 11], ELISA [12, 13], HPLC [14-16], capillary electrophoresis, chemiluminescence sensor, and so on, have been described. In these methods urine samples are mostly used for determination clenbuterol, however, animal tissues are fewer used [5, 10, 11, 14]. Animal tissues pre-treatment is more complex than that of urine because tissues are solid so a homogenisation step will be needed to allow access of the extracting solvent to the sample. Besides, clenbuterol must be isolated from the tissue proteins by hydrolysis–alkaline hydrolysis , acidic hydrolysis [10, 14], enzymatic hydrolysis [11, 20], for example. Because animal matrix contains components too much to remove well, two kinds of cartridge (C8 and SCX [21,22], C18 and SCX ) are often used for clean-up of clenbuterol residue or Bond Elut Certify column, a kind of mixed-phase cartridge, is often used for the procedures[6,23]. By using these solid-phase columns it not only can bring high cost, but also make pretreating procedure complex. And most of recoveries are lower than 70%. In this paper, we describe a simple, sensitive and lower cost method, as well as satisfied the with requirement of maximum residue limits (1mg/kg) and recoveries >70%. Because this method involved a simple clean-up by adjusting pH 11, following extraction by isopropanol-ethyl acetate, the extract got a better clean-up with only one SCX column.
We have studied the influence of the acidic hydrolysis and enzymatic hydrolysis on extracting efficiency, and compared one SCX column with C18 column and two columns of C18 combining with SCX. Finally we selected the acidic hydrolysis for extraction method, one SCX column for clean-up. The cost is lower, the time is more saved, and the procedure is simpler by using this method.
2.1 Chemicals and reagents
Clenbuterol HCl [2-tert.– butylamino-1-(4-amino-3,5-dichlorophenyl) ethanol hydrochloride] (purity ¡Ý 98.5%) was obtained from the national institute for the control of pharmaceutical and biological products (Beijing, China). N,O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA), trimethylchlorosilane, (TMCS) and heptafluorobutyric anhydride (HFBA) were obtained from SPUELCO (USA). Butylboronic acid (BBA) was obtained from Tokyo Kasei Kogyo Co. Ltd., (Tokyo, Japan), N,O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA) containing 1% of trimethylchlorosilane (TMCS) and b-Glucuronidase, From Helix pomatia were supplied by Sigma¨CAldrich (St Louis, MO, USA).
Ethyl acetate, isopropanol, methanol, and toluene were HPLC purity reagent from Merck (Darmstadt, Germany). Sodium chloride for analysis was pretreated for 4h at 650oC. Sodium hydroxide, sodium dihydrogen phosphate£¬ammonia and perchloric acid (super purity grade) were obtained from Beijing Chemicals Co. (Beijing, China). The reagents were analytical grade unless indicated otherwise.
Purified water was produced with a Milli Q system from Millipore (Massachusetts, USA).For sample clean up, 500mg (3mL) C18 and SCX columns from Spuelco (USA) were used.
2.2 Standard solution
The stock standard solution of clenbuterol was prepared at concentration of 1.0mg/mL by dissolving 10.2mg of clenbuterol HCl in 10.00mL methanol and was stable at < -18oC for six months.
Working solution were prepared by dilution of stock solution with methanol to give concentrations 10.0 ng/mL, 50.0 ng/mL, 100.0ng/mL and stored under refrigeration (0-4oC).Phosphate buffer, 0.1mol/L, pH 6.0, was prepared by dissolution of 12.0g NaH2PO4 in 1000mL of deionised water and adjusting the pH to 6.0 with 2mol/L NaOH solution.
Perchloric acid, 0.2mol/L, was prepared by dilution of 83mL perchloric acid with deionised water to 1000mL.
4% ammonia solution/methanol, was prepared by dilution of 4mL ammonia with methanol to 100mL.
Ammonium acetate buffer, pH 5.2, was prepared by dissolution of 1.45g NH4Ac in 1000mL of deionised water and adjusting the pH to 5.2 with acetic acid.
2.3. Instrumental analysis
An Ultraturrax homogenator (IKA, Staufen, Germany) was used for tissue homogenisation. Nitrogen Evaporator with OA-SYS Heating System was obtained from Organomation Associates, Inc. (N-EVAPTM 112, USA). SIGMA 3K30 Laboratory Centrifuges was from Germany. The vacuum manifold used for solid phase extraction (VISIPREPTM DL) procedure was obtained from SUPELCO (USA). HP 6890 gas chromatograph and HP 5973N mass selective detector (MSD) was obtained from Agilent Technologies (USA). GC was equipped with electronic pressure control unit and an autosampler (G 2613A). Instrument control, data acquisition and data processing were carried out with HP ChemStation plus Rev. A. 09. 01. The column used was an HP 5MS (30m length¡Á0.25mm i.d.¡Á0.25 mm film thickness) from Hewlett¨CPackard (CA, USA).
Samples were injected in the splitless mode (0.75 min delay) using helium as the carrier gas at the flow rate 1.0 ml/ min. The injector and transfer line temperatures were set at 220oC and 280oC, respectively. Oven temperature was programmed as follows: initial temperature 70oC for 1min, increasing at a rate of 25oC /min up to 150oC, held for1min, and then increasing at a rate of 6oC /min up to 280oC, maintained at 280oC for 5min. The mass spectrometer conditions were as follows: electron impact ionization voltage 70 eV for both SCAN and selected ion monitoring (SIM) mode, and with a solvent delay of 8 min. The ion source and quadrupoles temperature were 230oC and 150oC, respectively.
2.4 Sample preparation procedure
2.4.1 Extraction of clenbuterol from tissue
A 5.00g of tissue sample that was cut into small pieces was put into a 50mL polypropylene centrifuge tube with stopper, 20mL of 0.2mol/L perchloric acid was added, after that homogenised for 3min. Next, the sample was extracted in an ultrasonic bath for 20min, followed by 30min under 80oC water bath. After being taken out and cooled to room temperature, the sample was centrifugated under 4500g for 10min at 10oC. 10mL of the supernatant was purred into another centrifuge tube, and adjusted pH to 10-12 with 2mol/L sodium hydroxide. After about 5g of sodium chloride being added and mixed, the supernatant was extracted for 1min by a vortex blender using 10mL of mixture of isopropanol and ethyl acetate (4:6, v/v), centrifugated for 10min under 4500g. The extraction step was repeated twice. The extracts were then evaporated to dryness under a stream of nitrogen at 70oC. The residue was dissolved with 1.0mL of 0.1mol/L phosphate buffer (pH 6.0) and filtered over 0.45-mm filter unless the residues were dissolved completely.
A 5.00g of tissue sample was put into a 50mL polypropylene centrifuge tube with stopper, 20mL of 20mmol/L ammonium acetate buffer (pH 5.2) was added, homogenised for 5min, after that 50mL b-glucuronidase was added and blended. The enzymatic hydrolysis was performed at 37oC for 18 h. When cooled to room temperature, the sample was centrifugated under 4500g for 10min at 10oC. Next steps were carried out according to acidic hydrolysis procedures.
SCX columns were conditioned by washing consecutively with 6mL of methanol, 3mL of deionized water, 3mL of sodium dihydrogen phosphate buffer (pH 6.0) and 3mL of deionized water in turn. Next, the samples were loaded on the preconditioned columns. The columns were washed with 4ml deionized water and 4ml methanol and were dried under vacuum to remove as much residual solvent as possible. Then the samples were eluted with 6ml of ammonia solution/methanol (4:96, v/v), the eluent was then evaporated to dryness under a stream of nitrogen at 60oC.
The residue was dried in a desiccator for at least 20min before derivatisation. Then 50mL 1% TMCS/BSTFA (1:99, v/v) was added, vortex mixed and kept at 80oC for 60min. Next the reagent was evaporated, and the derivative was dissolved in 0.5mL toluene, vortex mixed and analyzed by GC-MS. The series standard solutions were derivatized simultaneously.
2.4.4 Standard curve
0.10, 0.20, 0.50, 1.00, 2.00mL of standard solution (10.0ng/mL) was taken, respectively, and the solvent was evaporated under the stream of nitrogen at 60oC water bath, and derivatized under the described conditions above. The concentration of 2.0, 4.0, 10.0, 20.0, 40.0 ng/mL derivatives were obtained and analysed by GC-MS. Calibration cure was constructed from response versus concentrations. Good linearity was observed for derivative, R= 0.7144C + 1.1798, r = 0.9975. The linear range was 2.0-50.0 ng/mL.
- RESULTS AND DISCUSSION
3.1 The conditions of extraction
Because the analyses are often combine with protein in the living, when extracted the samples require enzymatic hydrolysis [5, 11, 20]. Keskin considered that clenbuterol seemed to be excreted free in urine rather than conjugated . We did experiment of enzymatic hydrolysis and acidic hydrolysis with positive swine lungs, found there was no significant difference between two results (listed in the Table 1) (a=0.05, tcal=0.34<ttable=2.78). This result is similar to the literature .
However, the interfering compounds are more using enzymatic hydrolysis than using acidic hydrolysis. Moreover, pretreated time is longer using enzymatic hydrolysis. So it is optimum that the method of extraction using acidic hydrolysis.Table 1
Comparing acid hydrolysis with enzymatic hydrolysis (mg/kg)
3.2 Solid phase extraction
Solid-phase extraction has been widely in use to analyze clenbuterol in biological matrices. Two columns combining with or mixed-phase cartridges were often used in order to obtain optimum clean-up, like describing in the part of introduction. We have tried to contribute a simpler and lower cost clean-up method. In this experiment C18, SCX and C18+SCX columns were used for cleaned up the sample, respectively. Three columns were compared with each other. It was found that one SCX column was the same as C18+SCX and had good clean-up efficiency, but C18 was not satisfied. So we selected one SCX column to clean-up. Because acid extract was adjusted pH and extracted with isopropanol/ethyl acetate beforehand, SPE clean-up became simple.
3.3 The elution condition of SCX cartridge
The solution of phosphate buffer of clenbuterol was added onto the conditioned SCX column, then was washed and eluted under the conditions of describing in “2.4.2 solid-phase extraction procedure”. Every one milliliter of the eluent was collected separated, and dried, derivated and analyzed. The result showed in Table 2. It confirmed that clenbuterol was eluted completely by 6 mL of 4% ammonia/methanol.
Table 2 The results of eluent
|elutents||the first millimeter||the second millimeter||the third millimeter||the fourth millimeter||the fifth millimeter||the sixth millimeter|
|percents of elution (%)||ND||29.8||51.0||15.5||1.7||ND|
3.4 Derivatizing reagents
The standard solution was evaporated to dry under nitrogen, 50mL of 1% TMCS/BSTFA was added, vortex mixed and kept at 80oC for 60min. The reagent was evaporated under nitrogen, and the derivative redissolved in 0.5mL toluene, vortex mixed, and analyzed by GC-MS with both SCAN and selected ion monitoring (SIM) mode.
The standard solution was evaporated to dry under nitrogen, 50mL ethyl acetate was added and vortexed. Then 50mL BBA was added and vortex mixed, kept at 60oC for 60min. The solvent was evaporated under nitrogen, and the derivative redissolved in 0.5mL ethyl acetate, vortex mixed and analyzed by GC-MS with both SCAN and selected ion monitoring (SIM) mode.
The derivative reaction with 50mL HFBA was carried on room temperature for 30min. The other procedures were the same as above esterification. The acylation efficiency at room temperature is better than at 40oC, and derivatization reaction is better with ethyl acetate than without ethyl acetate.
Three derivative reagents were used, one which was BSTFA, a kind of silylation reagent, another was BBA, a kind of esterification, the third was HFBA, a kind of acylation. The results of derivatisation were given in Table 3 and Fig.1.
Fig. 1 Three derivatives of clenbuterol
The derivatives of clenbuterol-HFBA and clenbuterol-BBA (cyclic boronate derivatives of clenbuterol) are formed the most abundant ions in the high mass region, and there are strong characteristic. The sensitivity of clenbuterol-HFBA is lower than clenbuterol-BBA and clenbuterol-BSTFA. Furthermore, HFBA is easy to form acid, thus result in tailing factor. BBA is the same as HFBA. So, we have selected BSTFA as derivative reagent. Its derivative has lower limits of detection, less impurities, and it is of good volatile easy to remove.
Table 3 The data of three clenbuterol derivatives
|BSTFA /TMCS||348||15.56||86, 243, 262, 277||2||9.6||0.62|
|HFBA||472||15.63||363, 398, 439, 456||25||4.8||15.6|
|BBA||342||21.26||243, 245, 327, 329||5||24.3||0.62|
DR: derivatisation reagents; MW: molecular weight; RT: retention time; CI: Characteristic ions; C: concentration; StN.: Signal-to-Noise; LOD: limits of detection
3.5 Comparation BSTFA with 1% TMCS/BSTFA
According to the report  the mixture of TMCS and BSTFA (1:99, v/v) has stronger derivative effect than BSTFA alone. The result is good when making amino acid derivate. The amount of formation of N,O-TMS was less than 1% when clenbuterol was derived by BSTFA, but the amount of that is increased to 5% by 1% TMCS/BSTFA, and therefore 1% TMCS/BSTFA was through of having high ability of amido sylilation. The study was performed by comparing BSTFA with 1% TMCS/BSTFA though two concentration test, SCAN and SIM two modes determination, and found there was not different for formation of O-TMS.
3.6 Recovery, precision and limit of detection
The precision and recovery of the method were determined by analyzing 5.00g samples of swine tissue, which included meat, liver, lung, and kidney. Every kind of tissue was spiked with clenbuterol at three different concentrations of 1.0, 5.0, and 20.0 mg/ kg respectively, and the sample three repetitions were carried out for each fortification level. The sample was extracted, cleaned up and analyzed under the above conditions. Profiles are shown in Fig.2. The recoveries of the analytes such as meat, liver, lung, and kidney ranged from 70.4 % to 92.3, 73.4% to 98.8%, 79.4% to 107.0%, and 76.4% to 94.0%, respectively, and the relative standard deviations (RSDs) varied from 2.3% to 7.5%, 6.7% to 7.5%, 0.9% to 9.4%, 2.9% to 7.2%. As shown in Table 4, the recoveries are all above 70% for four kinds of tissue.
Table 4 Recoveries(%) and RSDs (%) in different tissues
|Spiked of levels
|Spiked meat (mg/kg)||Spiked liver (mg/kg)|
|Means of recovery||73.7||83.1||87.4||92.4||87.4||73.4|
|Spiked of levels
|Spiked kidney (mg/kg)||Spike lung (mg/kg)|
|Means of recovery||81.8||92.4||87.2||84.7||89.8||93.4|
Fig. 2 Total ion chromatograms of TMS derivative. (a) 5ng/mL clenbuterol standard; (b) kidney sample; (c) spiked kidney containing 1mg/kg clenbuterol; (d) lung sample; (e) spiked lung containing 1mg/kg clenbuterol; (f) liver sample; (g) spiked liver containing 1mg/kg clenbuterol; (h) meat sample; (i) spiked meat containing 1mg/kg clenbuterol.
The calculated limits of detection are 1mg/kg clenbuterol in the 5g tissue samples by a signal-to-noise ratio of 3:1. The limits of detection of four kinds of tissue are lower than 0.4mg/ kg. They are 0.34mg/ kg in meat, 0.23mg/ kg in liver, 0.27mg/ kg in lung, and 0.22mg/ kg in kidney.
The recovery of clenbuterol was checked by adding clenbuterol standard solution at three levels to untreated samples of four kinds of tissue. Blank samples from the same tissue without fortification were treated and analysed at the same time with spiked samples.
Complete recovery of the analytes through the extraction procedure was achieved at both concentrations with very good coefficients of variation.
The method proposed involved extraction with perchloric acid and a simple clean-up by extraction with isopropanol-ethyl acetate, and solid-phase extraction with SCX column. The precision and accuracy of the method were found to be suitable for routine analysis of clenbuterol. The method was successfully applied to the analysis of tissue samples after administration of clenbuterol. The limit of detection is good enough to find clenbuterol in tissues.