Bull. Vet. Inst. Pulawy 47, 139-144, 2003
GAS CHROMATOGRAPHY-MASS SPECTROMETRIC CONFIRMATORY METHOD FOR THE DETERMINATION OF CLENUTEROL RESIDUES IN ANIMAL URINE AND LIVER SAMPLES TOMASZ ŚNIEGOCKI, JAN ŻMUDZKI, ANDRZEJ POSYNIAK AND STANISŁAW SEMENIUK Department of Pharmacology and Toxicology, National Veterinary Research Institute, 24-100 Puławy, Poland e-mail:
[email protected] Received for publication December 20, 2002. A sensitive and specific gas chromatographic-mass spectrometric method for the determination of clenbuterol in urine and liver samples is described. Prepared for confirmatory purposes, this method includes solid phase extraction for clean up of biological samples, derivatization and analysis of the clenbuterol derivatives in the selected ion monitoring mode. During the studies, six derivatization procedures have been tested, of which BSTFA +1% TMCS gave the best results. This procedure was validated. The limit of the detection was established at the level of 0.5 ng/g in the liver and 0.5 ng/ml in urine, and the recoveries were better than 80%.
Key words: urine, liver, clenbuterol, GC-MS, confirmatory method.
Clenbuterol is a partitioning agent because of acceleration of animal growth and alternation of the animal carcass composition. However, concentrations of clenbuterol in the liver in the reported limit to be associated with toxicity to consumers (> 100 ng/g) were found in animals, which had received growth-promoting doses of the drug (5, 9, 14, 17). Illegal use of clenbuterol in meat production has prompted the development of test programmes, and the control has been performed by screening tests as well as by confirmatory methods. Detection of nanogram concentrations of this drug in animal tissues by several analytical techniques has been reported (2). Such techniques include enzyme immunoassay (EIA) that is used as screening methods (4, 11, 12, 14) and gas chromatography-mass spectrometry (GC-MS) as confirmatory methods (3, 6, 7, 10, 13, 16, 18, 19). The aim of this study was to develop a confirmatory analysis by GC-MS for clenbuterol residues in urine and liver samples of animal origin. Urine analysis allows to test animals in farm while the liver taken in slaughterhouse is monitored to check the presence of clenbuterol residues in meat for human consumption.
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Material and Methods Materials. Clenbuterol was obtained from Sigma Chemical Co. BSTFA (N, N-bis (trimethylsilyl trifluoroacedamid)) + 1% TMCS (trimethylchlorosilane) from Fluka. BSTFA, TMCS and MSTFA (N, -methyl –N, -trimethylsilyl trifluoroacedamid were from Supelco. Water was deionized (> 14 MΩ x cm); hexane, methanol and Bakerbond octadecyl C18 columns were from J.T. Baker. Potassium dihydrogenophosphate was purchased from Standart. Disodium hydrogenophosphate and sodium hydroxide were purchased from P.O.Ch. Gliwice. Stock solution and standards. Stock solutions of 1 mg/ml were prepared in methanol and stored in the dark at < -16°C. The working solutions at level of 0.2 μg/ml and 0.4 μg/ml for clenbuterol for GC-MS were prepared in methanol and stored in the dark at < 6°C no longer than two weeks. Gas chromatography-mass spectrometry. Trace GC 2000 series (Thermo Quest). Gas carrier: Helium. Chromatographic separation was performed in capillary column DB-1MS (J&W Scientific) 30 x 0.25 x 0.25 mm. The injection and transfer line were kept at 250°C and 280°C, respectively. The gas chromatograph oven was programmed from 70°C to 200°C at a rate of 18°C/min and subsequently to 250°C at 5°C/min and finally to 300°C at 20°C/min and stay for 8 min. The gas chromatograph was coupled to Finnigan Polaris (Thermo Quest) MS detector, operating in single ion mode (SIM) with the selection ions at m/z: 86, 243, 245, 262, 264, and 333. Extraction of the liver. A 10 g portion of minced liver was homogenized with 20 ml volume of acetonitrile. The tube containing the homogenate was centrifuged at 4000 g for 10 min at < 6°C. The supernatant was evaporated under vacuum; dry residue was dissolved in 20 ml phosphate buffer at pH = 7.6. The whole solution was cleaned up by SPE procedure. Extraction of urine. A 10 ml portion of urine sample was diluted with 10 ml of phosphate buffer at pH = 7.6 and centrifuged at 4000 g for 10 min at room temperature. The whole solution was cleaned up by SPE technique. Clean up. SPE C18 cartridges were preconditioned with 9 ml of methanol, 6 ml of water and finally with 2 ml of phosphate buffer. After percolation of the whole solutions, the columns were washed with 9 ml of water, 6 ml 20 % of methanol and 0.5 ml of methanol and dried under depression for 5 min. Clenbuterol was eluted with 5 ml of methanol. Derivatisation. The extracts were dried under gentle nitrogen stream at 45°C. The dry residue was dissolved in 300 μl of methanol and shaked vigorously with a vortex to retrieve the whole residue, and transfer the methanol in vial and dried under gentle nitrogen stream. One of the aliquots was derivatized using 50 μl BSTFA alone, and containing 1% or 10% of TMCS. The other was derivatized with 50 μl MSTFA alone, and containing 1% or 10% of TMCS. The vial was heated at 65°C for 1 h, and then reagent was evaporated. The residue was dissolved in 25 μl of hexane, and the solution in the volume of the 1 μl was injected in the gas chromatograph. Quantitation of clenbuterol. Samples of the liver were spiked with clenbuterol at levels of 1, 2 and 4 ng/g and urine samples were spiked at the same levels in ng per ml. All spiked samples were processed according to the procedure described above.
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Results Various derivatization procedures were compared. The best and comparable results were obtained when BSTFA + 1% TMCS or MSTFA + 1% TMCS have been used. The use of BSTFA or MSTFA alone was less effective. The addition TMCS at the level of 10% did not improve efficiency of derivatization step. The BSTFA + 1% TMCS was found as less laborious than MSTFA + 1% TMCS, and therefore it was used in the next steps of the studies. Fig. 1 shows GC-MS chromatograms of a standard solution containing derivatized of clenbuterol, blank urine sample and spike urine sample. In electron impact mode the spectrum of the clenbuterol derivative, obtained by derivatisation with BSTFA +1 % TMCS shows the following diagnostic ions at m/z: 86, 243, 245, 262, 264, 333 (Fig. 2). Tables 1 and 2 show the statistical validation data obtained for clenbuterol determinations in spiked liver and urine samples respectively. The detection limit in urine was 0.5 ng/ml and for the liver was established at the level of 0.5 ng/g. A D:\DATA POLARIS\...\Grudzien\61202ago01
02-12-06 12:16:26
20 1
Relative Abundance
RT: 12,00 - 13,99
100
NL: 4,91E4 TIC MS 61202ago01
Clenbuterol RT: 13,20
50
0 12,0
12,1
12,2
12,3
12,4
12,5
12,6
12,7
12,8
12,9
13,0 13,1 Time (min)
13,2
13,3
13,4
13,5
13,6
13,7
13,8
13,9
B D:\DATA POLARIS\...\271102 ago01
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blank
Relative Abundance
RT: 11,99 - 13,97 NL: 1,82E4 TIC MS 271102ago01
13,74
100
13,58
13,62
13,85
13,94
50 12,28 12,36 12,45 12,54 12,63 12,68 12,72 12,75
12,05 12,14
12,94 12,97 13,06
13,18
13,30 13,44 13,47
0 12,0
12,1
12,2
12,3
12,4
12,5
12,6
12,7
12,8
12,9 13,0 Time (min)
02-12-06 12:51:01
20 1
13,1
13,2
13,3
13,4
13,5
13,6
13,7
13,8
13,9
C D:\DATA POLARIS\...\Grudzien\61202ago02
Relative Abundance
RT: 11,99 - 13,99 NL: 2,81E4 TIC MS 61202ago02
150 Clenbuterol 100
RT: 13,20
50 0 12,0
12,1
12,2
12,3
12,4
12,5
12,6
12,7
12,8
12,9
13,0 13,1 Time (min)
13,2
13,3
13,4
13,5
13,6
13,7
13,8
Fig. 1. Typical GC-MS chromatograms of TMS clenbuterol derivatives. a) Standart peak at 13,20 min, b) Blank urine sample, c) Spike urine sample
13,9
142
D:\DATAPOLARIS\...\Grudzien\61202ago01
02-12-0612:16:26
201
262,0
Relative Abundance
100 80 243,1
60
264,0
245,0
40 20
333,0 86,1
245,7
0 100
120
140
160
180
200
220
240
260
280
300
320
m/z
Fig. 2. Mass spectrum of clenbuerol mono-TMS derivative. Table 1 Recovery and precision of analytical procedure for clenbuterol determination in the liver Found (mean ± SD) ng/g
Recovery %
6
0.83 ± 0.04
2
6
4
6
Added ng/g
n
1
RSD (%) Intra-day
Inter-day
85%
4.6
6.2
1.65 ± 0.06
83%
5.9
7.8
3.32 ± 0.09
87%
7.4
8.7
SD - Standard Deviation RSD - Relative Standard Deviation
Table 2 Recovery and precision of analytical procedure for clenbuterol determination in urine Found (mean ±SD) ng/g
Recovery %
6
0.91 ± 0.05
2
6
4
6
Added ng/g
n
1
RSD (%) Intra-day
Inter-day
91%
3.3
4.6
1.83 ± 0.07
92%
4.8
5.4
3.65 ± 0.09
94%
6.2
7.3
SD - Standard Deviation RSD - Relative Standard Deviation
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The recovery assessed by comparing the chromatographic peak height of clenbuterol standard solutions with that of extracted urine or liver spike with the some amount of clenbuterol was constantly higher than 80%. Discussion In our laboratory the urine and liver samples used for the detection of clenbuterol are routinely processed with a screening-confirmation protocol. In the first screening step separates aliquots of the samples are assayed with ELISA kits. In the second screening step, the sample extracts are analyzed by liquid chromatography with UV detection (14). Now, a new analytical procedure has been developed with gas chromatography coupled to spectrometry mass detector, as confirmatory technique. In the confirmatory step, new aliquots of the sample for clenbuterol are prepared as described in section: Material and Methods, and the compound of interest is derivatized with BSTFA + 1% TMCS and detect as TMS - products by GC-MS in EI mode. To fulfill the GC-MS confirmation criteria of the EU it is necessary to measure four independent mass fragments in the mass spectrum and the smallest fragment must be above 10% comparison with abundance of base fragment (1). In our studies four diagnostic ions at m/z: 243, 262, 264, 333 have been suitable for confirmational analysis (abundance above 10%) as it was obtained by other authors (3, 6, 10). In conclusion, the described GC-MS for the quantitative determination of clenbuterol in urine and liver samples shows good sensitivity and selectivity. The applied extraction and clean-up process leads to a “clean” residue devoid of most of the impurities. The whole procedure is simple and rapid, and available to the confirmatory of the clenbuterol residues in samples of animal origin after the illegal use of clenbuterol as partitioning agent. However, the applicability of new derivatizatoin reagents and improvement of selectivity by GC-MS in MS-MS mode is under development. The whole method is optimized for the determination of some other β-agonists - salbutamol, terbutaline, and tulobuterol. The procedure is validated according to new EU criteria (8), and results will be published separately. References 1.
2. 3.
4.
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