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Multi-Residue Determination of Eight Anabolic Steroids by GC-MS in Muscle Tissues from Pigs
Agricultural Sciences in China
February 2010
2010, 9(2): 306-312
Multi-Residue Determination of Eight Anabolic Steroids by GC-MS in Muscle Tissues from Pigs ZENG Dong-ping, LIN Cui-ping, ZENG Zhen-ling, HUANG Xian-hui and HE Li-min Laboratory of Veterinary Pharmacology, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510640, P.R. China
Abstract A gas chromatography-mass spectrometry (GC-MS) method to determine eight anabolic steroids (diethylstilbestrol, methyltestosterone, norethindrone, 17α-ethynylestradiol, estradiol, 6α-methyl-17α-hydroxy-progesterone, estradiol benzoate, and chlormadinone acetate) was developed. Muscle samples were extracted with liquid-liquid extraction and clean-up was performed in two steps, the extracts obtained were derivatized with heptafluorbutyric (HBF) anhydride and analyzed by GC-MS. In the above method, the linear scope was 2.5-50 g kg-1. The range of the recoveries was 78.5-148% for diethylstilbestrol, 70.8-109% for methyltestosterone, 69.8-87.2% for norethindrone, 67.7-120% for 17α-ethynylestradiol, 82.8-103% for estradiol, 70.3-99.2% for 6α-methyl-17α-hydroxy-progesterone, 73.0-104% for estradiol benzoate, and 72.991.8% for chlormadinone acetate. The range of the coefficients of variation within batches was 0.4-12%; the range of the coefficients of variation between batches was 6.4-11%. The limit of detections and the detection capability were 0.99 and 3.30 g kg-1 for diethylstilbestrol, 1.05 and 3.50 g kg-1 for methyltestosterone, 1.19 and 3.97 g kg-1 for norethindrone, 0.94 and 3.13 g kg-1 for 17α-ethynylestradiol, 1.45 and 4.83 g kg-1 for estradiol, 1.56 and 5.20 g kg-1 for 6α-methyl-17αhydroxy-progesterone, 1.92 and 6.40 g kg-1 for estradiol benzoate, and 2.41 and 8.03 g kg-1 for chlormadinone acetate, respectively. These results showed that the method was widely available, accurate, and sensitive. Key words: anabolic steroids, GC-MS, multi-residue determination, pigs
INTRODUCTION Anabolic steroids were a group of synthetic hormones which could promote the storage of protein and the growth of tissue (Li 2006). In the 1970s, anabolic steroids were widely used in food-animals to promote growth rate and improve feed conversion efficiency. But the further studies found that they had toxic or carcinogenic properties, so the european commissiom (EC) and the food and drugs administration (FDA) had banned their use in the fattening of slaughter animals, also in China (Sauer et al. 1998; McEvoy et al. 1998a,
b; Walshe et al. 1998). To control illegal treatments, it was essential to establish reliable analytical methods. A wide variety of procedures had been described for the determination of anabolic residue in tissue, faeces, urine, feed, and plasma (Sterk et al. 1998; Draisci et al. 1998). The European Communities had recommended several methods for the detection of hormonally active compounds, including high performance liquid chromatography (HPLC) (Zhou et al. 2005), high performance liquid chromatography-mass spectrometry (HPLC-MS) (Bean et al. 1997; Coldham et al. 1998; Barron et al. 1996), gas chromatography (GC) (Durant et al. 2002), gas chromatography-mass
This paper is translated from its Chinese version in Scientia Agricultura Sinica. Correspondence ZENG Dong-ping, Ph D, Tel: +86-20-85280237, E-mail: [email protected]
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(09)60098-5
Multi-Residue Determination of Eight Anabolic Steroids by GC-MS in Muscle Tissues from Pigs
spectrometry (GC-MS) (Casademont et al. 1996; Hartmann et al. 1997; Daeseleire et al. 1998; Dubois et al. 1998; Marchand et al. 2000; Promberger et al. 2001; Kuuranne and Steinhart 2003; Stolker et al. 2004), and immunoassay techniques such as enzyme linked immunosorbent assay (ELISA) had been developed for several compounds (Fitzpatrick et al. 2004). The aim of this study was to develop a GC-MS multiresidue method to confirm the presence of eight anabolic steroids (diethylstilbestrol, methyltestosterone, norethindrone, 17α-ethynylestradiol, estradiol, 6α-methyl17α-hydroxy-progesterone, estradiol benzoate, and chlormadinone acetate) after conversion of the heptafluorobutyric acid (HFBA) derivatives in muscle tissue.
307
prepared in methanol and stored at -20°C. Working solutions, which were prepared by a series of ten-fold dilutions of the stock solutions, were stored in the dark at approximately 4°C for a maximum period of 6 mon. Methanol-water solutions were prepared with methanol:water (4:1, v/v). The elution solutions were prepared with chloroform:acetone (4:1, v/v). The heptaffuorobutyril (HFB) derivatives solutions were prepared with HFBA:acetone (4:1, v/v).
Samples
MATERIALS AND METHODS
Samples of swine meat were collected and kept frozen (-20°C) until analysis by Animal and Poultry Products Quality Control Inspection and Testing Center (Guangzhou), Ministry of Agriculture, China.
Materials, chemicals and reagents
Methods
The synthetic steroids used in this study were methyltestosterone, estradiol and estradiol benzoate, from National Institute for the Control of Pharmaceutical and Biological Products, China. Diethylstilbestrol, norethindrone, 17α-ethynylestradiol, 6α-methyl-17αhydroxy-progesterone, and chlormadinone acetate were from Sigma-Aldrich Chemie (Guangzhou, China) and the HFBA anhydride from Supelco (Bellefonte, USA). Aether, methanol, petroleum ether II, chloroform, Nhexane, acetone, sodium acetate, and acetic acid of A.R. were from Guangzhou Chemical Reagent Factory, China.
The method of analysis was based on liquid-liquid extraction of the steroids, sample cleaned-up by solid phase extraction, dramatized with HFBA, and GC-MS analysis.
Liquid-liquid extraction
The GC-MS system was from Autosystem XLTurbomass PerkinElmer, USA. The solid phase extraction element from WAT058833 Waters, USA. The nitrogen evaporator model N-EVAPTM 111 from Organomation Associates, Inc., USA. The table centrifuge model Centrifuge 5 804 from Eppendorf, Germany. The solid phase extraction column model Si-SPE (500 mg per 3 mL), NH2-SPE (500 per mg 3 mL), and C18SPE (500 mg per 3 mL) from Supelco, USA.
A test sample of 50-100 g was thoroughly homogenized and a test portion of 5.0 g was weighed into a 50 mL centrifuge tube, standing for 1 min (Stolker et al. 2004). After addition of 10 mL ether (vortexed at 2 000 r/min for 1 min and centrifuged for 10 min at 7 000 r/min) (Wan et al. 2004), the mixture was evaporated in a water bath under a stream of nitrogen at 40°C. After the addition of 4 mL methanol : water (4:1, v/v) and 4 mL petroleum ether II (vortexed at 2 000 r/min for 1 min and centrifuged for 10 min at 3 000 r/min), the mixture was evaporated in a water bath under a stream of nitrogen at 55°C to a final volume of approximately 0.5 mL. After the addition of 2 mL of water, the mixture was extracted 4 mL ether. The extract was evaporated in a water bath under a stream of nitrogen at 40°C. The residue was dissolved in 0.5 mL chloroform and 5 mL hexane.
Solution preparation
Solid phase extraction
Stock solutions of 1 000 g mL-1 of the steroids were
A Si-SPE cartridge was conditioned with 5 mL hexane.
Equipments
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The aqueous extract was applied to the cartridge; the flask and cartridge were rinsed with 2.5 mL hexane. Then the NH 2-SPE cartridge were combined on the bottom of Si-SPE, conditioned with 5 mL hexane, then the cartridge was rinsed with 5 mL chloroform:acetone (4:1, v/v), the eluents were combined in a 10-mL tapered glass tube. The solvent was removed under a stream of nitrogen at 60°C.
Preparation of HFB derivatives After the residue was drying 10 min at 60°C, 50 L HFBA:acetone (1:4, v/v) was added. The vial was vortexed and incubated for 1 h at 60°C. Next, the reaction mixture was dissolved in 700 L methylbenzene, then 500 L ddH2O was added. The mixture was vortexed at 2 000 r/min for 1 min, centrifuged for 10 min at 3 000 r/min. The final solvent upper 600 L was injected into the GC-MS system (Impens et al. 2002).
Gas chromatography-mass spectrometry Chromatographic conditions: Injections were carried out in the splitless mode. In both cases, the carrier gas was high-purity helium at a flow rate of 1.0 mL min-1. The injector temperature was maintained at 280°C. The initial temperature of the column was 120°C and was programmed from 120°C (held for 2 min) to 250°C at 15°C min-1 (held 2 min) and then to 280°C at 4°C min-1 (held for 5 min). The injection volume was 2 L. Mass parameters, selected monitoring mode; electronic impact ionization; electron energy, 70 eV; ion source temperature, 200°C; emission current, 200 A; detector voltage, 524 V; interface temperature, 280°C.
Validation of method To calculate the accuracy and precision of the method, a meat sample (approx. 300 g) was homogenized thoroughly. Seven samples (5 g each) were prepared in a 50 mL centrifuge tube with addition of 100 mL a steroid standard (containing 0, 2.5, 5, 10, 20, 40, and 50 g kg-1). All the spiked samples were dealt with above method. The linearity of the detection was calculated between the concentrations and the peak ratio.
ZENG Dong-ping et al.
Each treatment had four replicates.
Recovery Recovery studies were performed on sample spikes at 0, 2.5, 10, and 50 g kg-1 level. The values were compared with the ratios obtained for a reference standard solution containing amounts of substances corresponding (n = 6). Each treatment had four replicates.
Detection and quantification The limit of detection was determined by analyzing sample fortified with increasing concentrations of the compounds (0, 1, 2, 4, and 5 g kg-1 for muscle tissue). A calibration curve was calculated and the limit of detection was calculated as 3 Sb/m, the limit of quantification was calculated as 10 Sb/m, where Sb is the standard deviation of the intercept on the y-axis and m is the slope of the calibration curve.
RESULTS AND DISCUSSION Chromatogram The derivatization method, carried out as above mentioned, is very easy to perform. It results in excellent derivatives of the steroids which are separated well in the gas chromatogram (Fig.1). The mass of the selected ions are presented in Table 1. GC-MS selectedion monitoring chromatograms of HFB-derivative for anabolic steroids are presented in Fig.2. GC-MS selected-ion monitoring chromatograms of HFB-derivative for control pork are presented in Fig.3. GC-MS selected-ion monitoring chromatograms of HFB-derivative for anabolic steroids in pork are presented in Fig.4. The linearity of the detection (r > 0.97) was checked as being between 2.5 and 50 g kg-1. The range of the recoveries was 78.5-148% for diethylstilbestrol, 70.8-109% for methyltestosterone, 69.8-87.2% for norethindrone, 67.7-120% for 17αethynylestradiol, 82.8-103% for estradiol, 70.3-99.2% for 6α-methyl-17α-hydroxy-progesteron-e, 73.0-104% for estradiol benzoate, and 72.9-91.8% for chlormadinone acetate. The range of the coefficients of variation within
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
309
Relative intensity
Relative intensity
Multi-Residue Determination of Eight Anabolic Steroids by GC-MS in Muscle Tissues from Pigs
Time (min)
Fig. 1 GC-MS full ion chromatogram of HFB-derivative for anabolic steroids (12.5 g mL -1 ). 1, cis-diethylstilbestrol; 2, transdiethylstilbestrol; 3, methyltestosterone; 4, norethindrone; 5, 17αethynylestradiol; 6, estradiol; 7, 6α-methyl-17α-hydroxyprogesterone; 8, estradiol benzoate; 9, chlormadinone acetate; retention time (17.00-20.00 min), column bleeding.
Table 1 Selected ions of the HFB derivatives Compound Diethylstilbestrol Methyltestosterone Norethindrone 17α-Ethynylestradiol Estradiol 6α-Methyl-17α-hydroxy-progesterone Estradiol benzoate Chlormadinone acetate
Relative intensity
Time (min)
m/z of selected ions 303, 341, 417, 355, 369, 465, 143, 461, 476, 115, 353, 446, 169, 356, 409, 133, 383, 479, 146, 540, 541 461, 497, 540
447 480 477 474 451 480
batches was 0.4-12%; the range of the coefficients of variation within batches was 6.4-11%. The limit of detections and the detection capability were 0.99 and 3.30 g kg-1 for diethylstilbestrol, 1.05 and 3.50 g kg-1 for methyltestosterone, 1.19 and 3.97 g kg-1 for norethindrone, 0.94 and 3.13 g kg-1 for 17α-ethynylestradiol, 1.45 and 4.83 g kg-1 for estradiol, 1.56 and 5.20 g kg-1 for 6α-methyl-17α-hydroxyprogesterone, 1.92 and 6.40 g kg -1 for estradiol benzoate, and 2.41 and 8.03 g kg-1 for chlormadinone acetate, respectively.
Sample preparation The sample preparation of the anabolic steroids were developed quite fast in the 1990s, a reliable, generally accepted isolation and purification procedure for the multi-residue determination of anabolic in urine and tissues of slaughtered animal was developed for TLC detection by Verbeke (1979). The advantage of this ref-
Time (min)
Fig. 2 GC-MS selected-ion monitoring chromatograms of HFBderivative for anabolic steroids (12.5 g mL-1). Retention time (min)1, cis-diethylstilbestro (9.58); 2, trans-diethylstilbestrol (10.34); 3, methyltestosterone (11.77); 4, norethindrone (12.21); 5, 17α-ethynylestradiol (12.61); 6, estradiol (13.43); 7, 6α-methyl17α-hydroxy-progesterone (15.43); 8, estradiol benzoate (16.62); 9, chlormadinone acetate (21.28). The same as below.
erence method is its applicability to androgenic, progestogenic, and estrogenic compounds with steroid or stilbene structrure. Disadvantageous are the tediousness of the extraction and clean-up procedure (2.5 d) and the amount of chemicals required (more than 900 mL solvents per sample, including toxicologically problematic ones like toluene and methylene chloride). To overcome some of the disadvantages, improved methods had been proposed by several authors who replaced the time-consuming self-preparation of chromatograph columns by the use of disposable solid-phase extraction (SPE) cartridges (Draisci et al. 2003). In the analysis of anabolic in urine, SPE-based methods were routinely used. The analysis of animal tissues seemed to be more difficult, however, so mostly combinations of liquid-liquid and solid-phase extraction methods were applied. Some authors proposed a
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
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ZENG Dong-ping et al.
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Time (min)
Relative intensity
Relative intensity
Time (min)
Time (min)
Time (min)
Fig. 3 GC-MS selected-ion monitoring chromatograms of HFBderivative for control pork. Retention time was not interfered with the blank samples.
Fig. 4 GC-MS selected-ion monitoring chromatograms of HFBderivative for anabolic steroids in pork.
combination of SPE and HPLC (Van et al. 2002). Another elegant solution was the clean-up with immunoaffinity solid phases. This approach was limited to a small number of steroid hormones, though, so it did not allow the determination of comprehensive hormone patterns (Barron et al. 1996). The aim of this study was to develop a rapid, economical, and reliable sample preparation which showed a wide applicability methods for the determination in meat, based on the combinations of liquid-liquid and solid-phase extraction methods by Hartmann and Steinhart (1997). More application of the revised European Union (EU) criteria was used for confirmation of anabolic steroids (Dubois et al. 1998; Daeseleire et al. 1998;Stolker et al. 2004 ).
Derivatization of steroids We chose a universally applicable derivatization method which was especially suitable for anabolic steroids. With addition of the catalyst HFBA to the derivatization reagent HFB, it resulted in excellent derivatives of the steroids which were separated well in the gas chromatogram. We also chosed the derivatization reagent TMS, BSTFA and so on, but it didn’t result good separation in gas chromatogram. HFBA reacted with 3-oxo groups of methyltestostero n e w i t h f o r m a t i o n o f 3 heptafluorobutyryl enol esters, but some 17hydroxy groups of methyltestosterone were not converted. In brief, the procedure yielded mono-HFB
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Multi-Residue Determination of Eight Anabolic Steroids by GC-MS in Muscle Tissues from Pigs
derivatives for methyltestosterone, while the rest of compounds yielded di-HFB derivatives. The stability of these derivatives was good, but derivatized samples were not kept longer than 3 d.
Selected ions of the HFB derivatives Both identification and quantitation were carried out by selected ion monitoring (SIM) of characteristic ions of the HFB derivatives. Individual scan of HFB derivatives was performed for each investigated anabolic standard to obtain the mass, fragmentation pattern. The most suitable ions (high ion intensity, high mass, and low background) were selected to perform the analysis with SIM mode.
Recovery and sensitivity evaluation
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Acknowledgements
References Barrón D, Barbosa J, Pascual J A, Segura J. 1996. Direct determination of anabolic steroids in human urine by on-line solidphase extraction/liquid chromatography/mass spectrometry. Journal of Mass Spectrometry, 31, 309-319. Bean K A, Henion J D. 1997. Direct determination of anabolic steroid conjugates in human urine by combined high-performance liquid chromatography and tandem mass spectrometry. Journal of Chromatography (B: Analytical Technologies in the Biomedical and Life Sciences), 690, 65-75. Casademont G, Perez B, Garcia Régueíro J A. 1996. Simultaneous determination, in calf urine, of twelve anabolic agents as heptafluorobutyryl derivatives by capillary gas chromatography-mass spectrometry. Journal of Chromatography (B: Analytical Technologies in the Biomedical and Life
Analytical recovery of the method was evaluated for each anabolic compound. The above mentioned showed that the range of the recoveries was 78.5-148% for diethylstilbestrol, 70.8-109% for methyltestosterone, 69.8-87.2% for norethindrone, 67.7-120% for 17αethynylestradiol, 82.8-103% for estradiol, 70.3-99.2% for 6α-methyl-17α-hydroxy-progesteron-e, 73.0-104% for estradiol benzoate, and 72.9-91.8% for chlormadinone acetate. The recoveries of the method were more reasonable than the other reports (Daeseleire et al. 1998) which the range was 26-65%. The limit of detections was 0.94-2.41 g kg-1, it was higher than the other repots (Daeseleire et al. 1998) which was 0.1-2.6 g kg-1. The following reason must be concerned, firstly the final volume in our study was 600 L, while the other laboratories were 25 L, secondly the m/z was quite low which must below 600 in our laboratory. So further study must be done in the future.
CONCLUSION In recent years we have developed multi-residue methods for the detection of anabolic steroids in muscle tissue. A few modifications were made so that the methods could be implemented in an accredited routine system.
Sciences), 686, 189-198. Coldham N G, Biancotto G, Montesissa C, Howells L C, Sauer M J. 1998. Utility of isolated hepatocytes and radio-HPLCMSn for the analysis of the metabolic fate of 19nortestosterone laurate in cattle. The Analyst, 123, 25892594. Daeseleire E, Vandeputte R, van Peteghem C. 1998. Validation of multi-residue methods for the detection of anabolic steroids by GC-MS in muscle tissues and urine samples from cattle. The Analyst, 123, 2595-2598. Draisci R, Palleschi L, Ferretti E, Lucentini L, Ouadri F D. 2003. Confirmatory analysis of 17[beta]-boldenone, 17[alpha]boldenone and androsta-1,4-diene-3,17-dione in bovine urine by liquid chromatography-tandem mass spectrometry. Journal of Chromatography (B: Analytical Technologies in the Biomedical and Life Sciences), 789, 219-226. Draisci R, Palleschi L, Ferretti E, Marchiafava C, Lucentini L, Cammarata P. 1998. Quantification of 17β-estradiol residues in bovine serum by liquid chromatography-tandem mass spectrometry with atmospheric pressure chemical ionization. The Analyst, 123, 2605-2609. Dubois M, Taillieu X, Colemonts Y, Lansival B, de Graeve J, Delahaut P. 1998. GC-MS determination of anabolic steroids after multi-immunoaffinity purification. The Analyst, 123, 2611-2616. Durant A A, Fente C A, Franco C M, Vázquez B I, Cepeda A. 2002. Gas chromatography-tandem mass spectrometry determination of 17α-ethinylestradiol residue in the hair of cattle. Application to treated animals. Journal of Agricultural and Food Chemistry, 50, 436-440.
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Fitzpatrick J, Manning B, O’Kennedy R. 2004. Enzyme-linked immunosorbent assay-based detection of free trenbolone in bovine bile. Journal of Agricultural and Food Chemistry, 52, 4351-4354. Hartmann S, Steinhart H. 1997. Simultaneous determination of anabolic and catabolic steroid hormones in meat by gas chromatography-mass spectrometry. Journal of Chromatography (B: Analytical Technologies in the Biomedical and Life Sciences), 704, 105-117. Impens S, de Wasch K, Comelis M, de Brabander H. 2002. Analysis on residues of estrogens, gestagens and androgens in kidney fat and meat with gas chromatography-tandem mass spectrometry. Journal of Chromatography (A), 970, 235247. Kuuranne T, Kotiaho T, Pedersen-Bjergaard S, Rasmussen K E, Leinonen A, Westwood S, Kostiainen R. 2003. Feasibility of a liquid-phase microextraction sample clean-up and liquid chromatographic/mass spectrometric screening method for selected anabolic steroid glucuronides in biological samples. Journal of Mass Spectrometry, 38, 16-26. Li D. 2006. Pharmacology. 5th ed. People’s Medical Publishing House, Beijing. pp. 293-307. (in Chinese) Marchand P, Le Bizec B, Gade C, Monteau F, André F. 2000.
tion of a multi-analyte method for the detection of anabolic steroids in bovine urine with liquid chromatography-tandem mass spectrometry. Journal of Chromatography (B: Analytical Technologies in the Biomedical and Life Sciences), 772, 211-217. Promberger A, Dornstauder E, Fruhwirth C, Schmid E R, Jungbauer A. 2001. Determination of estrogenic activity in beer by biological and chemical means. Journal of Agricultural and Food Chemistry, 49, 633-640. Sauer M J, Samuels T P W, Howells L G, Seymour M A, Nedderman A, Houghton E, Bellworthy S J, Anderson S, Coldham N G. 1998. Residues and metabolism of 19nortestosterone laurate in steers. The Analyst, 123, 26532660. Sterk S, Herbold H, Blokland M, van Rossum H, van Ginkel L, Stephany R. 1998. Nortestosterone: endogenous in urine of goats, sheep and mares? The Analyst, 123, 2633-2636. Stolker A A M, Linders S H M A, van Ginkel L A, Brinkman U A T. 2004. Application of the revised EU criteria for the confirmation of anabolic steroids in meat using GC-MS. Analytical and Bioanalytical Chemistry, 378, 1313-1321. Verbeke R. 1979. Sensitive multi-residue method for detection of anabolics in urine and in tissues of slaughtered animals. Journal of Chromatography, 177, 69-84.
Ultra trace detection of a wide range of anabolic steroids in meat by gas chromatography coupled to mass spectrometry. Journal of Chromatography (A), 867, 219-233.
Walshe M, O’Keeffe M, Le Bizec B. 1998. Studies on the determination of chlorotestosterone and its metabolites in bovine
McEvoy J D, McVeigh C E, McCaughey W J. 1998a. Residues of nortestosterone esters at injection sites. Part 1. Oral
urine. The Analyst, 123, 2687-2691. Wan M M, Chen Z L, Liu Y H, Tan D X. 2004. Determination of
bioavailability. The Analyst, 123, 2475-2478. McEvoy J D, McVeigh C E, McCaughey W J. 1998b. Residues
diethylstilbestrol in animal tissues by HPLC/ECD. Chinese Journal of Veterinary Drug, 38, 4-6. (in Chinese)
of nortestosterone esters at injection sites. Part 2. Behavioural effects. The Analyst, 123, 2479-2484.
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China Dairy Industry, 33, 56-58. (in Chinese) (Managing editor WANG Ning)
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
February 2010
2010, 9(2): 306-312
Multi-Residue Determination of Eight Anabolic Steroids by GC-MS in Muscle Tissues from Pigs ZENG Dong-ping, LIN Cui-ping, ZENG Zhen-ling, HUANG Xian-hui and HE Li-min Laboratory of Veterinary Pharmacology, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510640, P.R. China
Abstract A gas chromatography-mass spectrometry (GC-MS) method to determine eight anabolic steroids (diethylstilbestrol, methyltestosterone, norethindrone, 17α-ethynylestradiol, estradiol, 6α-methyl-17α-hydroxy-progesterone, estradiol benzoate, and chlormadinone acetate) was developed. Muscle samples were extracted with liquid-liquid extraction and clean-up was performed in two steps, the extracts obtained were derivatized with heptafluorbutyric (HBF) anhydride and analyzed by GC-MS. In the above method, the linear scope was 2.5-50 g kg-1. The range of the recoveries was 78.5-148% for diethylstilbestrol, 70.8-109% for methyltestosterone, 69.8-87.2% for norethindrone, 67.7-120% for 17α-ethynylestradiol, 82.8-103% for estradiol, 70.3-99.2% for 6α-methyl-17α-hydroxy-progesterone, 73.0-104% for estradiol benzoate, and 72.991.8% for chlormadinone acetate. The range of the coefficients of variation within batches was 0.4-12%; the range of the coefficients of variation between batches was 6.4-11%. The limit of detections and the detection capability were 0.99 and 3.30 g kg-1 for diethylstilbestrol, 1.05 and 3.50 g kg-1 for methyltestosterone, 1.19 and 3.97 g kg-1 for norethindrone, 0.94 and 3.13 g kg-1 for 17α-ethynylestradiol, 1.45 and 4.83 g kg-1 for estradiol, 1.56 and 5.20 g kg-1 for 6α-methyl-17αhydroxy-progesterone, 1.92 and 6.40 g kg-1 for estradiol benzoate, and 2.41 and 8.03 g kg-1 for chlormadinone acetate, respectively. These results showed that the method was widely available, accurate, and sensitive. Key words: anabolic steroids, GC-MS, multi-residue determination, pigs
INTRODUCTION Anabolic steroids were a group of synthetic hormones which could promote the storage of protein and the growth of tissue (Li 2006). In the 1970s, anabolic steroids were widely used in food-animals to promote growth rate and improve feed conversion efficiency. But the further studies found that they had toxic or carcinogenic properties, so the european commissiom (EC) and the food and drugs administration (FDA) had banned their use in the fattening of slaughter animals, also in China (Sauer et al. 1998; McEvoy et al. 1998a,
b; Walshe et al. 1998). To control illegal treatments, it was essential to establish reliable analytical methods. A wide variety of procedures had been described for the determination of anabolic residue in tissue, faeces, urine, feed, and plasma (Sterk et al. 1998; Draisci et al. 1998). The European Communities had recommended several methods for the detection of hormonally active compounds, including high performance liquid chromatography (HPLC) (Zhou et al. 2005), high performance liquid chromatography-mass spectrometry (HPLC-MS) (Bean et al. 1997; Coldham et al. 1998; Barron et al. 1996), gas chromatography (GC) (Durant et al. 2002), gas chromatography-mass
This paper is translated from its Chinese version in Scientia Agricultura Sinica. Correspondence ZENG Dong-ping, Ph D, Tel: +86-20-85280237, E-mail: [email protected]
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(09)60098-5
Multi-Residue Determination of Eight Anabolic Steroids by GC-MS in Muscle Tissues from Pigs
spectrometry (GC-MS) (Casademont et al. 1996; Hartmann et al. 1997; Daeseleire et al. 1998; Dubois et al. 1998; Marchand et al. 2000; Promberger et al. 2001; Kuuranne and Steinhart 2003; Stolker et al. 2004), and immunoassay techniques such as enzyme linked immunosorbent assay (ELISA) had been developed for several compounds (Fitzpatrick et al. 2004). The aim of this study was to develop a GC-MS multiresidue method to confirm the presence of eight anabolic steroids (diethylstilbestrol, methyltestosterone, norethindrone, 17α-ethynylestradiol, estradiol, 6α-methyl17α-hydroxy-progesterone, estradiol benzoate, and chlormadinone acetate) after conversion of the heptafluorobutyric acid (HFBA) derivatives in muscle tissue.
307
prepared in methanol and stored at -20°C. Working solutions, which were prepared by a series of ten-fold dilutions of the stock solutions, were stored in the dark at approximately 4°C for a maximum period of 6 mon. Methanol-water solutions were prepared with methanol:water (4:1, v/v). The elution solutions were prepared with chloroform:acetone (4:1, v/v). The heptaffuorobutyril (HFB) derivatives solutions were prepared with HFBA:acetone (4:1, v/v).
Samples
MATERIALS AND METHODS
Samples of swine meat were collected and kept frozen (-20°C) until analysis by Animal and Poultry Products Quality Control Inspection and Testing Center (Guangzhou), Ministry of Agriculture, China.
Materials, chemicals and reagents
Methods
The synthetic steroids used in this study were methyltestosterone, estradiol and estradiol benzoate, from National Institute for the Control of Pharmaceutical and Biological Products, China. Diethylstilbestrol, norethindrone, 17α-ethynylestradiol, 6α-methyl-17αhydroxy-progesterone, and chlormadinone acetate were from Sigma-Aldrich Chemie (Guangzhou, China) and the HFBA anhydride from Supelco (Bellefonte, USA). Aether, methanol, petroleum ether II, chloroform, Nhexane, acetone, sodium acetate, and acetic acid of A.R. were from Guangzhou Chemical Reagent Factory, China.
The method of analysis was based on liquid-liquid extraction of the steroids, sample cleaned-up by solid phase extraction, dramatized with HFBA, and GC-MS analysis.
Liquid-liquid extraction
The GC-MS system was from Autosystem XLTurbomass PerkinElmer, USA. The solid phase extraction element from WAT058833 Waters, USA. The nitrogen evaporator model N-EVAPTM 111 from Organomation Associates, Inc., USA. The table centrifuge model Centrifuge 5 804 from Eppendorf, Germany. The solid phase extraction column model Si-SPE (500 mg per 3 mL), NH2-SPE (500 per mg 3 mL), and C18SPE (500 mg per 3 mL) from Supelco, USA.
A test sample of 50-100 g was thoroughly homogenized and a test portion of 5.0 g was weighed into a 50 mL centrifuge tube, standing for 1 min (Stolker et al. 2004). After addition of 10 mL ether (vortexed at 2 000 r/min for 1 min and centrifuged for 10 min at 7 000 r/min) (Wan et al. 2004), the mixture was evaporated in a water bath under a stream of nitrogen at 40°C. After the addition of 4 mL methanol : water (4:1, v/v) and 4 mL petroleum ether II (vortexed at 2 000 r/min for 1 min and centrifuged for 10 min at 3 000 r/min), the mixture was evaporated in a water bath under a stream of nitrogen at 55°C to a final volume of approximately 0.5 mL. After the addition of 2 mL of water, the mixture was extracted 4 mL ether. The extract was evaporated in a water bath under a stream of nitrogen at 40°C. The residue was dissolved in 0.5 mL chloroform and 5 mL hexane.
Solution preparation
Solid phase extraction
Stock solutions of 1 000 g mL-1 of the steroids were
A Si-SPE cartridge was conditioned with 5 mL hexane.
Equipments
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The aqueous extract was applied to the cartridge; the flask and cartridge were rinsed with 2.5 mL hexane. Then the NH 2-SPE cartridge were combined on the bottom of Si-SPE, conditioned with 5 mL hexane, then the cartridge was rinsed with 5 mL chloroform:acetone (4:1, v/v), the eluents were combined in a 10-mL tapered glass tube. The solvent was removed under a stream of nitrogen at 60°C.
Preparation of HFB derivatives After the residue was drying 10 min at 60°C, 50 L HFBA:acetone (1:4, v/v) was added. The vial was vortexed and incubated for 1 h at 60°C. Next, the reaction mixture was dissolved in 700 L methylbenzene, then 500 L ddH2O was added. The mixture was vortexed at 2 000 r/min for 1 min, centrifuged for 10 min at 3 000 r/min. The final solvent upper 600 L was injected into the GC-MS system (Impens et al. 2002).
Gas chromatography-mass spectrometry Chromatographic conditions: Injections were carried out in the splitless mode. In both cases, the carrier gas was high-purity helium at a flow rate of 1.0 mL min-1. The injector temperature was maintained at 280°C. The initial temperature of the column was 120°C and was programmed from 120°C (held for 2 min) to 250°C at 15°C min-1 (held 2 min) and then to 280°C at 4°C min-1 (held for 5 min). The injection volume was 2 L. Mass parameters, selected monitoring mode; electronic impact ionization; electron energy, 70 eV; ion source temperature, 200°C; emission current, 200 A; detector voltage, 524 V; interface temperature, 280°C.
Validation of method To calculate the accuracy and precision of the method, a meat sample (approx. 300 g) was homogenized thoroughly. Seven samples (5 g each) were prepared in a 50 mL centrifuge tube with addition of 100 mL a steroid standard (containing 0, 2.5, 5, 10, 20, 40, and 50 g kg-1). All the spiked samples were dealt with above method. The linearity of the detection was calculated between the concentrations and the peak ratio.
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Each treatment had four replicates.
Recovery Recovery studies were performed on sample spikes at 0, 2.5, 10, and 50 g kg-1 level. The values were compared with the ratios obtained for a reference standard solution containing amounts of substances corresponding (n = 6). Each treatment had four replicates.
Detection and quantification The limit of detection was determined by analyzing sample fortified with increasing concentrations of the compounds (0, 1, 2, 4, and 5 g kg-1 for muscle tissue). A calibration curve was calculated and the limit of detection was calculated as 3 Sb/m, the limit of quantification was calculated as 10 Sb/m, where Sb is the standard deviation of the intercept on the y-axis and m is the slope of the calibration curve.
RESULTS AND DISCUSSION Chromatogram The derivatization method, carried out as above mentioned, is very easy to perform. It results in excellent derivatives of the steroids which are separated well in the gas chromatogram (Fig.1). The mass of the selected ions are presented in Table 1. GC-MS selectedion monitoring chromatograms of HFB-derivative for anabolic steroids are presented in Fig.2. GC-MS selected-ion monitoring chromatograms of HFB-derivative for control pork are presented in Fig.3. GC-MS selected-ion monitoring chromatograms of HFB-derivative for anabolic steroids in pork are presented in Fig.4. The linearity of the detection (r > 0.97) was checked as being between 2.5 and 50 g kg-1. The range of the recoveries was 78.5-148% for diethylstilbestrol, 70.8-109% for methyltestosterone, 69.8-87.2% for norethindrone, 67.7-120% for 17αethynylestradiol, 82.8-103% for estradiol, 70.3-99.2% for 6α-methyl-17α-hydroxy-progesteron-e, 73.0-104% for estradiol benzoate, and 72.9-91.8% for chlormadinone acetate. The range of the coefficients of variation within
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Multi-Residue Determination of Eight Anabolic Steroids by GC-MS in Muscle Tissues from Pigs
Time (min)
Fig. 1 GC-MS full ion chromatogram of HFB-derivative for anabolic steroids (12.5 g mL -1 ). 1, cis-diethylstilbestrol; 2, transdiethylstilbestrol; 3, methyltestosterone; 4, norethindrone; 5, 17αethynylestradiol; 6, estradiol; 7, 6α-methyl-17α-hydroxyprogesterone; 8, estradiol benzoate; 9, chlormadinone acetate; retention time (17.00-20.00 min), column bleeding.
Table 1 Selected ions of the HFB derivatives Compound Diethylstilbestrol Methyltestosterone Norethindrone 17α-Ethynylestradiol Estradiol 6α-Methyl-17α-hydroxy-progesterone Estradiol benzoate Chlormadinone acetate
Relative intensity
Time (min)
m/z of selected ions 303, 341, 417, 355, 369, 465, 143, 461, 476, 115, 353, 446, 169, 356, 409, 133, 383, 479, 146, 540, 541 461, 497, 540
447 480 477 474 451 480
batches was 0.4-12%; the range of the coefficients of variation within batches was 6.4-11%. The limit of detections and the detection capability were 0.99 and 3.30 g kg-1 for diethylstilbestrol, 1.05 and 3.50 g kg-1 for methyltestosterone, 1.19 and 3.97 g kg-1 for norethindrone, 0.94 and 3.13 g kg-1 for 17α-ethynylestradiol, 1.45 and 4.83 g kg-1 for estradiol, 1.56 and 5.20 g kg-1 for 6α-methyl-17α-hydroxyprogesterone, 1.92 and 6.40 g kg -1 for estradiol benzoate, and 2.41 and 8.03 g kg-1 for chlormadinone acetate, respectively.
Sample preparation The sample preparation of the anabolic steroids were developed quite fast in the 1990s, a reliable, generally accepted isolation and purification procedure for the multi-residue determination of anabolic in urine and tissues of slaughtered animal was developed for TLC detection by Verbeke (1979). The advantage of this ref-
Time (min)
Fig. 2 GC-MS selected-ion monitoring chromatograms of HFBderivative for anabolic steroids (12.5 g mL-1). Retention time (min)1, cis-diethylstilbestro (9.58); 2, trans-diethylstilbestrol (10.34); 3, methyltestosterone (11.77); 4, norethindrone (12.21); 5, 17α-ethynylestradiol (12.61); 6, estradiol (13.43); 7, 6α-methyl17α-hydroxy-progesterone (15.43); 8, estradiol benzoate (16.62); 9, chlormadinone acetate (21.28). The same as below.
erence method is its applicability to androgenic, progestogenic, and estrogenic compounds with steroid or stilbene structrure. Disadvantageous are the tediousness of the extraction and clean-up procedure (2.5 d) and the amount of chemicals required (more than 900 mL solvents per sample, including toxicologically problematic ones like toluene and methylene chloride). To overcome some of the disadvantages, improved methods had been proposed by several authors who replaced the time-consuming self-preparation of chromatograph columns by the use of disposable solid-phase extraction (SPE) cartridges (Draisci et al. 2003). In the analysis of anabolic in urine, SPE-based methods were routinely used. The analysis of animal tissues seemed to be more difficult, however, so mostly combinations of liquid-liquid and solid-phase extraction methods were applied. Some authors proposed a
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Time (min)
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Time (min)
Time (min)
Time (min)
Fig. 3 GC-MS selected-ion monitoring chromatograms of HFBderivative for control pork. Retention time was not interfered with the blank samples.
Fig. 4 GC-MS selected-ion monitoring chromatograms of HFBderivative for anabolic steroids in pork.
combination of SPE and HPLC (Van et al. 2002). Another elegant solution was the clean-up with immunoaffinity solid phases. This approach was limited to a small number of steroid hormones, though, so it did not allow the determination of comprehensive hormone patterns (Barron et al. 1996). The aim of this study was to develop a rapid, economical, and reliable sample preparation which showed a wide applicability methods for the determination in meat, based on the combinations of liquid-liquid and solid-phase extraction methods by Hartmann and Steinhart (1997). More application of the revised European Union (EU) criteria was used for confirmation of anabolic steroids (Dubois et al. 1998; Daeseleire et al. 1998;Stolker et al. 2004 ).
Derivatization of steroids We chose a universally applicable derivatization method which was especially suitable for anabolic steroids. With addition of the catalyst HFBA to the derivatization reagent HFB, it resulted in excellent derivatives of the steroids which were separated well in the gas chromatogram. We also chosed the derivatization reagent TMS, BSTFA and so on, but it didn’t result good separation in gas chromatogram. HFBA reacted with 3-oxo groups of methyltestostero n e w i t h f o r m a t i o n o f 3 heptafluorobutyryl enol esters, but some 17hydroxy groups of methyltestosterone were not converted. In brief, the procedure yielded mono-HFB
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Multi-Residue Determination of Eight Anabolic Steroids by GC-MS in Muscle Tissues from Pigs
derivatives for methyltestosterone, while the rest of compounds yielded di-HFB derivatives. The stability of these derivatives was good, but derivatized samples were not kept longer than 3 d.
Selected ions of the HFB derivatives Both identification and quantitation were carried out by selected ion monitoring (SIM) of characteristic ions of the HFB derivatives. Individual scan of HFB derivatives was performed for each investigated anabolic standard to obtain the mass, fragmentation pattern. The most suitable ions (high ion intensity, high mass, and low background) were selected to perform the analysis with SIM mode.
Recovery and sensitivity evaluation
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Acknowledgements
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Analytical recovery of the method was evaluated for each anabolic compound. The above mentioned showed that the range of the recoveries was 78.5-148% for diethylstilbestrol, 70.8-109% for methyltestosterone, 69.8-87.2% for norethindrone, 67.7-120% for 17αethynylestradiol, 82.8-103% for estradiol, 70.3-99.2% for 6α-methyl-17α-hydroxy-progesteron-e, 73.0-104% for estradiol benzoate, and 72.9-91.8% for chlormadinone acetate. The recoveries of the method were more reasonable than the other reports (Daeseleire et al. 1998) which the range was 26-65%. The limit of detections was 0.94-2.41 g kg-1, it was higher than the other repots (Daeseleire et al. 1998) which was 0.1-2.6 g kg-1. The following reason must be concerned, firstly the final volume in our study was 600 L, while the other laboratories were 25 L, secondly the m/z was quite low which must below 600 in our laboratory. So further study must be done in the future.
CONCLUSION In recent years we have developed multi-residue methods for the detection of anabolic steroids in muscle tissue. A few modifications were made so that the methods could be implemented in an accredited routine system.
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