Determination of anabolic steroids in muscle tissue by liquid chromatography–tandem mass spectrometry

Journal of Chromatography A, 1216 (2009) 8072–8079

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Determination of anabolic steroids in muscle tissue by liquid chromatography–tandem mass spectrometry George Kaklamanos a , Georgios Theodoridis b,∗ , Themistoklis Dabalis a a b

Veterinary Laboratory of Serres, Terma Omonoias, 62110, Serres, Greece Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University, 54124, Thessaloniki, Greece

a r t i c l e

i n f o

Article history: Available online 22 April 2009 Keywords: Steroid Anabolic Meat Validation Liquid chromatography–tandem mass spectrometry (LC–MS/MS)

a b s t r a c t A specific and sensitive multi-method based on liquid chromatography–tandem mass spectrometry using atmospheric pressure chemical ionization (LC–APCI–MS/MS) has been developed for the determination of 20 anabolic steroids in muscle tissue (diethylstilbestrol, ␤-estradiol, ethynylestradiol, ␣/␤-boldenone, ␣/␤-nortestosterone, methyltestosterone, ␤-trenbolone, triamcinolone acetonide, dexamethasone, flumethasone, ␣/␤-zearalenol, ␣/␤-zearalanol, zearalenone, melengestrol acetate, megestrol acetate and medroxyprogesterone acetate). The procedure involved hydrolysis, extraction with tert-butyl methyl ether, defattening and final clean-up with solid phase extraction (SPE) on Oasis HLB and Amino cartridges. The analytes were analyzed by reversed-phase LC–MS/MS, in positive and negative multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for the unambiguous confirmation of the hormones. The method was validated at the validation level of 0.5 ng/g. The accuracy and precision of the method were satisfactory. The decision limits CC␣ ranged from 0.03 to 0.14 ng/g while the detection capabilities CC␤ ranged from 0.05 to 0.24 ng/g. The developed method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in muscle tissue and can be used for residue control programs. © 2009 Elsevier B.V. All rights reserved.

1. Introduction A wide range of anabolic steroids has been used in animal fattening because of their capacity to increase weight gain and the improvements in feed conversion efficiency. However, for several years now the use of anabolic steroids in animal fattening is prohibited in the European Community [1] because of their toxic effects on public health. Therefore, monitoring for use of anabolic steroids is carried out through the National Plans of the individual Member States. For controls at retail level and for products imported in the EU, it is necessary to have analytical methods applicable to meat samples. Because of the complexity of the matrix of tissue in animal origin and the low minimum required performance limit (MRPL) levels established, it is necessary to have sensitive, selective and specific methods for the detection of the anabolic steroids, which must be in compliance with the criteria of the Commission Decision 2002/657/EC [2]. Over the years, various analytical procedures have been developed for the efficient clean-up of biological matrices, such as liquid–liquid extraction (LLE), solid phase microextraction (SPME),

∗ Corresponding author. Tel.: +30 2310 997718; fax: +30 2310 997719. E-mail address: [email protected] (G. Theodoridis). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.04.051

solid–liquid extraction (SLE) and solid phase extraction (SPE). In the protocols reported in the literature combinations of the abovementioned analytical procedures are used for the determination of anabolic steroids with satisfactory results. Alternatively a few papers report lipid removal by freezing filtration [3] and HPLC fractionation [4,5], or novel extraction approaches such as accelerated solvent extraction (ASE) or supercritical fluid extraction (SFE) are developed [6,7]. Also, multiresidue techniques such as those employing combination of chromatography and mass spectrometry have been developed for the determination of anabolic steroids in biological samples. Gas chromatography–mass spectrometry (GC–MS) is a sensitive, robust and suitable technique for the assay of hormones, but it is time-consuming because it requires derivatization to reduce analytes polarity and thermal instability and not all steroids are easily derivatized [3–6,8–16,15,17–22,27]. The combination of liquid chromatography with mass spectrometry (LC–MS/MS using electrospray ionization (ESI)) offers a rapid, simplified, specific and sensitive alternative to GC–MS methods involving simpler extraction procedures and removing the need for derivatization reactions [7,23–26,28–38]. For these reasons this technique has gained a wider application field and popularity over the last decade. In general the target specimen is serum, urine meat and hair samples no matter of the chromatographic method applied (GC or

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LC coupled to MS). Muscle tissue is a complex matrix, in which anabolic steroids must be detected at very low concentrations. Therefore, the development of analytical procedures for the determination of anabolic steroids in meat has always been a challenge and (as a result) the methodologies described in literature are usually rather complicated and time-consuming. Procedures reported involve complex processes such as combinations of HPLC fractionation [9], freezing lipid filtration [3] or melting lipid phase [8] several liquid–liquid extraction steps [8,19] with extraction on different SPE cartridges (up to three consecutive SPE steps [3,30]). An interesting approach is that of Le Bizec and co-workers [27] which have reported a methodology that separates the analyte group in four sub-groups following a process of LLE and three SPE steps before derivatization and GC–MS analysis. The scope of the present study was to develop a multi-method coupling liquid chromatography to tandem mass spectrometry for the detection of a big number of hormones from a wide range of chemical groups/families (stilbenes, steroids, corticosteroids, resorcyclic acid lactones, gestagens, etc.) in muscle tissue. In total 20 hormones are determined in bovine meat utilizing atmospheric pressure chemical ionization (APCI) in the positive and negative mode. The sample preparation procedure involved hydrolysis, extraction with tert-butyl methyl ether, defattening and final clean-up with SPE with Oasis HLB and Amino cartridges. The developed methodology gave satisfactory recoveries and clean final extracts. As a whole the method proved to be simple, reliable and reached the required sensitivity. Hence it provides a suitable means for the determination and confirmation of steroid residues in muscle tissue and can be used for residue control programs.

2. Experimental 2.1. Chemicals and reagents Methyltestosterone, medroxyprogesterone acetate, megestrol acetate, melengestrol acetate, ␣-zearalanol, ␤-zearalanol, ␣zearalenol, ␤-zearalenol, zearalenone were purchased from NARL (Pymble, NSW, Australia). ␤-Trenbolone, triamcinolone acetonide, dexamethasone, flumethasone, diethylstilbestrol were purchased from Cerilliant (Promochem, Wesel, Germany). ␤-Estradiol, ethynylestradiol, ␤-boldenone, ␣-boldenone, ␤-nortestosterone, ␣-nortestosterone, were purchased from Sigma (Sigma–Aldrich, Steinhem, Germany). In total seven internal standards were used, namely: 17␤-estradiol-d3, testosterone-d3, ␣/␤-zearalanol-d4, methyltestosterone-d3, megestrol acetate-d3, diethylstilbestrol-d6 and triamcinolone acetonide-d6, which were purchased from RIVM (Bilthoven, The Netherlands). Methanol (HPLC grade) and tris(hydroxymethyl)aminomethane were obtained from Merck (Darmstadt, Germany), TBME, hexane, acetone and protease Subtilisin were from Sigma (Sigma–Aldrich, Steinhem, Germany) and ammonia (25%) was from Panreac (Barcelona, Spain). Tris buffer 0.1 M (pH 9.5) was prepared by dissolving 12.1 g of Tris in 1000 ml of water. Ultrapure water was produced with a Pure Lab system (Sation 9000, Spain). 2% ammonium/water solution was prepared by adding 8 ml ammonium 25% in 92 ml of water. Oasis HLB (60 mg, 3 ml) cartridges were obtained from Waters (Milford, MA, USA) and Amino Supelclean NH2 cartridges from Supelco (Bellenfonte, IL, USA). Stock standard solutions (1 mg/ml) were prepared in methanol and stored at −20 ◦ C in the dark. Working solutions were prepared by appropriate dilution of the stock standard solutions with methanol and were stored at 4 ◦ C in the dark for a maximum period of six months.

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2.2. Samples Muscle samples collected from untreated bovine animals (male and female) at slaughterhouses were used as blank and, after fortification with the different steroids, as quality control samples. Meat samples from bovine animals collected as part of the national program for residue control in Greece, were assayed for the presence of the steroids. The samples were received in frozen condition and were kept frozen (−20 ◦ C) until analysis. 2.3. Instrumentation LC–MS/MS analysis was performed on a ThermoElectron TSQ Quantum AM mass spectrometer equipped with a Finnigan Surveyor MS pump Plus, a Finnigan Surveyor Autosampler plus and a Dell computer system with Xcalibur data acquisition software (ThermoElectron, San Jose, CA, USA). 2.4. LC–MS/MS analyses A reversed-phase Hypersil ODS column (150 mm × 4.6 mm i.d., 5 ␮m; ThermoElectron) was used for the analyses. The mobile phase was composed of deionized water as solvent A and methanol as solvent B. The gradient program used was as follows: 40% methanol as solvent B at the start (t = 0 min), increased linear to 70% (t = 12 min, held for 10 min), increased to 85% (t = 22.10 min, held for 1 min) and equilibrated for 3.5 min at the initial conditions. The flow rate was kept at 0.7 ml/min. Injection volume of 15 ␮l was selected. The ionization of each compound was tested in APCI positive and APCI negative multiple reaction monitoring (MRM) mode. The source conditions were optimized to obtain four identification points (two product ions) for each compound, according to the criteria of the Commission Decision 2002/657/EC. A capillary temperature of 300–360 ◦ C was employed. The nitrogen sheath and auxiliary gas flow rates were set at 10–50 and 0–5 arbitrary units, respectively. Vaporizer temperature was set at 450 ◦ C, the discharge current at 4–8 ␮A. The peak width for quadrupoles Q1 and Q3 was set at 0.70. The collision energy (CE) and tube lens were optimized for each compound (see Section 3.1 and Table 1). 2.5. Sample preparation For each meat sample, a mass of 100 g was homogenized and a test portion of 5.0 g was weighed into a 50 ml centrifuge tube. The mixture of internal standards was added at the concentration of 4 ng/g and was mixed with the test portions at least 30 min prior to the addition of 15 ml of 0.1 M Tris buffer (pH 9.5), containing 5 mg of Subtilisin. The mixture was incubated for 2 h at 52 ◦ C. After cooling down to room temperature, the mixture was extracted twice with 10 ml TBME (10 min rotating and centrifuged at 3000 rpm). The combined extracts were evaporated in a water bath (55 ◦ C) under a stream of nitrogen. After addition of 4 ml methanol/water (4/1, v/v) the mixture was washed twice with 6 and 4 ml of hexane for defattening. The tube was vortexed for 30 s and was subsequently centrifuged for 5 min at 3000 rpm. The hexane layers were decanted. The resulting solution was next evaporated (in a water bath at 55 ◦ C under a nitrogen stream) to reduce its volume to 0.5 ml. After the addition of 3 ml methanol/water (1/9, v/v), the mixture was loaded on an Oasis HLB cartridge, which was previously conditioned with 3 ml of methanol and 3 ml of water. After a washing step with 3 ml 5% methanol in 2% ammonium/water (v/v), 3 ml 40% methanol in 2% ammonium/water (v/v) and 3 ml water, the analytes were eluted with 3 ml of acetone:methanol (80/20, v/v). The extract was then loaded on an Amino cartridge, which was previously conditioned with 3 ml of acetone:methanol (80/20, v/v) and was directly collected. The final extract was evaporated to dryness

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Table 1 Precursor and most abundant product ions and their optimal collision energy. Compound

Rt (min)

Precursor ion [M+H]+

Product ions (m/z) a

CE (eV)

Flumethasone

12.46

411.15

253.192 121.126

25 39

␤-Zearalanol

12.73

323.20

123.154a 149.106

36 31

Dexamethasone

13.18

393.15

237.236a 147.160

30 25

Triamcinolone acetonide

13.32

435.18

213.166a 171.008

34 35

␤-Zearalenol

13.53

321.20

285.379a 267.313

16 21

␤-Trenbolone

13.94

271.17

253.112a 199.109

22 28

␤-Boldenone

14.35

287.20

121.115a 135.253

34 20

␤-Nortestosterone

14.93

275.20

109.128a 239.304

29 21

␣-Zearalanol

14.98

323.20

123.154a 149.106

36 31

␣-Zearalenol

15.47

321.20

285.379a 267.313

16 21

Zearalenone

16.27

319.15

185.109a 187.199

29 30

␣-Boldenone

16.28

287.20

121.115a 135.253

34 20

␣-Nortestosterone

16.73

275.20

109.128a 239.304

29 21

Methyltestosterone

17.81

303.20

109.111a 97.088

22 32

Megestrol acetate

20.35

385.24

224.329a 209.266

30 44

Medroxyprogesterone acetate

21.23

387.24

123.201a 285.477

30 23

Melengestrol acetate

21.93

397.24

279.329a 236.270

23 37

Compound

Rt (min)

Precursor ion [M−H]−

Product ions (m/z)

CE (eV)

Ethynylestradiol

15.45

295.19

145.308a 159.115

36 39

␤-Estradiol

15.47

271.18

145.170a 239.362

39 37

Diethylstilbestrol

15.69

267.15

222.127a 237.137

41 38

a

The most abundant ion (also used for analyte quantification).

in a water bath at 55 ◦ C under a stream of nitrogen. The residue was dissolved in 600 ␮l methanol, transferred to an injection vial, evaporated under a stream of nitrogen at 55 ◦ C to dryness, redissolved in 100 ␮l of methanol and analyzed on LC–MS/MS. The developed procedure for the extraction–purification of the anabolic steroids in meat samples is shown in Fig. 1. 3. Results and discussion 3.1. Liquid chromatography–mass spectrometry conditions The primary scope was the development of an effective methodology for the sensitive and efficient determination of the 20 hormones using LC–MS. The gradient LC program should facilitate separation of matrix constituents from the hormone molecules and also separation between isobaric analytes i.e. the ␤ and the ␣

forms of molecules such as nortestosterone, boldenone, zearalenol and zearalanol. The selected LC gradient accomplished these ends with baseline resolution of isobaric analytes (see Table 1 and Fig. 2 (subfigures A, B, G and H)). Compared to the literature the proposed methodology can separate a much larger number of steroids [24,30,32] to be determined in meat sample. A similar number of steroids (22 analytes) were determined in shorter analytical times by Blasco et al. [36] but with lower detection sensitivity (see also Section 3.3). Acquisition parameters of the mass spectrometer were optimized in ion spray mode by direct continuous pump infusion of standard working solutions of the analytes (10 ng/␮l) at a flow rate of 10 ␮l/min in the mass spectrometer. Data acquisition was performed preliminary on the standard compounds in full scan, to choose an abundant precursor ion [M+H]+ /[M−H]− . In the majority of the published works on this topic, electrospray ionization mode

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Fig. 1. Analytical procedure for the anabolic steroids in meat samples (hydrolysis, LLE, SPE).

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recoveries (ranging from 98% to 100%), in contrast to petroleum ether which gave recovery less than 89% in all analytes. The use of TBME for extraction of steroids has also been reported in previous papers [9,32]. After the extraction of the steroids we must remove the fat that remains in the sample extract. To facilitate this, liquid–liquid extraction procedures were tested. We needed a polar solvent in which the steroids would be soluble and a second nonpolar solvent in which the fat would partition. The choice of the two solvents determines the selectivity and the efficiency of LLE. Different combinations of methanol and acetonitril in water (polar solvent) with hexane and pentane (non-polar solvent) were tested. Mixtures of methanol or acetonitril in water ranging from 20/80 (v/v) to 80/20 (v/v) were tested. Losses for some analytes were observed when the organic content was lower than 50%. Finally a combination of methanol/water (4/1, v/v) with hexane gave a very good separation of the two phases and the highest steroid recoveries. A final SPE step is needed for effective clean-up of the muscle tissue samples. Solid phase extraction cartridges, including Discovery DSC-18 (500 mg, 3 ml, Supelco) and Oasis HLB (60 mg, 3 ml, Waters) were tested. For the washing step different combinations of methanol and water were tested. The best choice was found to be the application of mixtures of methanol/water (5/95, v/v) and methanol/water (40/60, v/v) for both cartridges. Increasing the methanol higher than 45% resulted in reduced recovery of some analytes (␣/␤-boldenone and lactones). This washing was found to enhance clean-up without eluting the steroids. Next the pH value of the washing step was studied; applying alkaline washing resulted in clean chromatograms, without additional interferences from the matrix. For the elution of the steroids methanol, acetonitril, acetone and combinations of them with water were tested. The combinations ranged from 20/80 (v/v) to 80/20 (v/v) of methanol with acetonitril and methanol with acetone. When using the Oasis cartridge the mixture acetone:methanol (80/20, v/v) as the elution solvent provided the highest recovery; for the Discovery DSC-18 cartridge methanol/water (80/20, v/v) provided the best results as the elution solvent. Overall the Oasis cartridge gave better recoveries and more satisfactory peak shapes at the final chromatogram and was thus finally selected for the rest of the study. 3.3. Validation

is applied [7,24–26,28–32,34–36,38]. APCI has been applied in the LC–MS/MS analysis of bovine urine and blood serum, increasing the detection sensitivity [33]. The present work is the first report in the utilization of APCI in LC–MS/MS analysis of steroids in meat. APCI was selected because it provided higher detection sensitivity. In preliminary studies, ESI provided slightly higher sensitivity (compared to APCI) for a small number of analytes (four hormones). However for multiresidue analysis the application of APCI resulted in higher signals for the 16 of the hormones and overall for the whole group of 20 analytes and was for this reason applied for the rest of the study. MS–MS product ion scans were then recorded in full scan. Finally, all the analyses were carried out by multiple reaction monitoring mode monitoring the product ions of the steroids in order to obtain higher detection specificity and sensitivity. Table 1 lists the precursor ions and the product ions of each compound with their optimum selected collision energy. The most abundant product ions were monitored for analyte quantitation. 3.2. Optimization of sample preparation An enzymatic digestion was applied to the muscle samples using Tris buffer and Subtilisin (protease) [9]. For the extraction of the steroids from the muscle tissue, TBME and petroleum ether were tested in different amounts. Extraction with TBME gave higher

In the present work validation of the developed methods is done according to the European Commission Decision 2002/657/EC [2]. Three experiments were performed on three different days, Exp1, Exp2 and Exp3. A homogeneous sample was made and divided in 63 sub-samples. 21 fortified samples were analyzed on each day for three consecutive days. The samples were fortified as follows: 1 sample not spiked (blank), 6 samples spiked at a level of 1* Validation Level (VL) which was set at the minimum required performance limit, 6 samples spiked at a level of 1.5*VL, 6 samples spiked at a level of 2*VL, 1 sample spiked at a level of 3*VL and 1 sample spiked at a level of 5*VL (n = 21). The validation level was 0.5 ng/g for all compounds. For the construction of the calibration curves the area of the selected ion of the analyte and internal standard are calculated and their ratio was used as the response variable. Utilization of a single internal standard was not expected to provide accurate results due to the wide range of molecular properties of analytes undergoing a series of treatments such as SPE and LC–MS analysis. For this reason a total of seven internal standards was used in order to enhance the accuracy of the mass spectrometry based quantitative determination and reduce analytical measurement uncertainties. A calibration curve was constructed by linear curve fitting using least squares linear regression calculation. Nine

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Fig. 2. MRM chromatogram (MS/MS) in APCI of a spiked meat sample containing: (A) ␤/␣-nortestosterone (14.93 and 16.73 min respectively); (B) ␤/␣-boldenone (14.35 and 16.28 min respectively); (C) megestrol acetate; (D) medroxyprogesterone acetate; (E) melengestrol acetate; (F) zearalenone; (G) ␤/␣-zearalenol (13.43 and 15.47 min respectively); (H) ␤/␣-zearalanol (12.73 and 14.98 min respectively); (I) ␤-estradiol; (J) ethynylestradiol; (K) ␤-trenbolone; (L) methyltestosterone; (M) dexamethasone; (N) flumethasone; (O) triamcinolone acetonide and (P) diethylstilbestrol at a concentration of 0.5 ng/g.

points were used for the calibration curve of the standard solutions at concentrations 0, 0.2, 0.5, 1, 1.5, 2, 3, 4 and 5 ng/g with the internal standards at concentration 4 ng/g. This dynamic range is equal or wider than the concentration ranges investigated in the literature [3,9,19,24,30,36]; linearity was good for all analytes in the whole concentration range, as proved by the correlation coefficients (r2 ) which were greater than 0.995 for all curves. Fig. 2 shows the chromatogram of a spiked sample of the steroids, containing the internal standards at a concentration of 4 ng/g in MRM mode. No interference was observed from matrix constituents, ensuring

the unambiguous detection and determination of the target analytes. From the three experiments on three different days the precision and accuracy were determined. Accuracy means the closeness of agreement between a result and the actual (true) value. Often accuracy is determined by measuring trueness and precision. In chemical analysis trueness can only be determined by assaying a certified reference material (CRM). If no CRM is available, the recovery can be determined, which means the percentage of the true concentration of a substance recovered during the analytical

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Table 2 Precision and accuracy data for the steroids obtained from the analysis of spiked meat samples on Exp1/2/3. Compound

Repeatability Exp1 Spiked (ng/g)

Exp2 Accuracy (%)

CV (%)

Accuracy (%)

Exp3 CV (%)

Accuracy (%)

CV (%)

Flumethasone

0.5 0.75 1

90.2 93.1 94.2

5.8 7.2 4.9

91.5 95.1 105.0

5.6 8.4 3.1

94.9 97.0 103.9

2.8 6.5 6.2

␤-Zearalanol

0.5 0.75 1

105.2 95.0 96.8

4.6 2.6 19.2

91.5 83.4 78.9

9.5 12.5 5.8

107.7 96.0 93.1

7.5 9.8 5.9

Dexamethasone

0.5 0.75 1

73.4 82.1 88.6

9.7 6.2 8.9

87.0 95.9 104.3

7.0 8.0 7.0

94.2 94.5 101.6

3.3 4.9 7.2

Triamcinolone acetonide

0.5 0.75 1

83.0 89.3 91.6

6.5 6.1 8.1

86.0 92.6 99.4

11.7 10.1 5.8

106.2 105.0 106.0

7.4 5.4 3.6

␤-Zearalenol

0.5 0.75 1

93.4 83.2 83.4

13.0 13.3 6.7

92.7 73.3 73.8

9.7 10.1 10.2

103.1 84.3 75.4

16.3 16.7 5.6

␤-Trenbolone

0.5 0.75 1

94.3 100.7 93.9

9.0 5.6 3.5

85.4 85.1 89.6

10.5 7.0 9.1

108.5 105.6 104.3

5.2 5.2 7.6

␤-Boldenone

0.5 0.75 1

101.4 97.2 97.0

3.7 8.0 1.9

110.9 104.1 100.4

6.3 3.1 6.1

112.5 111.8 106.0

4.6 4.7 3.6

␤-Nortestosterone

0.5 0.75 1

74.6 86.3 87.9

9.3 7.3 2.9

99.5 94.4 101.1

6.2 4.9 4.9

105.0 102.5 108.1

4.7 3.1 6.9

␣-Zearalenol

0.5 0.75 1

89.0 82.3 74.5

9.4 5.7 6.2

83.7 76.3 79.9

6.7 11.5 5.9

100.2 89.2 75.1

8.5 19.7 7.3

Zearalenone

0.5 0.75 1

116.5 102.6 108.5

21.2 16.5 10.5

99.6 115.2 101.5

3.2 9.8 8.8

101.9 111.0 114.5

4.8 10.3 11.5

␣-Boldenone

0.5 0.75 1

83.7 97.2 92.6

6.1 4.7 6.4

108.5 97.5 104.5

11.9 4.6 10.8

119.4 113.0 117.2

2.6 4.4 6.8

␣-Nortestosterone

0.5 0.75 1

86.0 101.1 101.5

6.0 4.6 2.7

110.2 108.0 109.5

7.2 6.1 5.4

112.6 101.6 105.8

5.9 2.1 4.7

Methyltestosterone

0.5 0.75 1

88.0 91.9 96.1

5.4 5.7 3.2

86.3 92.1 94.8

6.7 6.1 2.0

99.9 97.7 101.4

3.1 2.5 2.2

Megestrol acetate

0.5 0.75 1

98.1 90.2 86.6

3.1 2.1 2.3

96.5 91.6 92.2

4.3 2.9 4.3

98.9 98.0 97.5

3.5 2.0 2.5

Medroxyprogesterone acetate

0.5 0.75 1

71.5 72.3 70.1

13.7 6.6 7.7

73.3 70.4 74.5

14.7 5.2 5.6

76.7 75.8 76.5

3.9 5.0 8.0

Melengestrol acetate

0.5 0.75 1

95.0 82.3 80.5

8.4 4.3 2.4

90.5 87.1 83.8

5.3 3.2 4.4

99.4 91.9 88.0

3.9 3.7 4.1

Ethynylestradiol

0.5 0.75 1

79.7 78.1 86.6

6.4 11.5 4.4

72.1 74.6 78.3

8.2 8.6 7.8

85.3 93.6 89.8

8.2 13.2 6.8

␤-Estradiol

0.5 0.75 1

75.3 75.1 87.5

17.1 7.6 7.3

76.7 77.4 83.4

7.8 8.7 8.2

97.2 101.8 98.6

7.5 5.6 8.4

Diethylstilbestrol

0.5 0.75 1

107.2 105.5 118.0

14.9 7.4 7.3

119.0 105.5 118.0

6.1 7.4 7.3

96.7 101.0 99.6

7.3 12.7 3.7

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procedure. In the present study the recovery of the 20 hormones (three concentration levels tested) ranged from 78.7% to 119.4% (see Table 2). The coefficient of variation (CV) of the quantitative results (20 hormones, three concentration levels) ranged from 1.9% to 14.1% as shown in Table 2. These results indicated that the method has acceptable precision and satisfactory recoveries to be used on a routine basis. Next the specificity and ruggedness of the method was checked on a separate experiment (Exp4). Firstly, 20 different blank meat samples were analyzed to look for possible matrix interferences. No interfering ions and no false positive results were detected. Secondly, the same blank meat samples (20) were spiked at 1*VL, and the samples were assayed. No false negative results were observed and no significant matrix interferences were observed indicating that the specificity of the method is acceptable. In accordance with 2002/657/EC the decision limit (CC␣) is used instead of the detection limit and the detection capability (CC␤) is used instead of the limit of quantitation. CC␣ is the lowest concentration level of the analyte that can be detected in a sample with a chance of 1% of a false positive decision. The detection capability CC␤ is the smallest content of the analyte that may be detected, identified and/or quantified in a sample with an error probability less than or equal to 5%. From the calibration curves constructed for the spiked samples at the three experiments the values of the decision limits and detection capabilities for all analytes were calculated as shown in Table 3. This was further verified at 0.2 ng/g (0.4 VL). All analytes were clearly detected with a S/N ratio higher than 15 and were successfully confirmed using the ion ratios of the two product ions. The high S/N values give the evidence that detection and confirmation is possible near the CC␤ values. Compared to values reported in the literature for a similar number of analytes [36], the calculated CC␣ and CC␤ values are equal or (in most cases) lower, indicating high sensitivity of the reported methodology. For further validation the uncertainty of measurement for all compounds was calculated. The most important variables contributing to the uncertainty of measurement were the repeatability and the matrix effects. The matrix effect is determined by subtracting the repeatability of experiment 4 with the reproducibility variance of experiments 1–3. The calculated expanded uncertainties as a relative value (U%) for the anabolic steroids are shown in Table 4. The possible uncertainties from the preparation of the stock solutions (weighing/volumetric flask/chemical purity of the analyte Table 3 Calculated CC␣ and CC␤ for the analytes. Compound

CC␣ (ng/g)

CC␤ (ng/g)

Methyltestosterone Melengestrol acetate Megestrol acetate Medroxyprogesterone acetate ␣-Zearalanol ␤-Zearalanol ␣-Zearalenol ␤-Zearalenol zearalenone ␤-Trenbolone Triamcinolone acetonide Dexamethasone Flumethasone ␤-Boldenone ␣-Boldenone ␤-Nortestosterone ␣-Nortestosterone diethylstilbestrol ␤-Estradiol ethynylestradiol

0.03 0.06 0.05 0.08 0.09 0.09 0.14 0.09 0.11 0.09 0.06 0.06 0.05 0.05 0.07 0.07 0.09 0.08 0.07 0.10

0.05 0.10 0.08 0.13 0.15 0.15 0.24 0.15 0.18 0.15 0.10 0.10 0.08 0.09 0.13 0.12 0.16 0.14 0.12 0.17

Table 4 Expanded uncertainties (U%) for the analytes. Compound

U%

Methyltestosterone Melengestrol acetate Megestrol acetate Medroxyprogesterone acetate ␣-Zearalanol ␤-Zearalanol ␣-Zearalenol ␤-Zearalenol Zearalenone ␤-Trenbolone Triamcinolone acetonide Dexamethasone Flumethasone ␤-Boldenone ␣-Boldenone ␤-Nortestosterone ␣-Nortestosterone Diethylstilbestrol ␤-Estradiol Ethynylestradiol

11.67 11.23 8.98 14.02 25.40 22.95 26.06 25.40 30.95 23.02 22.06 19.40 12.30 14.60 28.77 24.95 18.08 24.56 30.26 29.05

standards and solvents), the pipettes and the construction of the calibration curves were investigated. The results showed that the combined standard uncertainties were not significant to the combined standard uncertainty from the reproducibility of the method. In accordance with the Commission Decision 2002/657/EC a sample can be confirmed as positive when the following criteria are met: (1) the relative retention time of the analyte (RRT) should correspond to that of the standard analyte, from a spiked sample, with a tolerance of ±2.5%, and (2) the relative intensities of the detected ions, expressed as a percentage of the intensity of the most intense ion, must correspond to those of the reference analyte, either from calibration standards or from incurred samples, at comparative concentrations and measured under the same condition, within the needed tolerances. The ion ratios of the two product ions (relative intensities >50%) of each analyte, signal 2/signal 1 (most abundant), must not exceed the tolerance of ±20%. All criteria were fulfilled for the analysis of the spiked meat samples with ion ratios ranging from 0.52 to 0.85 and not exceeding the tolerance of 20%. 3.4. Real samples analysis The developed method was applied to the analysis of 300 meat samples. These samples had been collected from veterinary directories of the Greek Ministry of Rural Development and Food in which our Laboratory is the National Reference Laboratory. All samples were processed according to the method described. The samples were analyzed and found not containing any of the monitored steroids. 4. Conclusions The aim of this work was to develop a specific, sensitive and reliable multi-method for the determination and confirmation of 20 anabolic steroids of various chemical sub-categories in bovine muscle. The analytical method has proven to be highly specific and sensitive facilitating efficient detection and determination of a wide range of hormone groups/families (stilbenes, steroids, corticosteroids, resorcyclic acid lactones, gestagens, etc.) with no interference from matrix constituents thus improving the efficiency and applicability of the method. Data obtained showed satisfactory precision and accuracy and results were validated and confirmed, according to the criteria of the European Commission Decision. Results did not exceed the needed tolerances confirming to the unambiguous detection of the analytes in the tested samples. Hence

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