Journal of Pharmaceutical and Biomedical Analysis 111 (2015) 138–146
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Determination of anabolic–androgenic steroid adulterants in counterfeit drugs by UHPLC–MS/MS So-Hyun Cho 1 , Hyoung Joon Park 1 , Ji Hyun Lee, Jung-Ah Do, Seok Heo, Jeong Hwa Jo, Sooyeul Cho ∗ Advanced Analysis Team, Toxicological Evaluation and Research Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Cheongju-si, Chungcheongbuk-do 363-700, Republic of Korea
a r t i c l e
i n f o
Article history: Received 16 January 2015 Received in revised form 17 March 2015 Accepted 19 March 2015 Available online 28 March 2015 Keywords: Anabolic–androgenic steroids Counterfeit drug UHPLC–MS/MS Adulterants Validation
a b s t r a c t Anabolic–androgenic steroids (AASs) have been illegally used in counterfeit drugs to improve the performance of athletes. In addition, AASs have been used for cosmetic purpose by non-athletes. To determine the presence of 26 AASs, an analysis method using ultra-liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) was developed and validated. The validated method was applied to 19 counterfeit drugs collected from the Internet and off-line markets during 2014. Nearly 50% (9/19) of the samples contained one of these 26 AASs. In addition, the concentration ranges of the AASs ranged from 0.09 to 119,228.57 mg/kg in the suspected samples. The determined AASs primarily consisted of testosterone and testosterone 17-propionate (26%) followed by boldenone (21%). These results indicate the adulteration of over-the-counter counterfeit drugs, and the continuous monitoring of counterfeit drugs or dubious dietary supplements containing anabolic steroids is warranted. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Anabolic–androgenic steroids (AASs) increase protein synthesis and build-up of muscle tissue. With increasing interest in aesthetic and strength gain, the use AASs has increased [1,2]. Despite the use of AASs being banned by the International Olympic Committee (IOC) in 1976, AAS use obtained through the black market by athletes has not declined [3]. According to the World Anti-Doping Agency (WADA), AASs are the most abused drugs in competitive sport [4]. More seriously, most AASs are used by non-athletes for cosmetic purposes, and most of the AASs are obtained on the black market [1,5,6]. AASs are typically produced in underground laboratories and distributed illegally through black market trade [7,8]. These counterfeit drugs contain the wrong ingredients and/or high level of contaminants and impurities where the source and/or identity of these adulterants are fraudulently and deliberately mislabeled [9]. The abuse of these AAS products may be potentially induced by adverse psychological effects, such as aggression, irritability, and personality disturbances in unsuspecting consumers [10,11].
∗ Corresponding author. Tel.: +82 43 719 5302; fax: +82 43 719 5300. E-mail address: [email protected] (S. Cho). 1 These authors contributed equally to the writing of this manuscript. http://dx.doi.org/10.1016/j.jpba.2015.03.018 0731-7085/© 2015 Elsevier B.V. All rights reserved.
Currently, these counterfeit drugs and dietary supplements with AASs are easily available through online markets or health clubs, which has resulted in an expansion in the use of these products [5,12,13]. Many people are at risk for potential adverse effects from the abuse of AASs [14]. Therefore, with an increasing use and number of clandestinely manufactured counterfeit drugs and dietary supplements, the control and regulation of illegal AAS products is necessary for public health and safety [5,12]. Various methods for the analysis of AASs in counterfeit products have been previously reported. The use of gaschromatography–mass spectrometry (GC–MS) to determine the presence of AASs in products obtained from the black market was reported by Ritsch and Musshoff [15] and Pellegrini et al. [16]. Poucke et al. [12] proposed a determination method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for screening for the presence of 49 AASs in dietary supplements. In addition, the identification of 17 AASs in confiscated drugs was performed by LC–MS/MS and GC/MS with a nitrogen–phosphorus specific detection (NPD) [17] and a LC with a photodiode array UV detector (PDA), and GC/MS was used for analysis of AASs in 76 black market drugs [18]. The determination of 21 AASs by atmospheric solids analysis probe (ASAP) mass spectrometry was introduced by Doue et al. [19]. In this study, simultaneous determination using ultra-liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS)
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Table 1 LC–MS/MS MRM transition parameters for the detection of 26 steroid compounds. Compound
Precursor ion (m/z)
Cone voltage (V)
19-Norandrostenione
273.16
26
1-Androstendione
287.22
26
Bolasterone
317.03
30
Boldenone
287.16
20
Boldenone (M)
289.20
22
Boldione
285.16
18
Calusterone
317.10
32
Clostebol
323.16
24
Danazol (M)
313.16
28
Fluoxymesterone
337.16
32
Formebolone (M)
347.22
17
Methylnortestosterone
289.22
28
Metenolone
303.29
30
Methandienone (M)
317.16
10
Nandrolone
275.16
24
Nandrolone (M1)
277.14
12
Nandrolone (M2)
277.09
12
Norbolethone
317.22
26
Norclostebol
309.10
26
Norethandrolone
303.22
26
Oral-turinabol (M)
351.16
12
Nandrolone decanoate
429.06
30
Testosterone
289.25
35
Testosterone 17-valerate
373.24
30
Testosterone 17-propionate
345.22
28
Methandrostenolone
301.09
35
a
Most abundant product ion with the quantification ion shown in bold font.
Product iona (m/z)
Collision energy (eV)
109.15 197.21 255.31 143.13 185.22 203.26 107.29 123.24 121.15 135.21 269.26 105.14 187.22 271.30 121.15 147.21 151.17 123.15 132.93 203.23 131.13 143.12 157.13 105.14 109.15 123.16 105.14 131.20 281.27 147.15 281.26 329.25 109.15 253.28 271.26 131.15 187.14 205.21 121.14 281.30 299.28 109.15 239.24 257.28 145.00 241.18 259.18 145.00 241.18 259.17 109.15 245.28 299.34 117.13 143.12 291.23 109.15 267.31 285.30 147.20 155.11 333.23 239.06 257.07 275.09 96.98 109.00 253.17 97.00 109.00 97.00 109.00 253.15 120.98 149.03
26 18 18 28 20 16 20 34 22 14 10 34 20 12 26 12 14 24 22 16 22 26 30 28 24 30 40 32 22 30 14 15 26 16 14 30 22 16 26 12 8 24 16 14 20 12 8 22 16 8 28 20 16 30 30 16 28 16 16 16 32 8 20 20 20 23 20 20 25 25 20 20 25 25 15
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Fig. 1. Chemical structures and structural formula of the 26 anabolic androgen steroids investigated.
Table 2 Calibration curve parameters and LOQs of 26 steroid compounds (n = 3). Compound
19-Norandrostenione 1-Androstendione Bolasterone Boldenone Boldenone (M) Boldione Calusterone Clostebol Danazol (M) Fluoxymesterone Formebolone (M) Methylnortestosterone Metenolone Methandienone (M) Nandrolone Nandrolone (M1) Nandrolone (M2) Norbolethone Norclostebol Norethandrolone Oral-turinabol (M) Nandrolone decanoate Testosterone Testosterone 17-valerate Testosterone 17-propionate Methandrostenolone
Curve parameters
LOQ (ng/mL)
Slope
Intercept
r
62.06 30.27 62.94 122.20 23.65 180.7 38.75 60.41 97.35 30.43 38.65 157.0 24.36 29.97 52.40 8.934 5.804 67.23 28.51 98.18 3.86 45.21 110.10 411.6 313.1 113.7
−930.5 −638.1 −518.6 −684.8 −671.1 −3327 114.6 −361.0 461.4 −209.1 −71.66 −3888 −172.0 1006 −863.1 −131.5 −120.1 4.731 −16.82 −978.1 218.8 553.5 −3056 14,317 480.7 2625
0.997 0.998 0.999 0.999 0.996 0.997 0.999 0.999 0.999 0.998 1 0.995 0.999 0.997 0.998 0.995 0.997 1 0.999 0.999 0.992 0.994 0.994 0.993 1 0.998
25.0 10.0 3.5 10.0 25.0 1.5 3.0 7.5 10.0 2.5 10.0 7.5 3.0 25.0 2.5 7.5 10.0 7.5 7.5 7.5 25.0 1.0 1.0 0.5 1.0 1.0
was developed and validated for screening for the presence of 26 AASs and quantification of the detected AASs in counterfeit drugs and dietary supplements. In addition, the developed method was applied to real products that are designed to improve muscle mass and strength. To the best of our knowledge, the UHPLC–MS/MS method has not been previously reported for the determination of our specific mixture of 26 AASs. 2. Materials and methods 2.1. Chemicals and standards stock solutions The studied compounds include 19-norandrostenedione, 1androstenedione, bolasterone, boldenone, boldenone metabolite (M), boldione, calusterone, clostebol, danazol M, fluoxymesterone, formebolone M, methylnortestosterone, metenolone, methandienone M, nandrolone, nandrolone M1, nandrolone M2, norbolethone, norclostebol, norethandrolone, oral-turinabol M, nandrolone decanoate, testosterone, testosterone 17-valerate, testosterone 17-propionate, and methandrostenolone were obtained from Steraloids (Newport, RI, USA) and NARL US Pharmacopeia (Rockville, MD, USA). The chemical structures of the 26 standards are shown in Fig. 1. Formic acid and acetonitrile for the HPLC were purchased from Sigma–Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Germany), respectively. Deionized water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). All of the solutions were degassed in a sonication bath for 30 min. Standard stock solutions (1000 g/mL) were prepared in HPLC-grade methanol and stored at 4 ◦ C. The mixtures
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Table 3 Intra- and inter-day assay precision and accuracy for 26 anabolic steroids (n = 3). Conc. (ng/mL)
19-Norandrostenione
1-Androstendione
Bolasterone
Boldenone
Boldenone (M)
Boldione
Calusterone
Clostebol
Danazol (M)
Fluoxymesterone
Formebolone (M)
Methylnortestosterone
Metenolone
Methandienone (M)
Nandrolone
Nandrolone (M1)
Nandrolone (M2)
Norbolethone
Norclostebol
Norethandrolone
Oral-turinabol (M)
Nandrolone decanoate
Testosterone Testosterone 17-valerate Testosterone 17-propionate Methandrostenolone
250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000
Intra-day
Inter-day
Accuracy (%)
Precision (RSD%)
Accuracy (%)
Precision (RSD%)
104.53 96.60 100.57 103.26 97.55 100.41 100.33 99.76 100.05 102.88 97.88 100.38 104.26 96.84 100.55 101.24 99.07 100.16 99.60 100.31 99.96 100.05 99.97 100.01 102.39 98.30 100.34 98.91 100.83 99.87 99.77 100.18 99.98 103.38 97.53 100.45 98.76 100.96 99.86 97.32 102.04 99.68 102.86 97.86 100.36 102.80 97.95 100.38 99.27 100.62 99.95 99.34 100.50 99.92 104.12 96.95 100.53 100.92 99.42 100.17 93.80 104.67 99.24 97.66 101.76 99.71 103.92 97.10 100.51 96.04 102.98 99.51 99.03 100.75 99.89 98.80 100.93 99.86
0.24 2.19 2.72 4.47 3.43 5.80 3.82 3.12 0.28 2.25 4.48 3.24 1.56 3.38 2.93 1.81 2.09 1.08 7.47 4.56 2.94 1.18 3.74 0.16 3.66 2.24 1.07 0.69 1.03 1.94 1.37 1.04 2.35 0.76 0.68 1.98 6.63 4.70 2.40 2.60 0.55 1.94 4.74 3.16 1.34 3.10 4.46 4.99 4.67 1.01 2.91 1.46 1.61 0.84 6.33 7.25 6.01 1.22 1.70 1.60 5.20 3.12 9.60 0.95 0.92 2.48 2.16 1.75 3.32 1.36 0.48 1.10 1.36 0.82 0.73 2.81 2.08 0.80
101.07 99.21 100.14 101.88 98.61 100.24 98.87 100.86 99.87 101.58 98.87 100.23 100.60 99.57 100.08 100.56 99.62 100.09 99.51 100.39 99.95 99.44 100.43 99.94 101.83 98.76 100.29 97.18 102.15 99.66 101.95 98.55 100.25 103.57 97.32 100.45 101.78 98.71 100.24 99.39 100.47 99.93 103.07 97.70 100.39 103.55 97.34 100.45 100.84 99.48 100.16 101.23 99.08 100.15 104.09 96.96 100.52 101.65 98.89 100.27 98.30 101.29 99.79 94.76 103.98 99.37 104.80 96.44 100.62 98.73 100.98 99.85 100.04 99.99 100.02 100.12 100.01 100.07
3.64 2.23 7.47 2.74 3.61 5.36 2.78 6.01 8.59 4.30 5.22 5.73 5.32 4.21 9.28 5.48 5.51 8.35 8.64 6.87 6.83 4.75 4.71 7.44 5.36 7.36 7.16 8.24 5.00 9.26 6.25 8.03 7.13 3.34 8.61 7.82 2.08 8.86 8.29 6.81 7.27 5.49 1.49 7.72 7.95 7.38 5.40 4.43 2.51 9.57 9.66 5.14 7.37 7.47 3.34 4.45 6.52 8.47 5.50 8.47 9.89 8.92 3.15 6.40 7.37 8.49 3.71 9.65 9.43 8.26 8.44 8.89 9.51 9.01 8.84 8.60 9.10 6.77
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Table 4 Recoveries of the 26 steroid compounds spiked in the solid and liquid type samples (n = 3). Conc. (ng/mL)
19-Norandrostenione
1-Androstendione
Bolasterone
Boldenone
Boldenone (M)
Boldione
Calusterone
Clostebol
Danazol (M)
Fluoxymesterone
Formebolone (M)
Methylnortestosterone
Metenolone
Methandienone (M)
Nandrolone
Nandrolone (M1)
Nandrolone (M2)
Norbolethone
Norclostebol
Norethandrolone
Oral-turinabol (M)
Nandrolone decanoate
Testosterone Testosterone 17-valerate Testosterone 17-propionate Methandrostenolone
100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000
Solid
Liquid
Recovery (%)
Precision (RSD%)
Recovery (%)
Precision (RSD%)
111.1 101.0 106.6 102.41 101.13 117.6 92.6 114.6 108.2 84.7 100.2 113.7 97.4 86.0 115.3 83.7 107.8 108.3 94.8 118.8 112.0 87.6 114.3 115.2 112.2 105.2 106.7 116.9 110.3 107.1 110.6 118.0 116.5 88.8 114.2 114.7 119.0 105.0 104.6 111.2 119.2 105.1 112.3 107.6 83.9 92.2 118.3 93.1 89.9 114.0 89.0 117.8 119.6 114.5 81.4 111.7 110.3 102.3 85.1 96.1 112.8 93.2 114.0 89.9 114.7 92.2 111.5 104.9 108.7 86.3 80.9 97.0 86.7 81.7 98.4 119.7 107.4 98.4
14.51 2.63 3.97 14.17 4.24 9.13 14.71 2.17 1.56 4.72 1.23 0.65 13.62 1.80 4.94 6.11 0.98 0.51 13.52 6.18 4.82 5.12 4.41 0.98 5.57 1.96 4.81 8.99 5.12 2.43 6.03 2.30 9.48 7.07 1.70 2.22 2.29 13.22 1.10 3.22 1.40 1.72 5.58 3.96 1.99 11.61 10.66 2.52 13.77 14.79 5.68 8.11 0.14 1.93 3.87 3.00 4.95 1.35 0.54 1.67 10.32 7.38 4.61 2.35 4.48 1.06 3.06 2.08 2.82 0.84 0.26 0.68 0.59 0.85 2.56 7.09 2.67 2.56
119.7 104.0 105.9 98.0 119.8 104.9 119.1 118.6 98.5 114.3 104.8 106.4 82.2 94.5 114.3 109.7 107.7 106.7 110.0 101.1 84.0 90.5 118.0 92.2 99.5 95.7 95.5 93.7 119.7 86.8 116.5 119.7 93.1 85.8 116.9 107.0 110.2 103.1 91.8 116.0 119.5 98.2 118.5 102.5 96.1 107.2 113.1 86.2 114.6 110.2 84.6 102.0 119.2 94.4 111.1 117.1 94.0 109.0 88.3 104.3 113.9 107.7 106.3 108.4 104.3 93.8 96.1 116.9 102.9 91.4 85.5 99.5 91.3 86.4 99.4 117.7 107.6 99.4
4.18 4.35 3.18 12.64 6.32 1.57 11.41 3.63 0.43 1.33 2.14 2.41 5.79 10.78 2.09 6.33 2.70 3.70 9.14 3.33 2.36 5.01 4.17 0.65 4.29 4.69 0.92 11.23 2.18 0.09 3.78 2.18 1.32 14.18 1.75 7.09 8.01 4.09 3.88 6.92 1.39 1.61 13.25 5.03 4.50 10.71 5.66 4.24 12.75 10.97 2.25 5.63 0.22 0.84 13.79 3.54 1.26 3.66 1.30 0.43 12.82 4.19 1.90 3.48 0.76 1.21 7.15 6.03 5.34 1.10 0.43 1.43 0.33 0.90 1.57 4.32 2.78 1.57
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Fig. 2. The extracted parent ion chromatograms of (a) sample spiked standard at LOQ level and (b) matrix-blank sample obtained by LC–MS/MS.
of the working standard solutions (1 g/mL) were prepared daily by diluting each stock solution in methanol. 2.2. Samples and extraction The counterfeit medicines and dietary supplements, which advertised the ability to increase muscle mass and strength, were
obtained from markets or websites. The 19 collected samples consisted of solids tablet (3) and liquid injectables (16). The samples (1 g) were extracted with 50 mL of methanol–distilled water (70:30, v/v) and degassed in a sonication bath for 30 min. The extract was filtered through a 0.22 m polyvinylidene difluoride (PVDF) membrane syringe filter (Millipore, Milford, MA, USA) and analyzed by UHPLC–MS/MS.
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Fig. 3. MRM chromatogram of (a) boldenone standard and (b) counterfeit sample containing boldenone.
2.3. Instrumental conditions The chromatographic system, which consisted of a Waters ACQUITY Ultra Performance Liquid (UPLC; Waters, Milford, MA, USA) with a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 m), was employed to separate the target compounds. The column temperature was maintained at 35 ◦ C, and the flow rate was maintained at 0.25 mL/min. The mobile phase consisted of 0.1% formic acid in water (solvent A,
v/v) and acetonitrile (solvent B, v/v). The initial composition of the mobile phase consisted of 20% solvent B for 3 min. The gradient elution involved a programmed linear gradient of 100% solvent B in 17 min, which was maintained for 3 min. The mobile phase was returned to the initial composition of the gradient program and equilibrated for 4.5 min. The total run time was 25 min, and the injection volume of the sample was 2 L. The mass spectra were recorded on a Waters Xevo TQ (Waters, Milford, MA, USA). The flow rates of the cone (N2 ) and desolvation gases were set to 50
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and 600 L/h, respectively. The capillary voltage was set to 2.7 kV, and the desolvation temperature was 500 ◦ C. All of the compounds were ionized via an electrospray ionization (ESI) source operated in positive mode. The screening of the 26 target compounds was performed at unit resolution in multiple reaction monitoring (MRM) mode. The MRM transitions of the analyte were monitored and selected to determine the optimum conditions. The MRM parameters of the 26 target compounds are shown in Table 1. 2.4. Method validation The method was validated by determining the selectivity, linearity, limit of quantification (LOQ), precision (intra- and inter-day), accuracy, and recovery. Some samples from the 10 sources (2 different tablets and 8 different liquid injectables), which contained none of the detectable target compounds, were used as “matrix-blank samples”. The matrix-blank samples (solid and liquid type) were analyzed to confirm the selectivity and determine the potential interference from intrinsic substances in the target substances. The calibration standard solutions were prepared three times at six concentration levels as follows: 5, 25, 100, 200, 500, and 1000 ng/mL for bolasterone, boldione, calusterone, fluoxumesterone, metenolone, nandrolone, nandrolone decanoate, testosterone, testosterone 17valerate, and testosterone 17-propionate; 10, 50, 100, 200, 500, and 1000 ng/mL for 1-androstendione, boldenone, clostebol, danazol M, formebolone M, methylnortestosterone, nandrolone M1, nandrolone M2, norbolethone, norclostebol and norethandrolone; and 25, 50, 100, 200, 500, and 1000 ng/mL for 19-norandrostenione, boldenone M, methandienone M and oral-turinabol M. The calibration curves were evaluated based on the correlation coefficients (r) of the following equation: y = ax + b (y: peak area, x: concentration, a: slope, b: intercept). Standard solutions at concentration with a signal-to-noise (S/N) ratio of >10 were spiked in the matrixblank samples to calculate the LOQ. The standard solutions spiked at three different concentrations (250, 500, and 1000 ng/mL) were used to determine the precision and accuracy of the method. The intra-day assays were performed by three replicate analyses performed on the same day, and the inter-day assays were repeated in triplicate on three different days. The precision was based on the relative standard deviation (RSD). The accuracy was evaluated by comparing the true concentration spiked into the matrix with the calculated concentration. The recovery was evaluated with blank samples (solid and liquid type) at three different concentrations (i.e., 100, 500, and 1000 ng/mL). The average percent recovery was calculated by comparing the peak area from spiked samples to that from standard solutions at equivalent concentrations. 3. Results and discussion The ESI mass spectra of the 26 target compounds revealed [M+H]+ ions (Q1) in the positive electrospray. The product ions (Q3) were produced by fragment Q1 in the collision cell. Based on the optimized MRM transitions (Table 1), the solutions containing a mixture of the 26 standards were determined by UHPLC/MS/MS system. The most sensitive product ion was used for quantitation of the AASs. The matrix-blank samples and matrix-blank samples spiked standard solutions were extracted and analyzed for selectivity. No interference peak was detected in the eluting positions of the anabolic steroid in matrix-blank chromatograms (Fig. 2). The calibration curve parameters are listed in Table 2. The calibration curves were analyzed in a range of 5–1000 ng/mL for the 26 AASs. The correlation coefficients (r) for the AASs were higher than 0.99. A standard solution mixture at a concentration of 1 g/mL was spiked into the matrix-blank sample, and the serial dilutions of the spiked sample were performed to calculate the LOQs at a signal-to-noise
145
Table 5 Number and concentration range of the detected compounds from the studied samples. Compound
Studied samples
Concentration range (mg/kg)
19-Norandrostenedione Boldenone Boldione Metenolone Nandrolone Methandienone (M) Testosterone Testosterone-17-valerate Testosterone 17-propionate
2 4 2 1 2 1 5 3 5
12.44–54.23 0.09–390.06 10.09–160.12 43.77 199.88–334.06 39.45 11.25–140.25 3.90–10.89 4.24–119228.57
ratio of more than 10. The LOQs for the 26 compounds were determined to be 0.5–25.0 ng/mL (Table 2). The accuracy and precision of the intra and inter-day assays at concentrations of 250, 500, and 1000 ng/mL are summarized in Table 3. The precision was within 9.60%, and the accuracy was determined to be 93.80–104.53% in the intra-batch assays. The precision for the inter-day assay was less than 9.89%. The inter-day assay accuracy was 94.76–104.80%. The results of the intra- and inter-day assay indicated excellent accuracy and reproducibility. In addition, the accuracy and precision data for the intra and inter-day assays met the required limits [20]. The recoveries of the AASs from the solid and liquid matrix-blank samples were 80.9–119.7% and 82.2–119.8%, respectively (Table 4). This validated method was suitable for screening AAS compounds in counterfeit drugs. The validated method was applied to the 19 samples to monitor for the presence of AASs (Fig. 3). Approximately 50% (9/19) of the samples were adulterated with AASs. In addition, some products contained more than two AAS compounds. The compounds detected in the samples are shown in Table 5. Testosterone and testosterone 17-propionate (26%) have the highest percentage among the detected compounds followed by boldenone (21%) and testosterone-17-valerate (16%). In addition, the concentrations of the detected AASs have a wide range from 0.09 (boldenone) to 119,228.57 (testosterone 17-propionate) mg/kg in the samples (Table 5). 4. Conclusions In this study, an UHPLC–MS/MS method was developed and validated to screen for AAS compounds in counterfeit drugs. The counterfeit drugs were obtained from the Korean market in 2014 and analyzed by the validated LC–MS/MS method. Nearly half of the samples were adulterated. 9 AASs (i.e., testosterone 17-propionate, testosterone, boldenone, testosterone 17-valarate, 19-norandrostenedione, boldione, nandrolone, metenolone, and methandienone (M)) were identified in the studied samples. With increasing interest in aesthetic appearance, many people have found that AASs help increase muscle mass and strength. Therefore, the traffickers and distributors of illegal drugs have added AASs, and people are exposed to potential risks from the side effect caused by the AASs. Therefore, continued monitoring of the presence of AASs in counterfeit drugs is essential for public health. Acknowledgments This research was supported by a grant from the Criminal Investigations Office of the Ministry of Food and Drug Safety (MFDS) in 2014. References [1] D.B.D.J. Neves, R.G.A. Marcheti, E.D. Caldas, Incidence of anabolic steroid counterfeiting in Brazil, Forensic Sci. Int. 228 (2013) 81–83.
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Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Determination of anabolic–androgenic steroid adulterants in counterfeit drugs by UHPLC–MS/MS So-Hyun Cho 1 , Hyoung Joon Park 1 , Ji Hyun Lee, Jung-Ah Do, Seok Heo, Jeong Hwa Jo, Sooyeul Cho ∗ Advanced Analysis Team, Toxicological Evaluation and Research Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Cheongju-si, Chungcheongbuk-do 363-700, Republic of Korea
a r t i c l e
i n f o
Article history: Received 16 January 2015 Received in revised form 17 March 2015 Accepted 19 March 2015 Available online 28 March 2015 Keywords: Anabolic–androgenic steroids Counterfeit drug UHPLC–MS/MS Adulterants Validation
a b s t r a c t Anabolic–androgenic steroids (AASs) have been illegally used in counterfeit drugs to improve the performance of athletes. In addition, AASs have been used for cosmetic purpose by non-athletes. To determine the presence of 26 AASs, an analysis method using ultra-liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) was developed and validated. The validated method was applied to 19 counterfeit drugs collected from the Internet and off-line markets during 2014. Nearly 50% (9/19) of the samples contained one of these 26 AASs. In addition, the concentration ranges of the AASs ranged from 0.09 to 119,228.57 mg/kg in the suspected samples. The determined AASs primarily consisted of testosterone and testosterone 17-propionate (26%) followed by boldenone (21%). These results indicate the adulteration of over-the-counter counterfeit drugs, and the continuous monitoring of counterfeit drugs or dubious dietary supplements containing anabolic steroids is warranted. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Anabolic–androgenic steroids (AASs) increase protein synthesis and build-up of muscle tissue. With increasing interest in aesthetic and strength gain, the use AASs has increased [1,2]. Despite the use of AASs being banned by the International Olympic Committee (IOC) in 1976, AAS use obtained through the black market by athletes has not declined [3]. According to the World Anti-Doping Agency (WADA), AASs are the most abused drugs in competitive sport [4]. More seriously, most AASs are used by non-athletes for cosmetic purposes, and most of the AASs are obtained on the black market [1,5,6]. AASs are typically produced in underground laboratories and distributed illegally through black market trade [7,8]. These counterfeit drugs contain the wrong ingredients and/or high level of contaminants and impurities where the source and/or identity of these adulterants are fraudulently and deliberately mislabeled [9]. The abuse of these AAS products may be potentially induced by adverse psychological effects, such as aggression, irritability, and personality disturbances in unsuspecting consumers [10,11].
∗ Corresponding author. Tel.: +82 43 719 5302; fax: +82 43 719 5300. E-mail address: [email protected] (S. Cho). 1 These authors contributed equally to the writing of this manuscript. http://dx.doi.org/10.1016/j.jpba.2015.03.018 0731-7085/© 2015 Elsevier B.V. All rights reserved.
Currently, these counterfeit drugs and dietary supplements with AASs are easily available through online markets or health clubs, which has resulted in an expansion in the use of these products [5,12,13]. Many people are at risk for potential adverse effects from the abuse of AASs [14]. Therefore, with an increasing use and number of clandestinely manufactured counterfeit drugs and dietary supplements, the control and regulation of illegal AAS products is necessary for public health and safety [5,12]. Various methods for the analysis of AASs in counterfeit products have been previously reported. The use of gaschromatography–mass spectrometry (GC–MS) to determine the presence of AASs in products obtained from the black market was reported by Ritsch and Musshoff [15] and Pellegrini et al. [16]. Poucke et al. [12] proposed a determination method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for screening for the presence of 49 AASs in dietary supplements. In addition, the identification of 17 AASs in confiscated drugs was performed by LC–MS/MS and GC/MS with a nitrogen–phosphorus specific detection (NPD) [17] and a LC with a photodiode array UV detector (PDA), and GC/MS was used for analysis of AASs in 76 black market drugs [18]. The determination of 21 AASs by atmospheric solids analysis probe (ASAP) mass spectrometry was introduced by Doue et al. [19]. In this study, simultaneous determination using ultra-liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS)
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139
Table 1 LC–MS/MS MRM transition parameters for the detection of 26 steroid compounds. Compound
Precursor ion (m/z)
Cone voltage (V)
19-Norandrostenione
273.16
26
1-Androstendione
287.22
26
Bolasterone
317.03
30
Boldenone
287.16
20
Boldenone (M)
289.20
22
Boldione
285.16
18
Calusterone
317.10
32
Clostebol
323.16
24
Danazol (M)
313.16
28
Fluoxymesterone
337.16
32
Formebolone (M)
347.22
17
Methylnortestosterone
289.22
28
Metenolone
303.29
30
Methandienone (M)
317.16
10
Nandrolone
275.16
24
Nandrolone (M1)
277.14
12
Nandrolone (M2)
277.09
12
Norbolethone
317.22
26
Norclostebol
309.10
26
Norethandrolone
303.22
26
Oral-turinabol (M)
351.16
12
Nandrolone decanoate
429.06
30
Testosterone
289.25
35
Testosterone 17-valerate
373.24
30
Testosterone 17-propionate
345.22
28
Methandrostenolone
301.09
35
a
Most abundant product ion with the quantification ion shown in bold font.
Product iona (m/z)
Collision energy (eV)
109.15 197.21 255.31 143.13 185.22 203.26 107.29 123.24 121.15 135.21 269.26 105.14 187.22 271.30 121.15 147.21 151.17 123.15 132.93 203.23 131.13 143.12 157.13 105.14 109.15 123.16 105.14 131.20 281.27 147.15 281.26 329.25 109.15 253.28 271.26 131.15 187.14 205.21 121.14 281.30 299.28 109.15 239.24 257.28 145.00 241.18 259.18 145.00 241.18 259.17 109.15 245.28 299.34 117.13 143.12 291.23 109.15 267.31 285.30 147.20 155.11 333.23 239.06 257.07 275.09 96.98 109.00 253.17 97.00 109.00 97.00 109.00 253.15 120.98 149.03
26 18 18 28 20 16 20 34 22 14 10 34 20 12 26 12 14 24 22 16 22 26 30 28 24 30 40 32 22 30 14 15 26 16 14 30 22 16 26 12 8 24 16 14 20 12 8 22 16 8 28 20 16 30 30 16 28 16 16 16 32 8 20 20 20 23 20 20 25 25 20 20 25 25 15
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Fig. 1. Chemical structures and structural formula of the 26 anabolic androgen steroids investigated.
Table 2 Calibration curve parameters and LOQs of 26 steroid compounds (n = 3). Compound
19-Norandrostenione 1-Androstendione Bolasterone Boldenone Boldenone (M) Boldione Calusterone Clostebol Danazol (M) Fluoxymesterone Formebolone (M) Methylnortestosterone Metenolone Methandienone (M) Nandrolone Nandrolone (M1) Nandrolone (M2) Norbolethone Norclostebol Norethandrolone Oral-turinabol (M) Nandrolone decanoate Testosterone Testosterone 17-valerate Testosterone 17-propionate Methandrostenolone
Curve parameters
LOQ (ng/mL)
Slope
Intercept
r
62.06 30.27 62.94 122.20 23.65 180.7 38.75 60.41 97.35 30.43 38.65 157.0 24.36 29.97 52.40 8.934 5.804 67.23 28.51 98.18 3.86 45.21 110.10 411.6 313.1 113.7
−930.5 −638.1 −518.6 −684.8 −671.1 −3327 114.6 −361.0 461.4 −209.1 −71.66 −3888 −172.0 1006 −863.1 −131.5 −120.1 4.731 −16.82 −978.1 218.8 553.5 −3056 14,317 480.7 2625
0.997 0.998 0.999 0.999 0.996 0.997 0.999 0.999 0.999 0.998 1 0.995 0.999 0.997 0.998 0.995 0.997 1 0.999 0.999 0.992 0.994 0.994 0.993 1 0.998
25.0 10.0 3.5 10.0 25.0 1.5 3.0 7.5 10.0 2.5 10.0 7.5 3.0 25.0 2.5 7.5 10.0 7.5 7.5 7.5 25.0 1.0 1.0 0.5 1.0 1.0
was developed and validated for screening for the presence of 26 AASs and quantification of the detected AASs in counterfeit drugs and dietary supplements. In addition, the developed method was applied to real products that are designed to improve muscle mass and strength. To the best of our knowledge, the UHPLC–MS/MS method has not been previously reported for the determination of our specific mixture of 26 AASs. 2. Materials and methods 2.1. Chemicals and standards stock solutions The studied compounds include 19-norandrostenedione, 1androstenedione, bolasterone, boldenone, boldenone metabolite (M), boldione, calusterone, clostebol, danazol M, fluoxymesterone, formebolone M, methylnortestosterone, metenolone, methandienone M, nandrolone, nandrolone M1, nandrolone M2, norbolethone, norclostebol, norethandrolone, oral-turinabol M, nandrolone decanoate, testosterone, testosterone 17-valerate, testosterone 17-propionate, and methandrostenolone were obtained from Steraloids (Newport, RI, USA) and NARL US Pharmacopeia (Rockville, MD, USA). The chemical structures of the 26 standards are shown in Fig. 1. Formic acid and acetonitrile for the HPLC were purchased from Sigma–Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Germany), respectively. Deionized water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). All of the solutions were degassed in a sonication bath for 30 min. Standard stock solutions (1000 g/mL) were prepared in HPLC-grade methanol and stored at 4 ◦ C. The mixtures
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Table 3 Intra- and inter-day assay precision and accuracy for 26 anabolic steroids (n = 3). Conc. (ng/mL)
19-Norandrostenione
1-Androstendione
Bolasterone
Boldenone
Boldenone (M)
Boldione
Calusterone
Clostebol
Danazol (M)
Fluoxymesterone
Formebolone (M)
Methylnortestosterone
Metenolone
Methandienone (M)
Nandrolone
Nandrolone (M1)
Nandrolone (M2)
Norbolethone
Norclostebol
Norethandrolone
Oral-turinabol (M)
Nandrolone decanoate
Testosterone Testosterone 17-valerate Testosterone 17-propionate Methandrostenolone
250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000
Intra-day
Inter-day
Accuracy (%)
Precision (RSD%)
Accuracy (%)
Precision (RSD%)
104.53 96.60 100.57 103.26 97.55 100.41 100.33 99.76 100.05 102.88 97.88 100.38 104.26 96.84 100.55 101.24 99.07 100.16 99.60 100.31 99.96 100.05 99.97 100.01 102.39 98.30 100.34 98.91 100.83 99.87 99.77 100.18 99.98 103.38 97.53 100.45 98.76 100.96 99.86 97.32 102.04 99.68 102.86 97.86 100.36 102.80 97.95 100.38 99.27 100.62 99.95 99.34 100.50 99.92 104.12 96.95 100.53 100.92 99.42 100.17 93.80 104.67 99.24 97.66 101.76 99.71 103.92 97.10 100.51 96.04 102.98 99.51 99.03 100.75 99.89 98.80 100.93 99.86
0.24 2.19 2.72 4.47 3.43 5.80 3.82 3.12 0.28 2.25 4.48 3.24 1.56 3.38 2.93 1.81 2.09 1.08 7.47 4.56 2.94 1.18 3.74 0.16 3.66 2.24 1.07 0.69 1.03 1.94 1.37 1.04 2.35 0.76 0.68 1.98 6.63 4.70 2.40 2.60 0.55 1.94 4.74 3.16 1.34 3.10 4.46 4.99 4.67 1.01 2.91 1.46 1.61 0.84 6.33 7.25 6.01 1.22 1.70 1.60 5.20 3.12 9.60 0.95 0.92 2.48 2.16 1.75 3.32 1.36 0.48 1.10 1.36 0.82 0.73 2.81 2.08 0.80
101.07 99.21 100.14 101.88 98.61 100.24 98.87 100.86 99.87 101.58 98.87 100.23 100.60 99.57 100.08 100.56 99.62 100.09 99.51 100.39 99.95 99.44 100.43 99.94 101.83 98.76 100.29 97.18 102.15 99.66 101.95 98.55 100.25 103.57 97.32 100.45 101.78 98.71 100.24 99.39 100.47 99.93 103.07 97.70 100.39 103.55 97.34 100.45 100.84 99.48 100.16 101.23 99.08 100.15 104.09 96.96 100.52 101.65 98.89 100.27 98.30 101.29 99.79 94.76 103.98 99.37 104.80 96.44 100.62 98.73 100.98 99.85 100.04 99.99 100.02 100.12 100.01 100.07
3.64 2.23 7.47 2.74 3.61 5.36 2.78 6.01 8.59 4.30 5.22 5.73 5.32 4.21 9.28 5.48 5.51 8.35 8.64 6.87 6.83 4.75 4.71 7.44 5.36 7.36 7.16 8.24 5.00 9.26 6.25 8.03 7.13 3.34 8.61 7.82 2.08 8.86 8.29 6.81 7.27 5.49 1.49 7.72 7.95 7.38 5.40 4.43 2.51 9.57 9.66 5.14 7.37 7.47 3.34 4.45 6.52 8.47 5.50 8.47 9.89 8.92 3.15 6.40 7.37 8.49 3.71 9.65 9.43 8.26 8.44 8.89 9.51 9.01 8.84 8.60 9.10 6.77
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Table 4 Recoveries of the 26 steroid compounds spiked in the solid and liquid type samples (n = 3). Conc. (ng/mL)
19-Norandrostenione
1-Androstendione
Bolasterone
Boldenone
Boldenone (M)
Boldione
Calusterone
Clostebol
Danazol (M)
Fluoxymesterone
Formebolone (M)
Methylnortestosterone
Metenolone
Methandienone (M)
Nandrolone
Nandrolone (M1)
Nandrolone (M2)
Norbolethone
Norclostebol
Norethandrolone
Oral-turinabol (M)
Nandrolone decanoate
Testosterone Testosterone 17-valerate Testosterone 17-propionate Methandrostenolone
100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000 100 500 1000
Solid
Liquid
Recovery (%)
Precision (RSD%)
Recovery (%)
Precision (RSD%)
111.1 101.0 106.6 102.41 101.13 117.6 92.6 114.6 108.2 84.7 100.2 113.7 97.4 86.0 115.3 83.7 107.8 108.3 94.8 118.8 112.0 87.6 114.3 115.2 112.2 105.2 106.7 116.9 110.3 107.1 110.6 118.0 116.5 88.8 114.2 114.7 119.0 105.0 104.6 111.2 119.2 105.1 112.3 107.6 83.9 92.2 118.3 93.1 89.9 114.0 89.0 117.8 119.6 114.5 81.4 111.7 110.3 102.3 85.1 96.1 112.8 93.2 114.0 89.9 114.7 92.2 111.5 104.9 108.7 86.3 80.9 97.0 86.7 81.7 98.4 119.7 107.4 98.4
14.51 2.63 3.97 14.17 4.24 9.13 14.71 2.17 1.56 4.72 1.23 0.65 13.62 1.80 4.94 6.11 0.98 0.51 13.52 6.18 4.82 5.12 4.41 0.98 5.57 1.96 4.81 8.99 5.12 2.43 6.03 2.30 9.48 7.07 1.70 2.22 2.29 13.22 1.10 3.22 1.40 1.72 5.58 3.96 1.99 11.61 10.66 2.52 13.77 14.79 5.68 8.11 0.14 1.93 3.87 3.00 4.95 1.35 0.54 1.67 10.32 7.38 4.61 2.35 4.48 1.06 3.06 2.08 2.82 0.84 0.26 0.68 0.59 0.85 2.56 7.09 2.67 2.56
119.7 104.0 105.9 98.0 119.8 104.9 119.1 118.6 98.5 114.3 104.8 106.4 82.2 94.5 114.3 109.7 107.7 106.7 110.0 101.1 84.0 90.5 118.0 92.2 99.5 95.7 95.5 93.7 119.7 86.8 116.5 119.7 93.1 85.8 116.9 107.0 110.2 103.1 91.8 116.0 119.5 98.2 118.5 102.5 96.1 107.2 113.1 86.2 114.6 110.2 84.6 102.0 119.2 94.4 111.1 117.1 94.0 109.0 88.3 104.3 113.9 107.7 106.3 108.4 104.3 93.8 96.1 116.9 102.9 91.4 85.5 99.5 91.3 86.4 99.4 117.7 107.6 99.4
4.18 4.35 3.18 12.64 6.32 1.57 11.41 3.63 0.43 1.33 2.14 2.41 5.79 10.78 2.09 6.33 2.70 3.70 9.14 3.33 2.36 5.01 4.17 0.65 4.29 4.69 0.92 11.23 2.18 0.09 3.78 2.18 1.32 14.18 1.75 7.09 8.01 4.09 3.88 6.92 1.39 1.61 13.25 5.03 4.50 10.71 5.66 4.24 12.75 10.97 2.25 5.63 0.22 0.84 13.79 3.54 1.26 3.66 1.30 0.43 12.82 4.19 1.90 3.48 0.76 1.21 7.15 6.03 5.34 1.10 0.43 1.43 0.33 0.90 1.57 4.32 2.78 1.57
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Fig. 2. The extracted parent ion chromatograms of (a) sample spiked standard at LOQ level and (b) matrix-blank sample obtained by LC–MS/MS.
of the working standard solutions (1 g/mL) were prepared daily by diluting each stock solution in methanol. 2.2. Samples and extraction The counterfeit medicines and dietary supplements, which advertised the ability to increase muscle mass and strength, were
obtained from markets or websites. The 19 collected samples consisted of solids tablet (3) and liquid injectables (16). The samples (1 g) were extracted with 50 mL of methanol–distilled water (70:30, v/v) and degassed in a sonication bath for 30 min. The extract was filtered through a 0.22 m polyvinylidene difluoride (PVDF) membrane syringe filter (Millipore, Milford, MA, USA) and analyzed by UHPLC–MS/MS.
144
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Fig. 3. MRM chromatogram of (a) boldenone standard and (b) counterfeit sample containing boldenone.
2.3. Instrumental conditions The chromatographic system, which consisted of a Waters ACQUITY Ultra Performance Liquid (UPLC; Waters, Milford, MA, USA) with a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 m), was employed to separate the target compounds. The column temperature was maintained at 35 ◦ C, and the flow rate was maintained at 0.25 mL/min. The mobile phase consisted of 0.1% formic acid in water (solvent A,
v/v) and acetonitrile (solvent B, v/v). The initial composition of the mobile phase consisted of 20% solvent B for 3 min. The gradient elution involved a programmed linear gradient of 100% solvent B in 17 min, which was maintained for 3 min. The mobile phase was returned to the initial composition of the gradient program and equilibrated for 4.5 min. The total run time was 25 min, and the injection volume of the sample was 2 L. The mass spectra were recorded on a Waters Xevo TQ (Waters, Milford, MA, USA). The flow rates of the cone (N2 ) and desolvation gases were set to 50
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and 600 L/h, respectively. The capillary voltage was set to 2.7 kV, and the desolvation temperature was 500 ◦ C. All of the compounds were ionized via an electrospray ionization (ESI) source operated in positive mode. The screening of the 26 target compounds was performed at unit resolution in multiple reaction monitoring (MRM) mode. The MRM transitions of the analyte were monitored and selected to determine the optimum conditions. The MRM parameters of the 26 target compounds are shown in Table 1. 2.4. Method validation The method was validated by determining the selectivity, linearity, limit of quantification (LOQ), precision (intra- and inter-day), accuracy, and recovery. Some samples from the 10 sources (2 different tablets and 8 different liquid injectables), which contained none of the detectable target compounds, were used as “matrix-blank samples”. The matrix-blank samples (solid and liquid type) were analyzed to confirm the selectivity and determine the potential interference from intrinsic substances in the target substances. The calibration standard solutions were prepared three times at six concentration levels as follows: 5, 25, 100, 200, 500, and 1000 ng/mL for bolasterone, boldione, calusterone, fluoxumesterone, metenolone, nandrolone, nandrolone decanoate, testosterone, testosterone 17valerate, and testosterone 17-propionate; 10, 50, 100, 200, 500, and 1000 ng/mL for 1-androstendione, boldenone, clostebol, danazol M, formebolone M, methylnortestosterone, nandrolone M1, nandrolone M2, norbolethone, norclostebol and norethandrolone; and 25, 50, 100, 200, 500, and 1000 ng/mL for 19-norandrostenione, boldenone M, methandienone M and oral-turinabol M. The calibration curves were evaluated based on the correlation coefficients (r) of the following equation: y = ax + b (y: peak area, x: concentration, a: slope, b: intercept). Standard solutions at concentration with a signal-to-noise (S/N) ratio of >10 were spiked in the matrixblank samples to calculate the LOQ. The standard solutions spiked at three different concentrations (250, 500, and 1000 ng/mL) were used to determine the precision and accuracy of the method. The intra-day assays were performed by three replicate analyses performed on the same day, and the inter-day assays were repeated in triplicate on three different days. The precision was based on the relative standard deviation (RSD). The accuracy was evaluated by comparing the true concentration spiked into the matrix with the calculated concentration. The recovery was evaluated with blank samples (solid and liquid type) at three different concentrations (i.e., 100, 500, and 1000 ng/mL). The average percent recovery was calculated by comparing the peak area from spiked samples to that from standard solutions at equivalent concentrations. 3. Results and discussion The ESI mass spectra of the 26 target compounds revealed [M+H]+ ions (Q1) in the positive electrospray. The product ions (Q3) were produced by fragment Q1 in the collision cell. Based on the optimized MRM transitions (Table 1), the solutions containing a mixture of the 26 standards were determined by UHPLC/MS/MS system. The most sensitive product ion was used for quantitation of the AASs. The matrix-blank samples and matrix-blank samples spiked standard solutions were extracted and analyzed for selectivity. No interference peak was detected in the eluting positions of the anabolic steroid in matrix-blank chromatograms (Fig. 2). The calibration curve parameters are listed in Table 2. The calibration curves were analyzed in a range of 5–1000 ng/mL for the 26 AASs. The correlation coefficients (r) for the AASs were higher than 0.99. A standard solution mixture at a concentration of 1 g/mL was spiked into the matrix-blank sample, and the serial dilutions of the spiked sample were performed to calculate the LOQs at a signal-to-noise
145
Table 5 Number and concentration range of the detected compounds from the studied samples. Compound
Studied samples
Concentration range (mg/kg)
19-Norandrostenedione Boldenone Boldione Metenolone Nandrolone Methandienone (M) Testosterone Testosterone-17-valerate Testosterone 17-propionate
2 4 2 1 2 1 5 3 5
12.44–54.23 0.09–390.06 10.09–160.12 43.77 199.88–334.06 39.45 11.25–140.25 3.90–10.89 4.24–119228.57
ratio of more than 10. The LOQs for the 26 compounds were determined to be 0.5–25.0 ng/mL (Table 2). The accuracy and precision of the intra and inter-day assays at concentrations of 250, 500, and 1000 ng/mL are summarized in Table 3. The precision was within 9.60%, and the accuracy was determined to be 93.80–104.53% in the intra-batch assays. The precision for the inter-day assay was less than 9.89%. The inter-day assay accuracy was 94.76–104.80%. The results of the intra- and inter-day assay indicated excellent accuracy and reproducibility. In addition, the accuracy and precision data for the intra and inter-day assays met the required limits [20]. The recoveries of the AASs from the solid and liquid matrix-blank samples were 80.9–119.7% and 82.2–119.8%, respectively (Table 4). This validated method was suitable for screening AAS compounds in counterfeit drugs. The validated method was applied to the 19 samples to monitor for the presence of AASs (Fig. 3). Approximately 50% (9/19) of the samples were adulterated with AASs. In addition, some products contained more than two AAS compounds. The compounds detected in the samples are shown in Table 5. Testosterone and testosterone 17-propionate (26%) have the highest percentage among the detected compounds followed by boldenone (21%) and testosterone-17-valerate (16%). In addition, the concentrations of the detected AASs have a wide range from 0.09 (boldenone) to 119,228.57 (testosterone 17-propionate) mg/kg in the samples (Table 5). 4. Conclusions In this study, an UHPLC–MS/MS method was developed and validated to screen for AAS compounds in counterfeit drugs. The counterfeit drugs were obtained from the Korean market in 2014 and analyzed by the validated LC–MS/MS method. Nearly half of the samples were adulterated. 9 AASs (i.e., testosterone 17-propionate, testosterone, boldenone, testosterone 17-valarate, 19-norandrostenedione, boldione, nandrolone, metenolone, and methandienone (M)) were identified in the studied samples. With increasing interest in aesthetic appearance, many people have found that AASs help increase muscle mass and strength. Therefore, the traffickers and distributors of illegal drugs have added AASs, and people are exposed to potential risks from the side effect caused by the AASs. Therefore, continued monitoring of the presence of AASs in counterfeit drugs is essential for public health. Acknowledgments This research was supported by a grant from the Criminal Investigations Office of the Ministry of Food and Drug Safety (MFDS) in 2014. References [1] D.B.D.J. Neves, R.G.A. Marcheti, E.D. Caldas, Incidence of anabolic steroid counterfeiting in Brazil, Forensic Sci. Int. 228 (2013) 81–83.
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