madman
Super Moderator
ABSTRACT
Introduction: The presence of anabolic-androgenic steroids (AAS) in illegal commercial products has been pointed as a global threat to public health. Due to the correlation with adverse toxicological effects, there is a growing interest in the implementation of straightforward methods for the determination of AAS in seized products. This work exploited the development of a mass spectrometry approach to characterize the illegal oil formulations containing AAS.
Methods: The optimization of sample preparation was performed through a simplex-centroid design and the best condition was described as follows: an aliquot of 5 μL of the sample was added with 995 μL of acetonitrile and water (75:25, v/v). The solution was vortexed and centrifuged. After that, 10 μL of supernatant were added with 35 μL of acetonitrile and water and internal standard (testosterone-d3, 1.25 ng). An aliquot of 5 μL was injected into the analytical system.
Results: The method developed was validated and successfully applied in 115 seized samples. Testosterone and its esters had the highest incidence, found in more than 50% of the samples. Besides that, drugs such as boldenone, methandienone, and trenbolone have also been found, where the low quality of the samples was evidenced by the wide variation in the concentration of the drugs, always quantified in sub-doses. Finally, at least one AAS was detected in each sample analyzed. The statistical results were grouped by principal components analysis, to better understand the profile of the seized samples.
Conclusion: This work successfully established a fast and simple method for the determination of AAS and can be applied to verify the profile of seized samples.
1. Introduction
Anabolic-androgenic steroids (AAS) are synthetic derivates of testosterone and therefore hold the potential to be utilized as a supplement to promote enhancement of strength, bodyweight, and performance [1,2]. In consequence, growth in the illegal market of AAS on the internet has been observed in recent years [3]. This fact can enable uncontrolled sales and distribution. In addition, the clandestine products may contain no active ingredients, different AAS from those stated on the label, or ingredients with no anabolic action [1]. The misuse of these compounds was associated with a series of adverse events, despite the individuals had a misperception that use is safe or that side effects are manageable [4]. Considering the adverse health effects, the consumption of AAS can lead to damage on several organ systems, including cardiovascular, hematologic, psychiatric and neuropsychologic, hormonal, and metabolic systems [4–8]. In this scenario, the early chemical characterization of consumed AAS products is crucial to improve toxicovigilance strategies.
The analytical procedures for the determination of counterfeit AAS products can be conducted by several analytical techniques. A number of studies in the literature describe the use of gas chromatography-combustion-isotope ratio mass spectrometry to differentiate between the endogenous of a synthetic preparation by measurement of their differing carbon isotope ( 13 C/12 C) ratio [9–11]. Other techniques can also be applied such as magnetic nuclear proton resonance [12], Fourier-transform infrared spectroscopy [13], direct analysis in real-time ionization mass spectrometry [14], and enzyme-linked immunoassays [15]. Gas chromatography coupled to mass spectrometry is widely applied in qualitative and quantitative methods being considered the gold standard due to the high sensitivity and specificity [1,16–20]. However, there are few studies for the determination of counterfeit AAS products applying ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) [21–24]. Furthermore, the use of chemometrics tools remains poorly explored to optimize sample preparation conditions. The use of a simplex-centroid design can be observed in the sample preparation step in seized products analysis [25]. The statistical model is based on a ternary mixture of solvents, in which different proportions were adopted to generate a mathematical model capable to define the best analytical condition. However, according to our knowledge, no reports were found in the literature describing the use of this optimization strategy to sample preparation of seized oil-based.
In addition, multivariate analysis can provide useful data to investigate the profile of seized samples. Therefore, this work exploited the development of a UHPLC-MS/MS approach to characterize the seized oil-based AAS products. The developed method was optimized and applied in 115 seized samples. Finally, the statistical results were grouped by principal components analysis, to better understand the profile of the seized samples.
3.2. Analysis of seized samples
The validated method was applied in 115 oil-based seized samples. We observed that all tested material contained at least one of the studied AAS. Among them, only one sample contained only one drug (methandienone), in the concentration of 880 µg/mL. All others were presented as mixtures of two or more AAS. The quantitative analysis showed a considerable variation in the concentrations, and in all samples, the compounds were detected at low concentrations compared with pharmaceutical products legally marketed for human or veterinary use. In our study, the most found drugs were testosterone and its esters, which in the form of mixtures were present in 60.87% (n = 70) of the samples followed by methandienone and trenbolone acetate, detected in 18.26% (n = 21) and 10.43% (n = 12) of the samples, respectively. The incidence of AAS in the samples is shown in Fig. 2. The low quality of the samples is evidenced by the wide variation in the concentration of AAS present. As an example, boldenone undecylenate was detected in two samples (930 µg/mL and 85 mg/mL).
4. Conclusion
In this study, a method for the determination of 12 AAS was proposed and implemented. The method validated by UHPLC-MS/ MS proved to be an excellent option for determining these drugs in injectable oil, with a rapid and reproductive analysis. The developed method was successfully applied in the analysis of 115 seized ampoules, without labeling. The statistical analysis by PCA helped in the understanding of the composition profile of the samples. Finally, the method established can be used as a safe alternative by inspection agencies, and the low quality of the material seized puts the health of consumers of black market originated AAS at risk.
Introduction: The presence of anabolic-androgenic steroids (AAS) in illegal commercial products has been pointed as a global threat to public health. Due to the correlation with adverse toxicological effects, there is a growing interest in the implementation of straightforward methods for the determination of AAS in seized products. This work exploited the development of a mass spectrometry approach to characterize the illegal oil formulations containing AAS.
Methods: The optimization of sample preparation was performed through a simplex-centroid design and the best condition was described as follows: an aliquot of 5 μL of the sample was added with 995 μL of acetonitrile and water (75:25, v/v). The solution was vortexed and centrifuged. After that, 10 μL of supernatant were added with 35 μL of acetonitrile and water and internal standard (testosterone-d3, 1.25 ng). An aliquot of 5 μL was injected into the analytical system.
Results: The method developed was validated and successfully applied in 115 seized samples. Testosterone and its esters had the highest incidence, found in more than 50% of the samples. Besides that, drugs such as boldenone, methandienone, and trenbolone have also been found, where the low quality of the samples was evidenced by the wide variation in the concentration of the drugs, always quantified in sub-doses. Finally, at least one AAS was detected in each sample analyzed. The statistical results were grouped by principal components analysis, to better understand the profile of the seized samples.
Conclusion: This work successfully established a fast and simple method for the determination of AAS and can be applied to verify the profile of seized samples.
1. Introduction
Anabolic-androgenic steroids (AAS) are synthetic derivates of testosterone and therefore hold the potential to be utilized as a supplement to promote enhancement of strength, bodyweight, and performance [1,2]. In consequence, growth in the illegal market of AAS on the internet has been observed in recent years [3]. This fact can enable uncontrolled sales and distribution. In addition, the clandestine products may contain no active ingredients, different AAS from those stated on the label, or ingredients with no anabolic action [1]. The misuse of these compounds was associated with a series of adverse events, despite the individuals had a misperception that use is safe or that side effects are manageable [4]. Considering the adverse health effects, the consumption of AAS can lead to damage on several organ systems, including cardiovascular, hematologic, psychiatric and neuropsychologic, hormonal, and metabolic systems [4–8]. In this scenario, the early chemical characterization of consumed AAS products is crucial to improve toxicovigilance strategies.
The analytical procedures for the determination of counterfeit AAS products can be conducted by several analytical techniques. A number of studies in the literature describe the use of gas chromatography-combustion-isotope ratio mass spectrometry to differentiate between the endogenous of a synthetic preparation by measurement of their differing carbon isotope ( 13 C/12 C) ratio [9–11]. Other techniques can also be applied such as magnetic nuclear proton resonance [12], Fourier-transform infrared spectroscopy [13], direct analysis in real-time ionization mass spectrometry [14], and enzyme-linked immunoassays [15]. Gas chromatography coupled to mass spectrometry is widely applied in qualitative and quantitative methods being considered the gold standard due to the high sensitivity and specificity [1,16–20]. However, there are few studies for the determination of counterfeit AAS products applying ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) [21–24]. Furthermore, the use of chemometrics tools remains poorly explored to optimize sample preparation conditions. The use of a simplex-centroid design can be observed in the sample preparation step in seized products analysis [25]. The statistical model is based on a ternary mixture of solvents, in which different proportions were adopted to generate a mathematical model capable to define the best analytical condition. However, according to our knowledge, no reports were found in the literature describing the use of this optimization strategy to sample preparation of seized oil-based.
In addition, multivariate analysis can provide useful data to investigate the profile of seized samples. Therefore, this work exploited the development of a UHPLC-MS/MS approach to characterize the seized oil-based AAS products. The developed method was optimized and applied in 115 seized samples. Finally, the statistical results were grouped by principal components analysis, to better understand the profile of the seized samples.
3.2. Analysis of seized samples
The validated method was applied in 115 oil-based seized samples. We observed that all tested material contained at least one of the studied AAS. Among them, only one sample contained only one drug (methandienone), in the concentration of 880 µg/mL. All others were presented as mixtures of two or more AAS. The quantitative analysis showed a considerable variation in the concentrations, and in all samples, the compounds were detected at low concentrations compared with pharmaceutical products legally marketed for human or veterinary use. In our study, the most found drugs were testosterone and its esters, which in the form of mixtures were present in 60.87% (n = 70) of the samples followed by methandienone and trenbolone acetate, detected in 18.26% (n = 21) and 10.43% (n = 12) of the samples, respectively. The incidence of AAS in the samples is shown in Fig. 2. The low quality of the samples is evidenced by the wide variation in the concentration of AAS present. As an example, boldenone undecylenate was detected in two samples (930 µg/mL and 85 mg/mL).
4. Conclusion
In this study, a method for the determination of 12 AAS was proposed and implemented. The method validated by UHPLC-MS/ MS proved to be an excellent option for determining these drugs in injectable oil, with a rapid and reproductive analysis. The developed method was successfully applied in the analysis of 115 seized ampoules, without labeling. The statistical analysis by PCA helped in the understanding of the composition profile of the samples. Finally, the method established can be used as a safe alternative by inspection agencies, and the low quality of the material seized puts the health of consumers of black market originated AAS at risk.
