1H NMR Determination of Adulteration of Anabolic Steroids in Seized Drugs

Steroids 138 (2018) 47–56 Contents lists available at ScienceDirect Steroids journal homepage: www.elsevier.com/locate/steroids 1H NMR determination of adulteration of anabolic steroids in seized drugs T Marcos Vinícius de Moura Ribeiro, Nivaldo Boralle, Lidiane Gaspareto Felippe, ⁎ Helena Redigolo Pezza, Leonardo Pezza Institute of Chemistry, São Paulo State University “Júlio de Mesquita Filho”, 14800-060 Araraquara, SP, Brazil ARTICLE INFO ABSTRACT Keywords: Counterfeiting and adulteration of pharmaceuticals is a prevalent problem worldwide and represents a major Anabolic steroids health risk to the population, with anabolic steroids being one of the main classes of drugs consumed and 1 H NMR obtained from dubious sources. In this work, we propose the use of the 1H NMR technique to evaluate for- Authenticity mulations containing anabolic steroids, with analysis of 40 samples of anabolic drugs that are used in injectable Adulteration and capsule forms. The samples analyzed presented the following active ingredients: testosterone propionate, Quantification testosterone phenylpropionate, testosterone isocaproate, testosterone decanoate, testosterone cypionate, testosterone undecanoate, stanozolol, drostanolone propionate, trenbolone acetate, oxymetholone, and methandrostenolone. The 1H NMR spectroscopic measurements were performed using a 600 MHz Bruker Avance III spectrometer, with deuterated chloroform (CDCl3) containing 0.1% TMS as solvent. Of the 40 samples analyzed, eight did not show the presence of the active principle stated on the label. Three types of adulteration were found in the analyzed samples: absence of the active ingredient, adulteration with other substances, and concentration values below those indicated on the label. Sildenafil citrate was found in four samples. The GC–MS technique was used to confirm the adulteration results found using 1H NMR. Quantitative determination by NMR was performed using internal standard and ERETIC 2 methods, and the results obtained were statistically the same. 1. Introduction achieving an athletic body in a short time, make use of anabolic steroids that are often prescribed by instructors, unscrupulous doctors, and According to the World Health Organization (WHO), a counterfeit physical education teachers with no knowledge in the area. This is due medicine is a product that is improperly packaged and labeled, in a to the ease of obtaining the substances, since they can be purchased on deliberate and fraudulent way, with its source or identity not being the black market, via the internet or across borders, without medical respected, and that can exhibit alterations and adulterations, compared prescription. Such substances are manufactured/falsified in clandestine to the original formula [1,2]. Drug counterfeiting and commercializa- laboratories and are packaged in ampoules, often unsterilized or mixed tion is an extremely lucrative activity, with a lower risk of repression by with other drugs [7–9]. Some users receive veterinary products based police authorities, relative to drug trafficking, and is directly linked to on steroids, for which there is no information available concerning the international criminal organizations [3,4] likely risks to humans [10]. In Brazil, the drugs most widely falsified are those most commonly The literature describes several methods for determination of the sought by the population and that command higher prices on the legal authenticity and quantification of anabolic steroids in different phar- market. These include erectile dysfunction, anabolic steroid, weight maceutical formulations, highlighting chromatographic methods in- loss, and anti-cancer formulations. These types of medicines are easily cluding gas chromatography [7,8,11,12] and liquid chromatography purchased via the internet, free fairs, and clandestine drugstores [3,5]. [9,13–15]. However, although these techniques are well established for The illegal production of medicines, with copying of their active in- this type of analysis, with good detection limits, they have a number of gredients (or simply omitting them) does not require expensive facil- disadvantages from the operational point of view, such as the need for ities or procedures. The profits are considerable, even when the pro- specific chromatographic columns, laborious sample preparation steps, ducts are sold at prices much lower than those of the original drugs [6]. and sometimes the use of derivatization reactions and toxic solvents. Anabolic steroids appear at the top of the list of counterfeit sub- Unlike other techniques, NMR generally does not require separation stances. People who wish to make fast weight and muscle gains, steps in order to quantitatively determine the analyte of interest. ⁎ Corresponding author at: Rua Prof. Francisco Degni 55, Jardim Quitandinha, 14800-060 Araraquara, SP, Brazil. E-mail address: [email protected] (L. Pezza). https://doi.org/10.1016/j.steroids.2018.07.002 Received 26 March 2018; Received in revised form 11 June 2018; Accepted 5 July 2018 Available online 10 July 2018 0039-128X/ © 2018 Elsevier Inc. All rights reserved. M.V.d.M. Ribeiro et al. Steroids 138 (2018) 47–56 Therefore, residual solvents, excipients, degraded products, and other Table 1 impurities in drugs can be identified and subsequently quantified. In the Qualitative analysis of the presence or absence of the active principle in the case of falsifications where there is the intentional addition of im- samples. purities or diluents, the quality of the medicinal product can be easily Active ingredient Form Sample Result (adulterated checked because the added organic components will show character- or authentic) istic signals in the 1H NMR spectra. The ability of NMR to detect un- known organic impurities means that it can be considered a universal Testosterone propionate Injectable 1 Authentic 2 Authentic detector capable of identifying counterfeit drugs [16]. NMR also stands 3 Authentic out for being a nondestructive technique that uses a small amount of 4 Authentic solvent, where the analyte of interest can be recovered by the solvent Testosterone propionate Testosterone Injectable 5 Adulterated drying process. The potential of this technique for drug analysis is re- phenylpropionate Testosterone 6 Adulterated flected in the fact that methods of quantitative analysis of drugs using isocaproate Testosterone 7 Adulterated NMR have already been made official and are listed in international decanoate 8 Adulterated pharmacopoeias, such as in the American and European Pharmaco- Testosterone cypionate Injectable 9 Authentic poeias [17–20]. 10 Authentic It is vital to be able to use simple analytical methods that are rapid, 11 Authentic 12 Authentic effective, and reliable for the quality control of medicines [21], espe- cially those that are widely used and of great commercial value, such as Testosterone undecanoate Capsule 13 Authentic 14 Authentic anabolic steroids. The objective of the present study was to propose the 15 Authentic 1 use of H NMR for evaluation of the quality of medications containing 16 Authentic anabolic steroids, seized by the Brazilian Federal Police, which are Stanozolol Injectable 17 Authentic widely available in the national marketplace. Qualitative and quanti- 18 Authentic tative analyses were performed using different analytical methods. 19 Authentic 20 Authentic 2. Materials and methods Capsule 21 Adulterated 22 Adulterated 23 Adulterated 2.1. Samples and reagents 24 Adulterated Drostanolone propionate Injectable 25 Authentic Analyses were made of 40 samples of drugs containing anabolic 26 Authentic steroids, seized and provided by the Brazilian Federal Police, with 27 Authentic different active ingredients: testosterone propionate (TPR), testosterone 28 Authentic phenylpropionate (TPH), testosterone isocaproate (TIS), testosterone Trenbolone acetate Injectable 29 Authentic decanoate (TDE), testosterone cypionate (TCY), testosterone un- 30 Authentic decanoate (TUN), stanozolol (STZ), drostanolone propionate (DPR), 31 Authentic 32 Authentic trenbolone acetate (TRA), oxymetholone (OXY), and methandrostenolone (MTH). Analytical standards for stanozolol (99.21%) and Oxymetholone Capsule 33 Authentic 34 Authentic testosterone propionate (99.88%) were used in quantitative assays. The 35 Authentic samples were solubilized using deuterated chloroform (CDCl3, 99.8%) 36 Authentic containing 0.1% tetramethylsilane (TMS). All the reagents used were Methandrostenolone Capsule 37 Authentic analytical grade and were purchased from Sigma-Aldrich. The spectral 38 Authentic profiles of the samples were obtained using solutions containing 39 Authentic − 5mgmL 1 of each active ingredient. 40 Authentic 2.2. 1H NMR analysis − concentrations of 5 mg mL 1. For the injectable samples, aliquots of The 1H NMR spectroscopic measurements were performed at 298 K 40–70 μL of the drugs (the volume used depended on the concentration with a 5 mm direct observation multinuclear probe (BBFO-Z plus indicated on the label) were transferred to 2 mL Eppendorf tubes, fol- SmartProbe Broadband Observe), using a Bruker Avance III HD 600 lowed by addition of CDCl3 (with 0.1% TMS) and stirring manually spectrometer operating at 14.1 Tesla (600.13 MHz for 1H). A time of until solubilization was complete. Aliquots (600 μL) of the prepared 17.15 ms was obtained for a 90° pulse. Subsequently, the longitudinal solutions were transferred to 5 mm NMR tubes. For preparation of the relaxation time (T1) was estimated using the inversion recovery pulse capsule

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