Hair analysis as a powerful tool for the identification of anabolic androgenic steroids and other performance and image enhancing drugs in a subject with tissues damages.

Abstract

Anabolic androgenic steroids (AAS) use is widespread not only among athletes and sportsmen but also in different populations, including young people who use them for aesthetic purposes, to boost muscles, and sculpt the body.1, 2 Although AAS should be used exclusively under medical supervision for specific pathologies, it is feasible for users to purchase them without a prescription online and in actual stores or gyms. According to research conducted in several countries, AAS are the most detected products among the seizures of performance and image enhancing drugs (PIED) sold on the black market.3-7 Many are the side effects related to the use of AAS on several organs and systems, including cardiovascular, reproductive, central nervous and musculoskeletal systems as well as renal, skin, and liver.8-13 In addition, AAS users are often prone to polydrug abuse to reduce the side effects and enhance the desired outcomes.1 Substances used in association with AAS belong to different chemical and pharmacological classes and include misused prescription drugs and illicit substances of abuse.13-17 Polyabuse makes it quite challenging to identify the causal relationship between a specific substance and its adverse effect. To complicate matters further, frequent users are not aware of the actual composition of the preparations they are taking when purchased from non-registered producers. In many instances, the medications purchased through illegal channels are unreliable because they may contain ingredients of low quality or in the wrong doses, be deliberately and fraudulently mislabeled with respect to their identity or source, have the wrong ingredients, or have low levels of active ingredients.3-7, 18 In this paper, the toxicological, histological, and medico-legal findings of an AAS user who died of a cause unrelated to AAS abuse (road accident) are reported, with a special focus on the usefulness of hair analysis to uncover PIED administration. A 46-year-old male died in a road accident while driving a motorcycle due to aggressive driving with reckless overtaking after an altercation with another driver. The man's height was 187 cm, and his weight was 98 kg (body mass index: 28.02). External inspection of the body showed the presence of hypertrophic muscle masses. During the autopsy, tissue samples were collected for histological investigation. In addition, urine, blood, vitreous humor, and head and pubic hair were sampled for toxicological analysis. A head hair sample (color: dark brown, length: 1.5 cm) was collected from the vertex posterior region. Several drugs, together with syringes, were found in the top box of his motorcycle. Among the pharmaceutical products found, some of them were manufactured by regular producers with marketing authorizations, whereas the others were unlabeled, not intact, and/or produced by unauthorized companies. The former group included Creon 1000® (pancrelipase), Gaviscon® (sodium alginate and sodium bicarbonate), Aulin® (nimesulide), Sildenafil Sandoz®, Cialis® (tadalafil), and Testovis® (testosterone propionate). The latter group included Oxa-Med, declared as oxandrolone 10 mg, containing three tablet fragments; Meta-med, declared as methandienone 10 mg, containing 57 tablets; Xanavar, declared as oxandrolone 10 mg, containing 90 tablets; Deca-med—nandrolone decanoate, a 10 mL vial containing an oily solution, branched-chain amino acids (BCAA) (tablets), and fragments of a blue tablet in an unlabeled packaging. Methandienone, oxandrolone, stanozolol, and trenbolone were from Steraloids, Newport, RI, USA. Testosterone enanthate, testosterone propionate, nandrolone decanoate, sildenafil, and testosterone-D3, were from Sigma-Aldrich, Saint Louis, MO, US. Caffeine, ephedrine, pseudoephedrine, and methamphetamine-D5 were from LGC Standards (Milano, Italy). Water, acetonitrile, formic acid, and methanol were purchased from Sigma-Aldrich (Milan, Italy); ammonium formate was from Agilent Technologies (Santa Clara, CA, USA). All reagents and solvents were of LC-MS grade. Urine, blood, and hair were also analyzed for common drugs of abuse and benzodiazepines using the methods described elsewhere.19, 20 In addition, blood and vitreous humor were tested for ethanol by headspace gas chromatography with a flame ionization detector (GC-FID). The hair samples were analyzed according to a method developed and published previously.16 Briefly, hair samples were washed three times with 0.5 mL of a solution of tween 80 (0.1% v/v in distilled water), rinsed twice with 0.5 mL of distilled water, once with 0.2 mL of acetone for 10 s, dried, and cut into small pieces with scissors. 30 mg of each sample was added with 100 μL of testosterone-D3 (1 μg/mL) as internal standard and left at 40°C overnight under sonication after the addition of 300 μL of methanol. 100 μL of the supernatant was then put in a microvial, and 10 μL was directly injected into the LC-HRMS system. The urine sample (100 μL) was diluted 1:1 with 0.1% formic acid aqueous solution and hydrolyzed with β-glucuronidase/arylsulfatase at 40°C for 8 h. Internal standards (testosterone-D3 and methamphetamine-D5 100 ng/mL) were added to the sample, and afterward, it was further diluted with 300 μl 0.1% formic acid solution. A blood sample (500 μL) was added with internal standards (testosterone-D3 and methamphetamine-D5 20 ng/mL) and 500 μL of methanol. The sample was then centrifuged for 15 min at 150000 x g. Afterward, the supernatant was recovered and injected into the LC-HRMS system. Validation parameters for hair analysis are reported elsewhere, whereas those for blood and urine for stanozolol, methandienone, trenbolone, testosterone, and sildenafil are shown in Table 1. LOD (ng/mL) LOQ (ng/mL) LOD (ng/mL) LOQ (ng/mL) Medicinal products were prepared in agreement with the methods described in previously published papers.21, 22 Samples were weighted, then internal standards and 5 mL of methanol were added. The specimen was vortexed and centrifuged. The methanolic phase was evaporated to dryness and reconstituted in 2 mL of a solution of sodium hydroxide 1 M. A mixture of pentane/ethyl ether was added for liquid/liquid extraction. After centrifugation, the organic phase was evaporated and reconstituted with methanol/water/formic acid 6:4:0.03. All samples were subsequently analyzed using LC-HRMS. The LC-HRMS system was composed of a Thermo UltiMate 3000 system equipped with an analytical column Thermo Acclaim RSLC 120 C18 (2.1 mm × 100 mm, 2.2 μm of particle size), coupled to a Thermo single-stage Orbitrap (Exactive) MS system, interfaced with a HESI source. The whole equipment and the column were from Thermo Fisher Scientific (Milan, Italy). Mobile phase A was ultrapure water with 5 mM ammonium formate and 0.1% formic acid, whereas mobile phase B was methanol/acetonitrile 1:1 with 0.1% of formic acid. The analytical column was maintained at 40°C. The injection volume was 10 μL, and the flow rate was set at 0.4 mL/min. Two mobile phase gradients were used, one for steroids and the other for stimulants and phosphodiesterase five inhibitors. Instrumental conditions and mobile phase gradients were reported elsewhere.21, 23 Data were acquired in full scan mode over a mass range of 110 to 800 m/z. The instrument operated in positive ion mode with a resolving power of 100.000 full width at half maximum (FWHM). A further set of experiments was performed with in-source collision-induced dissociation (CID) with the voltage set at 40 V, acquiring ions from 50 to 550 m/z, with a resolving power of 50.000 FWHM, obtaining the accurate masses of both precursor and fragment ions. The analysis for illicit drugs and benzodiazepines did not detect any substance in the blood, urine, or hair. Alcohol analysis was negative for both blood and vitreous humor. In hair specimens, different AAS were identified, in particular, stanozolol, trenbolone, testosterone enanthate, testosterone, and sildenafil. Concentrations detected in the head and pubic hair are depicted in Table 2. In the urine sample, the main stanozolol metabolite 3-hydroxystanozolol and desmethylsildenafil were detected. Caffeine was present in both hair and urine samples. None of the above-mentioned substances was detected in the blood sample. Figure 1 depicts the LC-HRMS ionic chromatogram of stanozolol, methandienone, trenbolone, testosterone enanthate, testosterone, and sildenafil in the pubic hair sample. The analysis of the products showed the presence of other substances with respect to those declared on the label, when present, sometimes in conjunction with the ones reported. Methandienone and stanozolol were identified in the three tablet fragments named Oxa-Med instead of oxandrolone. The preparation Metha-Med contained methandienone together with stanozolol. In the Xanavar tablets, methandienone and stanozolol were identified instead of oxandrolone. Analysis of the Deca-Med solution evidenced the presence of testosterone propionate, testosterone decanoate, and nandrolone decanoate. In BCAA tablets, amino acids, leucine and valine, and vitamins belonging to the B group, together with caffeine, ephedrine, and pseudoephedrine, were identified. A forensic autopsy showed prominent skeletal muscle hypertrophy with multiorgan congestion. Cardiomegaly (450 g) and fatty liver were found. The main myocardial findings consisted of left ventricular hypertrophy, and the histology showed interstitial and perivascular fibrosis within the left ventricle (Figure 2). Microscopic examination revealed small droplets steatosis of hepatic parenchyma with the focal expansion of portal tracts with mixed chronic inflammation (Figure 3). Moreover glomerulomegaly with limited foci of interstitial fibrosis was identified (Figure 4). In literature, the use of AAS has been associated with liver alteration and pathological modifications in heart morphology, such as cardiac hypertrophy and dilated cardiomyopathy. Indeed, left ventricular hypertrophy, often associated with fibrosis and myocytolysis, was often identified in post-mortem findings on AAS abusers who died of sudden cardiac death.24-26 Also, renal alterations are described.9, 10 In the present case, the death was due to the injuries caused by accident, but anatomopathological and histological findings showed organ and tissue alterations probably attributable to the use of AAS. Urine and hair analysis confirmed that the subject had a history of AAS and sildenafil use. Hair analysis showed that the man used at least four different AAS in the last months. The concentrations measured in head hair were comparable or higher to the ones found by other authors for chronic users. In particular, stanozolol was detected in the hair from 2 up to 881 pg/mg,27-29 methandienone levels in hair ranged from 3 to 206 pg/mg,11, 30, 31, 29 and testosterone enanthate from 0.6 up to 18.8 ng/mg.32 Also, testosterone was in a concentration higher than those reported for endogenous contribution, which ranged from 0.3 to 11.81 pg/mg.33-36 Despite the reported endogenous level, testosterone was recently detected in concentrations below 1 ng/mg, even in confirmed users. Favretto et al. reported a testosterone hair concentration of 18 pg/mg for a testosterone undecanoate user,13 whereas Kintz et al. described the case of a hair sample with testosterone at 140 pg/mg together with testosterone propionate and decanoate.11 Anyway, the presence of the ester is an unequivocal sign of the exogenous origin of the substance. Trenbolone was detected in the present case only in pubic hair. Recent publications reported this substance's concentrations in hair from 9 to 143 pg/mg.11, 12, 36 These values are below our method LOD. Few papers reported hair concentration levels for sildenafil. The detected concentrations were 7.5 and 38 pg/mg for DFSA cases,37, 38 for which there was a single sildenafil intake or at least a limited use of the substance. Sildenafil was identified at 0.177 ng/mg in a post-mortem case.39 Higher concentrations of sildenafil, up to 1.7 and 19.8 ng/mg, were detected by Saisho et al. for chronic users (1 tablet with 50 mg of sildenafil once every 10 days for at least 6 months).40 Although it is not possible to relate the concentration in hair specimens with the taken amount, the sildenafil level detected in the presented case (5.8 ng/mg in head hair) appeared to be similar to the latter results reported in the literature. The concentrations of all the substances detected, AAS and sildenafil, were always higher in pubic hair with respect to the ones in head hair, as expected. This different distribution for stanozolol and other AAS in hair collected from different anatomic regions was also documented in previous publications.35, 41, 42 This may be due to different growth rates, the variable ratio between the anagen stage and the catagen and telogen stages, or higher contribution in drug incorporation by sweat or contamination by urine. Among the AAS identified in biological specimens, methandienone and stanozolol have also been identified in the tablets found in the motorcycle case. As reported by other authors, the content of medical preparations analyzed differed, for both type of product(s) and their concentrations, with respect to that stated on the labels.3-7, 19 This issue can pose a serious additional risk to health, firstly because of the toxicity and adverse side effects connected with the use of these substances, especially if they are taken without medical supervision. The 17-alpha-alkylated anabolic steroids, such as stanozolol and methandienone, have been associated with cases of liver pathologies unrelated to the route of administration.2 In particular, stanozolol assumption was linked to several hepatic diseases such as bland cholestasis, lesions in centrilobular hepatocytes, hepatic lipidosis, ultrastructural alterations in the canaliculi, and degenerative changes in mitochondrial and lysosomes.43-46 Secondly, the use of these preparations can pose a threat to health because of the possible drug–drug interaction with other medicaments.47 Polyabuse among AAS users is not infrequent. This population is prone to mix not only in different steroids but also steroids and other PIED. The main purpose is to boost steroids' effects while reducing the undesired ones. In particular, phosphodiesterase-5 inhibitors such as sildenafil are used to improve erectile function and the reduction of sexual desire. The possible effect on the health of this association is still unknown. The use of many different anabolic steroids would have gone unnoticed without hair analysis, which allowed the detection of methandienone, stanozolol, testosterone enanthate, trenbolone, and sildenafil. In the urine sample, in fact, were only present the stanozolol and sildenafil metabolites, whereas, in the blood sample, none of the substances were detected. This demonstrates the usefulness of hair analysis also in the case of performance and image-enhancing drugs, as highlighted by other authors' recent findings.11-13 Histopathological and toxicological findings for an AAS user, who died after the consequences of a road accident, showed the adverse effects likely due to AAS abuse on various organs. The heart, liver, and renal alterations were consistent with a wide and repeated use of AAS, as reported in the literature. Hair analysis demonstrated a continuative use of various AAS and sildenafil. This fact would have gone unnoticed without the hair analysis, given the unrelated cause of death and their absence in blood and urine. This case demonstrates the usefulness of hair analysis to obtain a complete evaluation of each forensic case. The analyses performed on falsified medicines in the victim's possession confirmed that the actual qualitative content lacks consistency with what was declared on the label. Open Access Funding provided by Universita Cattolica del Sacro Cuore within the CRUI-CARE Agreement.