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Powered by Sports drug testing and toxicology TOP ARTICLES SUPPLEMENT CONTENTS REVIEW: Applications and challenges in using LC–MS/MS assays for quantitative doping analysis Bioanalysis Vol. 8 Issue 12 REVIEW: Current status and recent advantages in derivatization procedures in human doping control Bioanalysis Vol. 7 Issue 19 REVIEW: Advances in the detection of designer steroids in anti-doping Bioanalysis Vol. 6 Issue 6 Review For reprint orders, please contact [email protected] 8 Review 2016/05/28 Applications and challenges in using LC–MS/MS assays for quantitative doping analysis Bioanalysis LC–MS/MS is useful for qualitative and quantitative analysis of ‘doped’ biological Zhanliang Wang‡,1, samples from athletes. LC–MS/MS-based assays at low-mass resolution allow fast Jianghai Lu*,‡,2, and sensitive screening and quantification of targeted analytes that are based on Yinong Zhang1, Ye Tian2, 2 ,2 preselected diagnostic precursor–product ion pairs. Whereas LC coupled with high- Hong Yuan & Youxuan Xu** 1Food & Drug Anti-doping Laboratory, resolution/high-accuracy MS can be used for identification and quantification, both China Anti-Doping Agency, 1st Anding have advantages and challenges for routine analysis. Here, we review the literature Road, ChaoYang District, Beijing 100029, regarding various quantification methods for measuring prohibited substances in PR China athletes as they pertain to World Anti-Doping Agency regulations. 2National Anti-doping Laboratory, China Anti-Doping Agency, 1st Anding Road, First draft submitted: 5 February 2016; Accepted for publication: 29 April 2016; ChaoYang District, Beijing 100029, PR China Published online: 31 May 2016 *Author for correspondence: Tel.: +86 108 437 6282 Keywords: doping analysis • LC–MS/MS • quantification Fax: +86 106 498 0526 [email protected] **Author for correspondence: LC–MS/MS can be used to measure trace its for threshold substances) [2] established [email protected] compounds with high sensitivity and selec- that ten substances had urinary thresholds ‡Authors contributed equally tivity, and it is suitable for quantitation and for competitive sports: 19-norandrosterone identification of banned substances in biolog- (19-NA), 11-nor-Δ9-tetrahydrocannabinol- ical samples. Operation of the technique in 9-carboxylic acid (THCA), salbutamol, multiple reaction monitoring (MRM) mode formoterol, glycerol, morphine, cathine permits rapid screening of multiple [1] small (d-norpseudoephedrine), ephedrine, methy- molecules in a single run, so LC–MS/MS lephedrine and pseudoephedrine. If found is a preferred technique for routine athletic in greater concentrations than WADA lim- doping analysis. its (threshold + Uc Max – maximum accept- 12 Advances in measuring high molecu- able combined standard uncertainty val- lar weight peptides and proteins for doping ues), such compounds are reported as an control have been made: high-resolution MS ‘adverse analytical finding’ (AAF). Based enhances the signal-to-noise ratio and speci- on global WADA accredited laboratories 2016 ficity for compound identification by provid- and relevant rounds of the WADA Exter- ing elemental fragment composition. Thus, nal Quality Assessment Scheme, the Uc Max isobaric fragments with different elemental represents a 95% CI finding. Additionally, compositions from different molecular por- to ensure that all WADA-accredited labo- tions can be differentiated. Based on the ratories can uniformly report prohibited exact mass detected, high-resolution MS substances, metabolite(s) or marker(s) as (HRMS)/MS was confirmed to be well suited well as provide detection within thresholds, for quantification and offers a better signal- standardized testing methods have estab- to-noise ratio compared with low-resolution lished. The WADA Technical Document LC–MS/MS. 2015 minimum required performance lev- The World Anti-Doping Agency (WADA) els (MRPL) [3] requires that all laboratories Technical Document 2014 DL (decision lim- attain a minimal capacity for screening and part of 10.4155/bio-2016-0030 © 2016 Future Science Ltd Bioanalysis (2016) 8(12), 1307–1322 ISSN 1757-6180 1307 Review Wang, Lu, Zhang, Tian, Yuan & Xu confirmation of doping compounds by establishing Technical Document 2014 for Decision Limits [2] that thresholds for AAFs. Therefore, as required by WADA ‘the threshold concentration is based on the sum of the TD2014DL and WADA Technical Document 2015 glucuronide conjugate (expressed as the free drug) and MRPL, quantitative analysis of the banned substance free drug concentrations,’ some suggest that 19-NA against a threshold and estimation of the substance sulfates and glucuronides can be measured in human concentrations (semiquantification) are used for dop- urine [5–9] but that these methods have less LOQ at ing tests. As defined by the WADA International Stan- the ng/ml level and omit hydrolysis. In addition, these dard for Laboratories (§5.2.4.4), any testing results methods show the detection difficulty in free form obtained from hair, nails, oral fluid or other biological 19-NA. Thus, the ratio between the glucuro- and material shall not be used to counter AAFs or atypi- sulfoconjugate derivatives of 19-NA and 19-noretio- cal findings in the urine. Thus, this review is chiefly cholanolone (19-NET) do not offer more information focused on methods to assay urine samples (see Table 1 about the origin of 19-NA or 19-NET. To discriminate for methods). endogenous versus exogenous 19-NA or 19-NET [5] ion suppression for each exceeds 50%, with higher RSD. Quantification of banned substances with Meanwhile 19-NA and 19-NET separation was not & without thresholds preferred even with a relatively reasonable retention 19-norandrosterone time of 13.57 and 14.02 min, respectively. Thus, screen- Confirmation of illicit use of nandrolone or other ing may not be problematic with sufficient sensitivity 19-norsteroids is based on confirming that the main for MRPL, but for quantification, ion suppression and urinary metabolite 19-NA (derived from hydrolysis separation may increase data variation. with β-glucuronidase) exceeds 2 ng/ml [4]. The pres- ence of 19-NA in the range of 2–10 ng/ml or as high THCA as 15 ng/ml can be confirmed by GC–MS/(MS) and LC–MS/MS methods for THCA quantification in LC–MS/MS, GC/C/IRMS is employed to distinguish urine, plasma, hair and oral fluids are summarized the origination of 19-NA which is endogenous or exog- in Table 2. THCA and its glucuronide conjugate are enous. Normally, quantification of 19-NA includes a major urinary cannabinoid metabolites and the WADA deuterated internal standard (e.g., d4–19-NA-glucuro- threshold for THCA is 150 ng/ml. Dilute-and-shoot nide), a calibration curve that includes the estimated method development and validation for LC–MS/MS sample concentration or a single calibration point at to measure urinary THCA has been described in the 10 or 15 ng/ml plus appropriate negative and positive literature [10–12] and this approach permitted more rapid quality control samples. Although required by WADA sample preparation with fewer steps prior to LC–MS/ Table 1. Decision limits for the confirmatory quantification of threshold substances. Threshold substance Threshold (T) Max. combined standard Decision limit Ref. Nonthreshold (MRPL) uncertainty (uc Max) at T (DL) Absolute Relative 19-Norandrosterone 2.0 ng/ml 0.3 ng/ml 15 2.5 ng/ml [4–9] THCA 150 ng/ml 15 ng/ml 10 180 ng/ml [10–21] Salbutamol 1.0 μg/ml 0.1 μg/ml 10 1.2 μg/ml [22–24] Formoterol 40 ng/ml 6.0 ng/ml 15 50 ng/ml [10,25–27] Glycerol 4.3 mg/ml 0.65 mg/ml 15 5.3 mg/ml [28–30] Morphine 1.0 μg/ml 0.15 μg/ml 15 1.3 μg/ml [31–41] Cathine 5.0 μg/ml 0.5 μg/ml 10 6.0 μg/ml [22] Ephedrine 10 μg/ml 0.5 μg/ml 5 11 μg/ml [42–51] Methylephedrine 10 μg/ml 0.5 μg/ml 5 11 μg/ml Pseudoephedrine 150 μg/ml 7.5 ug/ml 5 170 μg/ml Clenbuterol† 0.2 ng/ml [67–73] Glucocorticoids† 30 ng/ml [74–76] †Nonthreshold substances. MRPL: Minimum required performance levels. 1308 Bioanalysis (2016) 8(12) future science group Table 2. Summary of assay characteristics dedicated to THCA from urine, blood, hair and oral fluid. Analytes Matrix IS Sample Stationary Mobile phase Quantification LOD (ng/ml) Linearity Ref. preparation phase mode range (ng/ml) THCA Urine D9-THCA Hydrolysis C18, RP, 5 μm 0.1% formic acid/ ESI+ 6.1 50–400 [10] Dilution 0.1% formic acid ACN MRM THCA Urine D9-THCA Hydrolysis C8, RP, 5 μm 0.001%HAc,1 mM NH4OAc/ ESI+, ESI- 0.5 5–40 [11] Dilution 0.001%HAc,1 mM NH4OAc MeOH MRM THCA Urine D3-THCA Hydrolysis C18, RP, 1.7 μm 0.1% formic acid/ ESI- 10 5–3000 [12] Filtration 0.1% formic acid MeOH MRM THCA Urine D9-THCA SLE C18, RP, 5 μmm 10 mM NH4OAc, pH = 6.15/ ESI- 0.5 1–100 [13] THCAG THC column 15% MeOH-ACN MRM 0.5 5–500 Applications &challenges in using LC–MS/MS assays for quantitative doping analysis 5 others THCA Whole D3-THC PP C18, RP, 5 μmm 0.1% formic acid/ ESI+/- 0.1 0.25–50 [14] THC blood D3-THCA SPE 0.1% formic acid ACN MRM THCA Whole D3-THCA PP C18, RP, ACN/water/formic acid, ESI+ 1.5 2.5–100 [15] 2 others blood On-line SPE 30/70/0.01, v/v MRM THCA Whole D3-THCA SPE C18, RP, 1.8 μm 2 mM NH4CO2,0.1% formic acid/ ESI+ 0.5 0.5–100 [16] 2 others blood MeOH MRM THCAG Whole D9-THCA PP C18, RP, 5 μm 10 mM NH4OAc, pH = 6.15/ ESI- 1 5–250 [17] THCA blood SPE 15% MeOH-ACN MRM 1 1–100 4 others † † THCA Hair D3-THCA MPE C18, RP, 3 m 5 mM NH4OAc 0.5% formic acid/ ESI- 0.1 0.1–5 [18] ACN MRM THCA- Oral D3-THC SPE C18, RP, 1.8 μm 5 mM NH4CO2, pH = 6.4/ ESI+ 0.01 0.01–1 [19] derivative fluid 0.5% formic acid ACN MRM THCA Oral D3-THCA PP C18, RP, 3 μm 10 mM NH4OAc, pH = 6/ HRMS 0.015 0.015–0.5 [20] 3 others fluid SPE ACN HESI-II www.future-science.com THCA Oral D9-THCA SPE C18, RP, 3 μm 0.01% acetic acid/ ESI- 0.009 0.012–1.02 [21] fluid 0.01% acetic acid MeOH MRM †pg/mg.
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    1. Medical Journal of Clinical Trials & Case Studies ISSN: 2578-4838 Hepatotoxicity Associated with Methylstenbolone and Stanozolol Abuse Vargas KEA*, Guaraná TA, Biccas BN, Agoglia LV, Carvalho ACG, Case Report Gismondi R and Esberard EBC Volume 2 Issue 5 Received Date: July 27, 2018 Department of Gastroenterology/Hepatology, Department of Clinical Medicine, and Published Date: September 03, 2018 Department of Pathology, Antônio Pedro University Hospital, Federal Fluminense DOI: 10.23880/mjccs-16000176 University, Rio de Janeiro, Brazil *Corresponding author: Vargas Karen Elizabeth Arce, Department of Gastroenterology/Hepatology, Department of Clinical Medicine, and Department of Pathology, Antônio Pedro University Hospital, Federal Fluminense University, Rio de Janeiro, Ernani do Amaral Peixoto Avenue, 935. Ap.901 / Cep.24020043, Brazil, Tel: 005521981584624; Email: [email protected] Abstract Background & Objectives: Drug hepatotoxicity is a major cause of liver disease. Many drugs are well known to induce liver damage. Some toxic products, like anabolic androgenic steroids, that are pharmaceutical preparations since they contain pharmaceutically active substance, are available as nutritional supplements. Many patients are used to consume these like dietary stuff. Methods: We introduce a case series of two patients who developed hepatic damage after the consumption of anabolic- androgenic steroids, accompanied by a detailed bibliographic research on this topic. Results: We present two young men who developed significant liver damage, both with hyperbilirubinemia pattern after consumption of anabolic-androgenic steroids. This was associated with considerable morbidity, although both recovered without liver transplantation. The two anabolic-androgenic steroids were being marketed as dietary supplements. Conclusions: Although not well controlled substances in Brazil, anabolic-androgenic steroids are cause of severe hepatotoxicity.
  • A10 Anabolic Steroids Hardcore Info

    A10 Anabolic Steroids Hardcore Info

    CONTENTS GENERAL INFORMATION 3 Anabolic steroids – What are they? 4 How do they Work? – Aromatisation 5 More molecules – More problems 6 The side effects of anabolic steroids 7 Women and anabolic steroids 8 Injecting steroids 9 Abscesses – Needle Exchanges 10 Intramuscular injection 11 Injection sites 12 Oral steroids – Cycles – Stacking 13 Diet 14 Where do steroids come from? Spotting a counterfeit 15 Drug Information – Drug dosage STEROIDS 16 Anadrol – Andriol 17 Anavar – Deca-Durabolin 18 Dynabolon – Durabolin – Dianabol 19 Esiclene – Equipoise 20 Primobolan Depot – Proviron – Primobolan orals – Pronobol 21 Sustanon – Stromba, Strombaject – Testosterone Cypionate Testosterone Enanthate 22 Testosterone Propionate – Testosterone Suspension 23 Trenbolone Acetate – Winstrol OTHER DRUGS 24 Aldactone – Arimidex 25 Clenbuterol – Cytomel 26 Ephedrine Hydrochloride – GHB 27 Growth Hormone 28 Insulin 30 Insulin-Like Growth Factor-1 – Human Chorionic Gonadotrophin 31 Tamoxifen – Nubain – Recreational Drugs 32 Steroids and the Law 34 Glossary ANABOLIC STEROIDS People use anabolic steroids for various reasons, some use them to build muscle for their job, others just want to look good and some use them to help them in sport or body building. Whatever the reason, care needs to be taken so that as little harm is done to the body as possible because despite having muscle building effects they also have serious side effects especially when used incorrectly. WHAT ARE THEY? Anabolic steroids are man made versions of the hormone testosterone. Testosterone is the chemical in men responsible for facial hair, deepening of the voice and sex organ development, basically the masculine things Steroids are in a man. used in medicine to treat anaemia, muscle weakness after These masculine effects surgery etc, vascular are called the androgenic disorders and effects of testosterone.