Feasibility of a liquid-phase microextraction sample clean-up and liquid chromatographic/mass spectrometric screening method for selected anabolic steroid glucuronides in biological samples

2 Jan 2003

Feasibility of a liquid-phase microextraction sample clean-up and liquid chromatographic/mass spectrometric screening method for selected anabolic steroid glucuronides in biological samples / Tiia Kuuranne, Tapio Kotiaho, Stig Pedersen-Bjergaard, Knut Einar Rasmussen, Antti Leinonen, Steven Westwood, Risto Kostiainen. - (Journal of Mass Spectrometry 38 (2003) 1 (January); p. 16-26)

  • PMID: 12526002
  • DOI: 10.1002/jms.393


Anabolic androgenic steroids (AAS) are metabolized extensively in the human body, resulting mainly in the formation of glucuronide conjugates. Current detection methods for AAS are based on gas chromatographic/mass spectrometric (GC/MS) analysis of the hydrolyzed steroid aglycones. These analyses require laborious sample preparation steps and are therefore time consuming. Our interest was to develop a rapid and straightforward method for intact steroid glucuronides in biological samples, using liquid-phase microextraction (LPME) sample clean-up and concentration method combined with liquid chromatographic/tandem mass spectrometric (LC/MS/MS) analysis. The applicability of LPME was optimized for 13 steroid glucuronides, and compared with conventional liquid-liquid extraction (LLE) and solid-phase extraction (SPE) procedures. An LC/MS/MS method was developed for the quantitative detection of AAS glucuronides, using a deuterium-labeled steroid glucuronide as the internal standard. LPME, owing to its high specificity, was shown to be better suited than conventional LLE and SPE for the clean-up of urinary AAS glucuronides. The LPME/LC/MS/MS method was fast and reliable, offering acceptable reproducibility and linearity with detection limits in the range 2-20 ng ml(-1) for most of the selected AAS glucuronides. The method was successfully applied to in vitro metabolic studies, and also tested with an authentic forensic urine sample. For a urine matrix the method still has some unsolved problems with specificity, which should be overcome before the method can be reliably used for doping analysis, but still offering additional and complementary data for current GC/MS analyses.

Speeding up the screening of steroids in urine: development of a user-friendly library

11 Sep 2013

Speeding up the screening of steroids in urine : development of a user-friendly library / M. Galesio, H. López-Fdez, M. Reboiro-Jato, Silvana Gómez-Meire, D. Glez-Peña, F. Fdez-Riverola, Carlos Lodeiro, M.E. Diniz, J.L. Capelo. - (Steroids 78 (2013) 12-13 (11 December); p. 1226-1232)

  • PMID: 24036418
  • DOI: 10.1016/j.steroids.2013.08.014


This work presents a novel database search engine - MLibrary - designed to assist the user in the detection and identification of androgenic anabolic steroids (AAS) and its metabolites by matrix assisted laser desorption/ionization (MALDI) and mass spectrometry-based strategies. The detection of the AAS in the samples was accomplished by searching (i) the mass spectrometric (MS) spectra against the library developed to identify possible positives and (ii) by comparison of the tandem mass spectrometric (MS/MS) spectra produced after fragmentation of the possible positives with a complete set of spectra that have previously been assigned to the software. The urinary screening for anabolic agents plays a major role in anti-doping laboratories as they represent the most abused drug class in sports. With the help of the MLibrary software application, the use of MALDI techniques for doping control is simplified and the time for evaluation and interpretation of the results is reduced. To do so, the search engine takes as input several MALDI-TOF-MS and MALDI-TOF-MS/MS spectra. It aids the researcher in an automatic mode by identifying possible positives in a single MS analysis and then confirming their presence in tandem MS analysis by comparing the experimental tandem mass spectrometric data with the database. Furthermore, the search engine can, potentially, be further expanded to other compounds in addition to AASs. The applicability of the MLibrary tool is shown through the analysis of spiked urine samples.

Efficacy of needle-free administration of recombinant human growth hormone in adults with growth hormone deficiency

2 Feb 2006

Efficacy of needle-free administration of recombinant human growth hormone in adults with growth hormone deficiency Alberto M. Pereira, Agatha A. van der Klaauw, Hans P.F. Koppeschaar, Jan W.A. Smit, Sjoerd W. van Thiel, Jaap van Doorn, Nienke R. Biermasz, Ferdinand Roelfsema, Johannes A. Romijn. - (British Journal of Clinical Pharmacology 61 (2006) 4 (April); p. 451-455)

  • PMID: 16542206
  • PMCID: PMC1885043
  • DOI: 10.1111/j.1365-2125.2006.02585.x


Aim: Needle-free administration of recombinant human growth hormone (rhGH) is effective in the treatment of growth hormone deficiency (GHD) in children, but has not been studied in adult patients. Therefore, we evaluated the efficacy of needle-free administration of rhGH in adults with GHD.

Methods: Insulin-like growth factor-I (IGF-I) concentrations were compared in newly diagnosed patients with GHD (n = 21) and in patients previously treated by subcutaneous injection of rhGH (switchers, n = 34), at baseline, 12 months and 24 months.

Results: In the new patients, IGF-I standard deviation scores (SDS) increased from - 1.82 +/- 0.46 to + 0.75 +/- 0.33 at 12 months and to + 0.65 +/- 0.41 at 24 months (P < or = 0.001 vs. baseline). In switchers, IGF-I SDS remained unchanged with values of + 0.98 +/- 0.32 at baseline, + 0.87 +/- 0.23 at 12 months and + 0.73 +/- 0.29 at 24 months (P = 0.696 vs. baseline). In new patients, the rhGH dose was 0.46 +/- 0.03 mg day(-1) at 12 months and 0.47 +/- 0.03 mg day(-1) at 24 months. In switchers, the rhGH dose was 0.53 +/- 0.04 mg day(-1) at baseline (s.c. injection), 0.52 +/- 0.03 mg day(-1) at 12 months and 0.48 +/- 0.03 mg day(-1) at 24 months (NS between the different time points). There was no difference in the dose of rhGH at 12 and 24 months between the two groups. Side-effects were generally minor and consisted of local tissue reactions.

Conclusion: Administration of rhGH by needle-free, transdermal injection is effective in maintaining IGF-I concentrations in the normal range for age in adults with GHD, and is as effective as traditional subcutaneous injection of rhGH.

Alternative long-term markers for the detection of methyltestosterone misuse

2 Nov 2012

Alternative long-term markers for the detection of methyltestosterone misuse / C Gómez, O.J. Pozo, J. Marcos, J. Segura, R. Ventur. - (Steroids 78 (2013) 1 (January); p. 44-52)

  • PMID: 23127819
  • DOI: 10.1016/j.steroids.2012.10.008


Methyltestosterone (MT) is one of the most frequently detected anabolic androgenic steroids in doping control analysis. MT misuse is commonly detected by the identification of its two main metabolites excreted as glucuronide conjugates, 17α-methyl-5α-androstan-3α,17β-diol and 17α-methyl-5β-androstan-3α,17β-diol. The detection of these metabolites is normally performed by gas chromatography-mass spectrometry, after previous hydrolysis with β-glucuronidase enzymes, extraction and derivatization steps. The aim of the present work was to study the sulphate fraction of MT and to evaluate their potential to improve the detection of the misuse of the drug in sports. MT was administered to healthy volunteers and urine samples were collected up to 30days after administration. After an extraction with ethyl acetate, urine extracts were analysed by liquid chromatography tandem mass spectrometry using electrospray ionisation in negative mode by monitoring the transition m/z 385 to m/z 97. Three diol sulphate metabolites (S1, S2 and S3) were detected. Potential structures for these metabolites were proposed after solvolysis and mass spectrometric experiments: S1, 17α-methyl-5β-androstan-3α,17β-diol 3α-sulphate; S2, 17β-methyl-5α-androstan-3α,17α-diol 3α-sulphate; and S3, 17β-methyl-5β-androstan-3α,17α-diol 3α-sulphate. Synthesis of reference compounds will be required in order to confirm the structures. The retrospectivity of these sulphate metabolites in the detection of MT misuse was compared with the obtained with previously described metabolites. Metabolite S2 was detected up to 21days after MT administration, improving between 2 and 3 times the retrospectivity of the detection compared to the last long-term metabolite of MT previously described, 17α-hydroxy-17β-methylandrostan-4,6-dien-3-one.

Current status and bioanalytical challenges in the detection of unknown anabolic androgenic steroids in doping control analysis

1 Nov 2013

Current status and bioanalytical challenges in the detection of unknown anabolic androgenic steroids in doping control analysis / Oscar J Pozo, Nik De Brabanter, Andreu Fabregat, Jordi Segura, Rosa Ventura, Peter Van Eenoo, Koen Deventer. -  (Bioanalysis 5 (2013) 21 (November); p. 2661-2677)

  • PMID: 24180506
  • DOI: 10.4155/bio.13.242


Androgenic anabolic steroids (AAS) are prohibited in sports due to their anabolic effects. Doping control laboratories usually face the screening of AAS misuse by target methods based on MS detection. Although these methods allow for the sensitive and specific detection of targeted compounds and metabolites, the rest remain undetectable. This fact opens a door for cheaters, since different AAS can be synthesized in order to evade doping control tests. This situation was evidenced in 2003 with the discovery of the designer steroid tetrahydrogestrinone. One decade after this discovery, the detection of unknown AAS still remains one of the main analytical challenges in the doping control field. In this manuscript, the current situation in the detection of unknown AAS is reviewed. Although important steps have been made in order to minimize this analytical problem and different analytical strategies have been proposed, there are still some drawbacks related to each approach.

Effect of Bimagrumab vs Placebo on Body Fat Mass Among Adults With Type 2 Diabetes and Obesity: A Phase 2 Randomized Clinical Trial

13 Jan 2021

Effect of Bimagrumab vs Placebo on Body Fat Mass Among Adults With Type 2 Diabetes and Obesity: A Phase 2 Randomized Clinical Trial / Steven B. Heymsfield, Laura A. Coleman, Ram Miller, Daniel S. Rooks, Didier Laurent, Olivier Petricoul, Jens Praestgaard, Therese Swan, Thomas Wade, Robert G. Perry, Bret H. Goodpaster, Ronenn Roubenoff. - (JAMA Netword Open 4 (2021) 1 (13 January); p. 1-13)

  • PMID: 33439265
  • PMCID: PMC7807292
  • DOI: 10.1001/jamanetworkopen.2020.33457


Importance: Antibody blockade of activin type II receptor (ActRII) signaling stimulates skeletal muscle growth. Previous clinical studies suggest that ActRII inhibition with the monoclonal antibody bimagrumab also promotes excess adipose tissue loss and improves insulin resistance.

Objective: To evaluate the efficacy and safety of bimagrumab on body composition and glycemic control in adults with type 2 diabetes and overweight and obesity.

Design, setting, and participants: This double-masked, placebo-controlled, 48-week, phase 2 randomized clinical trial was conducted among adults with type 2 diabetes, body mass index between 28 and 40, and glycated hemoglobin (HbA1c) levels between 6.5% and 10.0% at 9 US and UK sites. The trial was conducted from February 2017 to May 2019. Only participants who completed a full treatment regimen were included in analysis.

Interventions: Patients were randomized to intravenous infusion of bimagrumab (10 mg/kg up to 1200 mg in 5% dextrose solution) or placebo (5% dextrose solution) treatment every 4 weeks for 48 weeks; both groups received diet and exercise counseling.

Main outcomes and measures: The primary end point was least square mean change from baseline to week 48 in total body fat mass (FM); secondary and exploratory end points were lean mass (LM), waist circumference (WC), HbA1c level, and body weight (BW) changes from baseline to week 48.

Results: A total of 75 patients were randomized to bimagrumab (n = 37; 23 [62.2%] women) or placebo (n = 38; 12 [31.6%] women); 58 (77.3%) completed the 48-week study. Patients at baseline had a mean (SD) age of 60.4 (7.7) years; mean (SD) BMI of 32.9 (3.4); mean (SD) BW of 93.6 (14.9) kg; mean (SD) FM of 35.4 (7.5) kg; and mean (SD) HbA1c level of 7.8% (1.0%). Changes at week 48 for bimagrumab vs placebo were as follows: FM, -20.5% (-7.5 kg [80% CI, -8.3 to -6.6 kg]) vs -0.5% (-0.18 kg [80% CI, -0.99 to 0.63 kg]) (P < .001); LM, 3.6% (1.70 kg [80% CI, 1.1 to 2.3 kg]) vs -0.8% (-0.4 kg [80% CI, -1.0 to 0.1 kg]) (P < .001); WC, -9.0 cm (80% CI, -10.3 to -7.7 cm) vs 0.5 cm (80% CI, -0.8 to 1.7 cm) (P < .001); HbA1c level, -0.76 percentage points (80% CI, -1.05 to -0.48 percentage points) vs -0.04 percentage points (80% CI, -0.23 to 0.31 percentage points) (P = .005); and BW, -6.5% (-5.9 kg [80% CI, -7.1 to -4.7 kg]) vs -0.8% (-0.8 kg [80% CI, -1.9 to 0.3 kg]) (P < .001). Bimagrumab's safety and tolerability profile was consistent with prior studies.

Conclusions and relevance: In this phase 2 randomized clinical trial, ActRII blockade with bimagrumab led to significant loss of FM, gain in LM, and metabolic improvements during 48 weeks in patients with overweight or obesity who had type 2 diabetes. ActRII pathway inhibition may provide a novel approach for the pharmacologic management of excess adiposity and accompanying metabolic disturbances.

Analytical progresses of the World Anti-Doping Agency Olympic laboratories: a 2016 update from London to Rio

26 Sep 2016

Analytical progresses of the World Anti-Doping Agency Olympic laboratories : a 2016 update from London to Rio / Ioanna Athanasiadou, Sven Voss, Emmanouil Lyris, Amina Aljaber, Mohammed Alsayrafi, Costas Georgakopoulos. - (Bioanalysis 8 (2016) 21 (November); p. 2265-2279)

  • PMID: 27665839
  • DOI: 10.4155/bio-2016-0185


The 2016 Olympic and Paralympic Games, the biggest event in human sports, was held in Rio de Janeiro with more than 10,500 athletes from 206 countries over the world competing for the highest of sports honors, an Olympic medal. With the hope that the Olympic ideal accompanies all aspects of the XXXI Olympiad, WADA accredited antidoping laboratories use the spearhead of analytical technology as a powerful tool in the fight against doping. This review summarizes the main analytical developments applied in antidoping testing methodology combined with the main amendments on the WADA regulations regarding analytical testing starting from the 2012 London Olympics until the 2016 Olympic Games in Rio de Janeiro.

Chemical derivatization to enhance ionization of anabolic steroids in LC-MS for doping-control analysis

29 Oct 2012

Chemical derivatization to enhance ionization of anabolic steroids in LC-MS for doping-control analysis / Ioanna Athanasiadou, Yiannis S. Angelis, Emmanouil Lyris, Costas Georgakopoulos. - (TrAC Trends in Analytical Chemistry 42 (2013) January; p. 137-156)

  • DOI: org/10.1016/j.trac.2012.10.003


Anabolic androgenic steroids are the most frequently misused compounds in human sports, despite their prohibition by the International Olympic Committee and the World Anti-Doping Agency. In equestrian sports, this class of substances has also been prohibited by the national equestrian federations and the Federation Equestre Internationale.

It is well known that the applicability of liquid chromatography-mass spectrometry (LC-MS) in the detection of these substances presents sensitivity limitations. The absence of acidic or basic groups or conjugated oxo-systems in the structures of some anabolic steroids explains their ionization difficulties in soft atmospheric pressure ionization (API) sources [e.g., electrospray ionization (ESI), atmospheric pressure chemical ionization or atmospheric pressure photoionization].

The goal of chemical derivatization is conversion of a poorly ionizable or non-ionizable compound into an easily detectable one by changing its chemical and physical properties and it is considered as a useful strategy for the ionization enhancement of anabolic steroids in API-MS techniques.

This review underlines the efforts made in the human and animal doping-control fields to develop and to apply derivatization methods for enhancing ESI efficiency of banned anabolic steroids, so improving their detection in LC-MS analysis according to their established minimum required performance limits.

Nonprescription Steroids on the Internet

17 Jan 2012

Nonprescription Steroids on the Internet / Christen L. McDonald, Douglas B. Marlowe, Nicholas S. Patapis, David S. Festinger, Robert F. Forman. - (Substance Use & Misuse 47 (2012) 3 (February) ; p. 329-341)

  • DOI: 10.3109/10826084.2011.630225
  • PMCID: PMC3252473
  • PMID: 22080724


This study evaluated the degree to which anabolic-androgenic steroids are proffered for sale over the Internet and how they are characterized on popular websites. Searches for specific steroid product labels (e.g., Dianabol) between March and June, 2006 revealed that approximately half of the websites advocated their “safe” use, and roughly one-third offered to sell them without prescriptions. The websites frequently presented misinformation about steroids and minimized their dangers. Less than 5% of the websites presented accurate health risk information about steroids or provided information to abusers seeking to discontinue their steroid use. Implications for education, prevention, treatment and policy are discussed.

Detection of SARMs in doping control analysis

27 Feb 2017

Detection of SARMs in doping control analysis / Mario Thevis, Wilhelm Schänzer. - (Molecular and Cellular Endocrinology 464 (2018) 15 March; p. 34-45)

  • PMID: 28137616
  • DOI: 10.1016/j.mce.2017.01.040


The class of selective androgen receptor modulators (SARMs) has been the subject of intense and dedicated clinical research over the past two decades. Potential therapeutic applications of SARMs are manifold and focus particularly on the treatment of conditions manifesting in muscle loss such as general sarcopenia, cancer-associated cachexia, muscular dystrophy, etc. Consequently, based on the substantial muscle- and bone-anabolic properties of SARMs, these agents constitute substances with significant potential for misuse in sport and have therefore been added to the Word Anti-Doping Agency's (WADA's) Prohibited List in 2008. Since then, numerous adverse analytical findings have been reported for various different SARMs, which has underlined the importance of proactive and preventive anti-doping measures concerning emerging drugs such as these anabolic agents, which have evidently been misused in sport despite the fact that none of these SARMs has yet received full clinical approval. In this review, analytical data on SARMs generated in the context of research conducted for sports drug testing purposes are summarized and state-of-the-art test methods aiming at intact drugs as well as diagnostic urinary metabolites are discussed. Doping control analytical approaches predominantly rely on chromatography hyphenated to mass spectrometry, which have allowed for appropriately covering the considerable variety of pharmacophores present in SARMs such as the non-steroidal representatives ACP-105, BMS-564929, GLPG0492 (DT-200), LG-121071, LGD-2226, LGD-4033/VK 5211, ostarine/enobosarm, RAD-140, S-40503, etc. as well as steroidal compounds such as MK-0773 and YK-11.

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