Nawed Deshmukh

Quantitative analysis of mephedrone using liquid chromatography tandem mass spectroscopy: Application to human hair

Recent abuse of designer drugs such as mephedrone has presented a requirement for sensitive, reliable and reproducible methods for the detection of these controlled drugs in different matrices. This study focuses on a fully developed validated method for the quantitative analysis of mephedrone and its two metabolites 4-methylephedrine and 4-methylnorephedrine in human hair. The calibration curve was found to be linear in the range 5-100 pg/mg for mephedrone and 10-150 pg/mg for 4-methylephedrine and 4-methylnorephedrine. The method was successfully validated for the intraday precision, interday precision, limit of detection, accuracy and extraction recovery. Five out of 154 hair samples were confirmed to be positive for mephedrone. Due to the structural similarities to other methcathinones and amphetamines, one can propose the metabolism for mephedrone based on a similar pathway that has been previously used for these psychoactive drugs. The outlined method can be valuable for the future detection of mephedrone and its two metabolites in hair.

Incongruence in Doping Related Attitudes, Beliefs and Opinions in the Context of Discordant Behavioural Data: In Which Measure Do We Trust?

Background Social psychology research on doping and outcome based evaluation of primary anti-doping prevention and intervention programmes have been dominated by self-reports. Having confidence in the validity and reliability of such data is vital. Methodology/Principal Findings The sample of 82 athletes from 30 sports (52.4% female, mean age: 21.48±2.86 years) was split into quasi-experimental groups based on i) self-admitted previous experience with prohibited performance enhancing drugs (PED) and ii) the presence of at least one prohibited PED in hair covering up to 6 months prior to data collection. Participants responded to questionnaires assessing a range of social cognitive determinants of doping via self-reports; and completed a modified version of the Brief Implicit Association Test (BIAT) assessing implicit attitudes to doping relative to the acceptable nutritional supplements (NS). Social projection regarding NS was used as control. PEDs were detected in hair samples from 10 athletes (12% prevalence), none of whom admitted doping use. This group of ‘deniers’ was characterised by a dissociation between explicit (verbal declarations) and implicit (BIAT) responding, while convergence was observed in the ‘clean’ athlete group. This dissociation, if replicated, may act as a cognitive marker of the denier group, with promising applications of the combined explicit-implicit cognitive protocol as a proxy in lieu of biochemical detection methods in social science research. Overall, discrepancies in the relationship between declared doping-related opinion and implicit doping attitudes were observed between the groups, with control measures remaining unaffected. Questionnaire responses showed a pattern consistent with self-reported doping use. Conclusions/Significance Following our preliminary work, this study provides further evidence that both self-reports on behaviour and social cognitive measures could be affected by some form of response bias. This can question the validity of self-reports, with reliability remaining unaffected. Triangulation of various assessment methods is recommended.

Detection of testosterone and epitestosterone in human hair using liquid chromatography–tandem mass spectrometry

The feasibility of using hair analysis as a complimentary test in doping control has received increased attention in the scientific community. The aim of the study was to take a step forward to this goal and develop a method that, for the first time, is able to detect testosterone (T) and epitestosterone (E) in human hair, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and alkali digestion followed by extraction using pentane. The method was linear within the quantification range of 0.25-100 pg/mg for T and 0.5-100 pg/mg for E, with determination coefficient (r²) values >0.9987. The limits of detection for T and E were 0.1 pg/mg and 0.25 pg/mg respectively. The accuracy, precision and extraction recovery of the assay were satisfactory for the detection of T and E when ca. 50 mg hair was processed. The validated method was successfully applied for the analysis of 75 hair samples collected from healthy volunteers (65.3% males), with the concentration of T between 0.7-11.81 pg/mg and 0.33-6.05 pg/mg and the concentration of E between 0.63-8.27 pg/mg and 0.52-3.88 pg/mg in males and in females respectively. In males, the T levels were significantly higher (p=0.020) but there was no difference in the E levels (p=0.359). However, E was not detectable in 34 samples (of which 19 were females). The T and E levels showed linear correlation (r=0.698, p<0.001) with average T/E ratio of 1.32±0.7. The newly developed analytical method was rapid, facile, sensitive, selective, reproducible and reliable for determining the levels of T and E in hair and thus for calculating the T/E ratio in hair.

Recovery and Adaptation From Repeated Intermittent-Sprint Exercise

PURPOSE This investigation aimed to ascertain a detailed physiological profile of recovery from intermittent-sprint exercise of athletes familiar with the exercise and to investigate if athletes receive a protective effect on markers of exercise-induced muscle damage (EIMD), inflammation, and oxidative stress after a repeated exposure to an identical bout of intermittent-sprint exercise. METHODS Eight well-trained male team-sport athletes of National League or English University Premier Division standard (mean ± SD age 23 ± 3 y, VO2max 54.8 ± 4.6 mL ·kg-1 · min-1) completed the Loughborough Intermittent Shuttle Test (LIST) on 2 occasions, separated by 14 d. Maximal isometric voluntary contraction (MIVC), countermovement jump (CMJ), creatine kinase (CK), C-reactive protein (CRP), interleukin-6 (IL-6), F2-isoprostanes, and muscle soreness (DOMS) were measured before and up to 72 h after the initial and repeated LISTs. RESULTS MIVC, CMJ, CK, IL-6, and DOMS all showed main effects for time (P < .05) after the LIST, indicating that EIMD was present. DOMS peaked at 24 h after LIST 1 (110 ± 53 mm), was attenuated after LIST 2 (56 ± 39 mm), and was the only dependent variable to demonstrate a reduction in the second bout (P = .008). All other markers indicated that EIMD did not differ between bouts. CONCLUSION Well-trained games players experienced EIMD after exposure to both exercise tests, despite being accustomed to the exercise type. This suggests that well-trained athletes receive a very limited protective effect from the first bout.

Potentially harmful advantage to athletes: a putative connection between UGT2B17 gene deletion polymorphism and renal disorders with prolonged use of anabolic androgenic steroids

Background and objectiveWith prolonged use of anabolic androgenic steroids (AAS), occasional incidents of renal disorders have been observed. Independently, it has also been established that there are considerable inter-individual and inter-ethnic differences, in particular with reference to the uridine diphosphate-glucuronosyltransferase 2B17 (UGT2B17) gene, in metabolising these compounds. This report postulates the association of deletion polymorphism in the UGT2B17 gene with the occurrence of renal disorders on chronic exposure to AAS.Presentation of the hypothesisThe major deactivation and elimination pathway of AASs is through glucuronide conjugation, chiefly catalyzed by the UGT2B17 enzyme, followed by excretion in urine. Excretion of steroids is affected in individuals with a deletion mutation in the UGT2B17 gene. We hypothesize that UGT2B17 deficient individuals are more vulnerable to developing renal disorders with prolonged use of AAS owing to increases in body mass index and possible direct toxic effects of steroids on the kidneys. Elevated serum levels of biologically active steroids due to inadequate elimination can lead to prolonged muscle build up. An increase in body mass index may cause renal injuries due to sustained elevated glomerular pressure and flow rate.Testing the hypothesisIn the absence of controlled clinical trials in humans, observational studies can be carried out. Real time PCR with allelic discrimination should be employed to examine the prevalence of different UGT2B17 genotypes in patients with impaired renal function and AAS abuse. In individuals with the UGT2B17 deletion polymorphism, blood tests, biofluid analyses, urinalysis, and hair analyses following the administration of an anabolic steroid can be used to determine the fate of the substance once in the body.Implications of the hypothesisIf the hypothesis is upheld, anabolic steroid users with a deletion mutation in the UGT2B17 gene may be exposed to an increased risk of developing renal disorders. In the current detecting - sanctioning anti-doping system, athletes motivated by the potential to evade detection owing to their unique genetic make-up could subject themselves to a serious health consequence. More research on AAS metabolism in the presence of UGT2B17 gene deletion is required. Benefit - harm evaluations in therapeutic use of anabolic steroids should also consider this potential link between UGT2B17 gene deletion polymorphism and renal disorders.

Determination of stanozolol and 3′-hydroxystanozolol in rat hair, urine and serum using liquid chromatography tandem mass spectrometry

BackgroundAnabolic androgenic steroids, such as stanozolol, are typically misused by athletes during preparation for competition. Out-of-competition testing presents a unique challenge in the current anti-doping detection system owing to logistic reasons. Analysing hair for the presence of a prohibited drug offers a feasible solution for covering the wider window in out-of-competition testing. To assist in vivo studies aiming to establish a relationship between drug levels detected in hair, urine and blood, sensitive methods for the determination of stanozolol and its major metabolite 3′-hydroxystanozolol were developed in pigmented hair, urine and serum, using brown Norway rats as a model system and liquid chromatography tandem mass spectrometry (LC-MS/MS).ResultsFor method development, spiked drug free rat hair, blood and urine samples were used. The newly developed method was then applied to hair, urine and serum samples from five brown Norway rats after treatment (intraperitoneal) with stanozolol for six consecutive days at 5.0 mg/kg/day. The assay for each matrix was linear within the quantification range with determination coefficient (r2) values above 0.995. The respective assay was capable of detecting 0.125 pg/mg stanozolol and 0.25 pg/mg 3′-hydroxystanozolol with 50 mg hair; 0.063 ng/mL stanozolol and 0.125 ng/mL 3′-hydroxystanozolol with 100 μL of urine or serum. The accuracy, precision and extraction recoveries of the assays were satisfactory for the detection of both compounds in all three matrices. The average concentrations of stanozolol and 3′-hydroxystanozolol, were as follows: hair = 70.18 ± 22.32 pg/mg and 13.01 ± 3.43 pg/mg; urine = 4.34 ± 6.54 ng/mL and 9.39 ± 7.42 ng/mL; serum = 7.75 ± 3.58 ng/mL and 7.16 ± 1.97 ng/mL, respectively.ConclusionsThe developed methods are sensitive, specific and reproducible for the determination of stanozolol and 3′-hydroxystanozolol in rat hair, urine and serum. These methods can be used for in vivo studies further investigating stanozolol metabolism, but also could be extended for doping testing. Owing to the complementary nature of these tests, with urine and serum giving information on recent drug use and hair providing retrospective information on habitual use, it is suggested that blood or urine tests could accompany hair analysis and thus avoid false doping results.

LC-MS/MS-Based Assay for Free and Deconjugated Testosterone and Epitestosterone in Rat Urine and Serum

Testosterone and epitestosterone are mainly excreted as glucuronides. The aim of this study was to develop and validate a method using liquid chromatography tandem mass spectrometry (LC-MS/MS) to analyse testosterone and epitestosterone in rat serum and urine to assist in vivo studies on steroid metabolism. The method was developed by spiking charcoal stripped rat plasma and urine with the analytes. The developed method was then applied to serum (n=6) and urine samples (n=6) from young male brown Norway rats to determine testosterone and epitestosterone concentrations. The assay showed linearity within quantification range coefficient (r2) values above 0.991. Optimum conditions were determined for the deconjugation of glucuronidated testosterone and epitestosterone along with the internal standard stanozolol D3. Accuracy, precision and extraction recovery for both compounds was satisfactory in both matrices. The method was capable of quantifying 0.250 ng/mL concentrations of testosterone and epitestosterone in 100 μL of serum and urine. The average concentrations of free and deconjugated testosterone and epitestosterone found in the rat samples were: urine–201.68 ± 90.16 ng/mL and 85.37 ± 21.20 ng/mL; serum– 363.40 ± 11.615 ng/mL and 1.75 ± 0.118 ng/mL, respectively. This method is sensitive, specific and reproducible for the determination of free and deconjugated testosterone and epitestosterone in rat serum and urine. The method can be used for in vivo analysis for further investigations of testosterone and epitestosterone concentrations in studies monitoring endocrine dysfunctions and doping.

Inhibitory Effects of Diclofenac on Steroid Glucuronidation In Vivo Do Not Affect Hair-Based Doping Tests for Stanozolol

In vitro studies show that diclofenac inhibits enzymatic steroid glucuronidation. This study was designed to investigate the influence of diclofenac on the excretion of stanozolol and 3′-hydroxystanozolol via analyses in hair, blood and urine in vivo in a rat study. Brown Norway rats were administered with stanozolol (weeks 1–3) and diclofenac (weeks 1–6). Weekly assessment of steroid levels in hair was complemented with spot urine and serum tests. Levels of both stanozolol and 3′-hydroxystanozolol steadily increased in hair during stanozolol treatment and decreased post-treatment, but remained readily detectable for 6 weeks. In contrast, compared to control rats, diclofenac significantly reduced urinary excretion of 3′-hydroxystanozolol which was undetectable in most samples. This is the first report of diclofenac altering steroid metabolism in vivo, detrimentally affecting detection in urine, but not in hair, which holds considerable advantages over urinalysis for anti-doping tests.