Accurate measurement of androgen after androgen esters: problems created by ex vivo esterase effects and LC-MS/MS interference.

BACKGROUND: Ex vivo androgen prodrug conversion by blood esterases after oral androgen ester administration may result in an overestimation of the measured blood androgens. OBJECTIVE: We investigated whether blood collection tubes with esterase inhibitors decreased the conversion of testosterone undecanoate (TU) and dimethandrolone undecanoate (DMAU) to their active metabolites, testosterone (T), and dimethandrolone (DMA), providing a more accurate assessment of circulating T/DMA levels. METHODS: Blood was collected in tubes with/without esterase inhibitors from: (i) four healthy and four hypogonadal men receiving no androgens and spiked ex vivo with TU/DMAU; (ii) four men taking oral TU (Andriol® ); and (iii) eight hypogonadal men dosed with oral 316 mg TU and 15 healthy men with 200 mg DMAU. T/DMA levels were measured by LC-MS/MS. RESULTS: Sodium fluoride (NaF, an esterase inhibitor) decreased measured T levels by 14.2% in men not receiving TU. Increasing amounts of TU/DMAU added to blood collected into plain tubes resulted in a concentration-dependent overestimation of T/DMA that was reduced by collecting blood into NaF tubes (by 30-85%), and keeping samples at 4 °C and minimizing time prior to centrifugation. After oral TU/DMAU administration to men, when TU/DMAU levels were >15/10 ng/mL, respectively, blood collected in NaF tubes yielded lower measured T concentrations by 15-30% and DMA by 22% due to an additional inhibitory effect of NaF on blood esterases. CONCLUSION: NaF directly lowers plasma T/DMA levels measured by LC-MS/MS and also inhibits blood esterase activity. Overestimation of T/DMA in blood collected in tubes without NaF after oral TU/DMAU administration is important for pharmacokinetics studies in drug development clinical trials but may have limited impact in clinical practice/utilization because the differences between measured and true androgen values are modest and the wide therapeutic androgen efficacy ranges obviate the need for highly accurate androgen measurements during therapy.

Determination of testosterone esters and nortestosterone esters in animal blood serum by LC-MS/MS.

A sensitive and robust confirmatory method for determination of steroid esters in blood serum is essential for reliable monitoring of possible illegal use of steroid hormones as growth promoters in meat production. A previously used sample preparation methodology was improved. The procedure consists of protein precipitation and removal of phospholipids by dispersive SPE Supel™ QuE Z-Sep (Sigma-Aldrich) followed by clean-up on alumina column and LC-MS/MS measurement. The modified method has been validated according to Commission Decision 2002/657/EC. Validation parameters for determination of six testosterone esters and five nortestosterone esters in bovine and porcine blood serum are presented in this article. Decision limits for all analytes were observed in the range 10-20 pg mL-1. The method described is considerably robust for bovine and porcine serum analyses and can be applied both for screening and confirmatory determination in routine residue monitoring.

The effect of nandrolone treatment with and without enforced swimming on histological and biochemical changes in the heart and coronary artery of male rats.

OBJECTIVE: Chronic anabolic androgenic steroid (AAS) consumption increases incidence of cardiovascular abnormalities in athletes and mechanisms underlying those abnormalities continue to be investigated. This study examines whether nandrolone consumption induced cardiac and coronary artery wall abnormalities via oxidative stress. It was also designed to determine whether enforced swimming augmented possible cardiotoxic effects of nandrolone in rat heart. METHODS: Twenty-four male Wistar rats were divided into 3 groups: control, nandrolone, and nandrolone with enforced swimming. Nandrolone group received 10 mg/kg body weight nandrolone 3 times a week for 6 weeks. Nandrolone group with enforced swimming received the same amount of nandrolone and was forced to swim with excess weight of 20% body weight. RESULTS: After 6 weeks of treatment, results indicated proliferation of heart muscle and coronary smooth muscle cells and lipid peroxidation; significant rise in levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), nicotinamide adenine dinucleotide phosphate oxidase, homocysteine (Hcy), apolipoprotein B, low-density lipoprotein, and cholesterol, as well as severe fibrosis in heart tissue and around coronary arteries of nandrolone and nandrolone with enforced swimming groups compared with control group. CONCLUSION: These findings strongly support idea that nandrolone intake by sedentary rats and exercised rats induced heart abnormality mediated by oxidative stress, which was manifest in increased lipid peroxidation, Hcy, and 8-OHdG in heart tissue.

Where does hydrolysis of nandrolone decanoate occur in the human body after release from an oil depot?

Long-term therapy of nandrolone (N) is recommended to increase mineral density and muscle strength. Using a parenteral sustained release drug formulation with nandrolone decanoate (ND), therapeutic N levels can be achieved and maintained. Until now, it is unknown if hydrolysis of ND into N occurs in tissue at the injection site or after systemic absorption. Therefore, hydrolysis studies were conducted to investigate the location and rate of ND hydrolysis after its release from the oil depot. ND hydrolysis was studied in porcine tissues, to mimic the human muscular and subcutaneous tissues. Additionally, the ND hydrolysis was studied in human whole blood, plasma and serum at a concentration range of 23.3-233.3µM. ND hydrolysis only occurred in human whole blood. The hydrolysis did not start immediately, but after a lag time. The mean lag time for all studied concentrations was 34.9±2.5min. Because of a slow penetration into tissue, hydrolysis of ND is found to be very low in surrounding tissue. Therefore the local generation of the active compound is clinically irrelevant. It is argued that after injection of the oil depot, ND molecules will be transported via the lymphatic system towards lymph nodes. From here, it will enter the central circulation and within half an hour it will hydrolyse to the active N compound.

Fundamental understanding of drug absorption from a parenteral oil depot.

Oil depots are parenteral drug formulations meant for sustained release of lipophilic compounds. Until now, a comprehensive understanding of the mechanism of drug absorption from oil depots is lacking. The aim of this paper was to fill this gap. A clinical study with healthy volunteers was conducted. An oil depot with nandrolone decanoate and benzyl alcohol was subcutaneously administered in the upper arm of female volunteers. Pharmacokinetic profiles of both substances were related to each other and to literature data. Benzyl alcohol absorbs much more rapidly than nandrolone. In detail, it appears that benzyl alcohol enters the central compartment directly, while nandrolone decanoate is recovered in serum after a lag time. This lag time is also seen in literature data, although not reported explicitly. The absorption of nandrolone is enhanced by the presence of benzyl alcohol. This is most likely an effect of altered oil viscosity and partition coefficient between the oil and aqueous phase. The absorption rate constant of compounds is found to be related to the logP of the solubilized prodrug. The absorption rate is however not only determined by the physico-chemical properties of the formulation but also by the tissue properties. Here, it is argued that lymphatic flow must be considered as a relevant parameter.

Investigation of vitamin B6 inadequacy, induced by exposure to the anti-B6 factor 1-amino D-proline, on plasma lipophilic metabolites of rats: a metabolomics approach.

PURPOSE: Vitamin B6 status in the body is affected by several factors including dietary supply of the antivitamin B6 factor, 1-amino D-proline (1ADP), which is present in flaxseed. Owing to the prevalence of moderate B6 deficiency in the general population, a co-occurrence of 1ADP may lead to a further deterioration of B6 status. To this end, we applied a nontargeted metabolomics approach to identify potential plasma lipophilic biomarkers of deleterious effect of 1ADP on moderately vitamin B6-deficient rats using a high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. METHODS: Twenty-four rats were fed with a semi-purified diet containing pyridoxine·HCl (PN·HCl) either 7 mg/kg diet (optimal B6) or 0.7 mg/kg diet (moderate B6). The rats were divided into four treatments (n = 6), and one treatment in each B6 diet group was also fed ad libitum with 10 mg/kg diet of synthetic 1ADP. After 5 weeks of study, plasma was collected from the rats and lipophilic metabolites were extracted using acetonitrile as a solvent for analysis. RESULTS: Ten potential plasma lipophilic biomarkers were identified out of >2500 detected entities, which showed significant differences between the treatments. Plasma glycocholic acid, glycoursodeoxycholic acid, murocholic acid, N-docosahexaenoyl GABA, N-arachidonoyl GABA, lumula, nandrolone and orthothymotinic acid concentrations were significantly elevated, while plasma cystamine and 3-methyleneoxindole concentrations were significantly reduced as a result of either low B6 status or 1ADP or their interaction. CONCLUSION: Changes in these metabolites revealed a potential defect in pathways linked with the biosynthesis and metabolism of bile acid components, N-acyl amino acids, analgesic androgens, anti-inflammatory and neuroprotective molecules. We also noted that the changes in these biomarkers can be alleviated by the application of adequate vitamin B6.

Selective androgen receptor modulator activity of a steroidal antiandrogen TSAA-291 and its cofactor recruitment profile.

Selective androgen receptor modulators (SARMs) specifically bind to the androgen receptor and exert agonistic or antagonistic effects on target organs. In this study, we investigated the SARM activity of TSAA-291, previously known as a steroidal antiandrogen, in mice because TSAA-291 was found to possess partial androgen receptor agonist activity in reporter assays. In addition, to clarify the mechanism underlying its tissue selectivity, we performed comprehensive cofactor recruitment analysis of androgen receptor using TSAA-291 and dihydrotestosterone (DHT), an endogenous androgen. The androgen receptor agonistic activity of TSAA-291 was more obvious in reporter assays using skeletal muscle cells than in those using prostate cells. In castrated mice, TSAA-291 increased the weight of the levator ani muscle without increasing the weight of the prostate and seminal vesicle. Comprehensive cofactor recruitment analysis via mammalian two-hybrid methods revealed that among a total of 112 cofactors, 12 cofactors including the protein inhibitor of activated STAT 1 (PIAS1) were differently recruited to androgen receptor in the presence of TSAA-291 and DHT. Prostate displayed higher PIAS1 expression than skeletal muscle. Forced expression of the PIAS1 augmented the transcriptional activity of the androgen receptor, and silencing of PIAS1 by siRNAs suppressed the secretion of prostate-specific antigen, an androgen responsive marker. Our results demonstrate that TSAA-291 has SARM activity and suggest that TSAA-291 may induce different conformational changes of the androgen receptor and recruitment profiles of cofactors such as PIAS1, compared with DHT, to exert tissue-specific activity.

A simple, rapid and sensitive liquid chromatography-tandem mass spectrometry method for the determination of dienogest in human plasma and its pharmacokinetic applications under fasting.

A simple, rapid and sensitive analytical method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) detection with positive ion electrospray ionization was developed for the determination of dienogest in human K2 EDTA plasma using levonorgestrel d6 as an internal standard (IS). Dienogest and IS were extracted from human plasma using simple liquid-liquid extraction. Chromatographic separation was achieved on a Zorbax XDB-Phenyl column (4.6 × 75 mm, 3.5 µm) under isocratic conditions using acetonitrile-5 mm ammonium acetate (70:30, v/v) at a flow rate of 0.60 mL/min. The protonated precursor to product ion transitions monitored for dienogest and IS were at m/z 312.30 → 135.30 and 319.00 → 251.30, respectively. The method was validated with a linearity range of 1.003-200.896 ng/mL having a total analysis time for each chromatograph of 3.0 min. The method has shown tremendous reproducibility with intra- and inter-day precision (coefficient of variation) <3.97 and 6.10%, respectively, and accuracy within ±4.0% of nominal values. The validated method was applied to a pharmacokinetic study in human plasma samples generated after administration of a single oral dose of 2.0 mg dienogest tablets to healthy female volunteers and was proved to be highly reliable for the analysis of clinical samples.

Single, escalating dose pharmacokinetics, safety and food effects of a new oral androgen dimethandrolone undecanoate in man: a prototype oral male hormonal contraceptive.

The novel androgen, dimethandrolone (DMA) has both androgenic and progestational activities, properties that may maximize gonadotropin suppression. We assessed the pharmacokinetics of dimethandrolone undecanoate (DMAU), an orally bioavailable, longer acting ester of DMA, for male contraceptive development. Our objective was to examine the safety and pharmacokinetics of single, escalating doses of DMAU (powder in capsule formulation) administered orally with or without food in healthy men. We conducted a randomized, double-blind Phase 1 study. For each dose of DMAU (25-800 mg), 10 male volunteers received DMAU and two received placebo at two academic medical centres. DMAU was administered both fasting and after a high-fat meal (200-800 mg doses). Serial serum samples were collected over 24 h following each dose. DMAU was well tolerated without significant effects on vital signs, safety laboratory tests or electrocardiograms. When administered while fasting, serum DMA (active compound) was detectable in only 4/10 participants after the 800 mg dose. When administered with a 50% fat meal, serum DMA was detectable in all participants given 200 mg DMAU and showed a dose-incremental increase up to 800 mg, with peak levels 4-8 h after taking the dose. Serum gonadotropins and sex hormone concentrations were significantly suppressed 12 h after DMAU administration with food at doses above 200 mg. This first-in-man study demonstrated that a single, oral dose of DMAU up to 800 mg is safe. A high-fat meal markedly improved DMAU/DMA pharmacokinetics.

Pharmacokinetic-pharmacodynamic study of subcutaneous injection of depot nandrolone decanoate using dried blood spots sampling coupled with ultrapressure liquid chromatography tandem mass spectrometry assays.

CONTEXT: Testosterone (T) and nandrolone (N) esters require deep im injections by medical personnel but these often deposit injectate into sc fat so that more convenient sc self-administration may be feasible. OBJECTIVE: To investigate the feasibility and pharmacology of sc injection of N decanoate in healthy men using dried blood spot (DBS) for frequent blood sampling without clinic visits. SETTING AND DESIGN: Healthy male volunteers received 100 mg N decanoate by a single sc injection. Finger-prick capillary blood was spotted onto filter paper before injection daily at home for 21 d and stored at room temperature. Venous whole blood was also spotted onto filter paper before and weekly for 3 wk after injection. DBS were extracted for assay of N and T by liquid chromatography tandem mass spectrometry in a single batch with serum concentrations estimated with adjustment for capillary blood sample volume and hematocrit to define peak (N) or nadir (T) time and concentration from individual daily measurements. RESULTS: Daily serum N peaked 2.50 ± 0.25 (SEM) ng/mL at a median (range) of 6 (4-13) days causing a reduction in serum T from 3.50 ± 0.57 ng/mL at baseline to a nadir of 0.38 ± 0.13 (SEM) ng/mL (89 ± 3% suppression) at a median (range) of 8 (5-16) days. Simultaneously sampled capillary, venous whole blood, and serum gave almost identical results for serum T and N. Finger-pricks and sc injections were well tolerated. CONCLUSIONS: This study demonstrates that A) DBS sampling with liquid chromatography mass spectrometry steroid analysis achieves frequent time sampling in the community without requiring clinic visits, venesection, or frozen serum storage, and B) androgen esters in an oil vehicle can be delivered effectively by sc injection, thus avoiding the need for medically supervised deep-im injections.

Sudden or unnatural deaths involving anabolic-androgenic steroids.

Anabolic-androgenic steroids (AASs) are frequently misused. To determine causes of death, characteristics, toxicology, and pathology of AAS positive cases, all cases (n = 24) presenting to the New South Wales Department of Forensic Medicine (1995-2012) were retrieved. All were male, and the mean age was 31.7 years. Deaths were mainly due to accidental drug toxicity (62.5%), then suicide (16.7%) and homicide (12.5%). Abnormal testosterone/epitestosterone ratios were reported in 62.5%, followed by metabolites of nandrolone (58.3%), stanozolol (33.3%), and methandienone (20.8%). In 23 of 24 cases, substances other than steroids were detected, most commonly psychostimulants (66.7%). In nearly half, testicular atrophy was noted, as was testicular fibrosis and arrested spermatogenesis. Left ventricular hypertrophy was noted in 30.4%, and moderate to severe narrowing of the coronary arteries in 26.1%. To summarize, the typical case was a male polydrug user aged in their thirties, with death due to drug toxicity. Extensive cardiovascular disease was particularly notable.

Monitoring the endogenous steroid profile disruption in urine and blood upon nandrolone administration: An efficient and innovative strategy to screen for nandrolone abuse in entire male horses.

Nandrolone (17ß-hydroxy-4-estren-3-one) is amongst the most misused endogenous steroid hormones in entire male horses. The detection of such a substance is challenging with regard to its endogenous presence. The current international threshold level for nandrolone misuse is based on the urinary concentration ratio of 5α-estrane-3ß,17α-diol (EAD) to 5(10)-estrene-3ß,17α-diol (EED). This ratio, however, can be influenced by a number of factors due to existing intra- and inter-variability standing, respectively, for the variation occurring in endogenous steroids concentration levels in a single subject and the variation in those same concentration levels observed between different subjects. Targeting an efficient detection of nandrolone misuse in entire male horses, an analytical strategy was set up in order to profile a group of endogenous steroids in nandrolone-treated and non-treated equines. Experiment plasma and urine samples were steadily collected over more than three months from a stallion administered with nandrolone laurate (1 mg/kg). Control plasma and urine samples were collected monthly from seven non-treated stallions over a one-year period. A large panel of steroids of interest (n = 23) were extracted from equine urine and plasma samples using a C18 cartridge. Following a methanolysis step, liquid-liquid and solid-phase extractions purifications were performed before derivatization and analysis on gas chromatography-tandem mass spectrometry (GC-MS/MS) for quantification. Statistical processing of the collected data permitted to establish statistical models capable of discriminating control samples from those collected during the three months following administration. Furthermore, these statistical models succeeded in predicting the compliance status of additional samples collected from racing horses.

Pharmacokinetic study of single and multiple oral administrations of 2 mg dienogest in healthy Korean women.

BACKGROUND: The progestin dienogest was developed for oral contraception, endometriosis treatment and menopause management. Dienogest's pharmacokinetics have been primarily studied in Caucasian women. This study evaluated the single- and multiple-dose pharmacokinetics of dienogest in Korean women. STUDY DESIGN: Sixteen healthy Korean adult women received a single administration of 2 mg dienogest, followed by multiple once-daily administrations for 14 days. The single-dose administration and the final dose of the multiple administrations were each followed by blood sampling over 60 h. RESULTS: The mean (SD) maximum serum concentration after multiple doses of dienogest was slightly increased compared with that after a single dose [from 51.6 (9.6) to 56.6 (11.9) ng/mL], as was the area under the concentration-time curves (AUC)0-24h [from 503 (56.3) to 613 (90.7) ng ∙ h/mL]. The linearity factor calculated by AUCs of single and multiple doses is 1.00 ± 0.14, and the terminal half-life remained unchanged when single dosing and multiple dosing were compared. CONCLUSIONS: The present study described the single- and multiple-dose pharmacokinetic profiles of dienogest in Korean women and showed linear pharmacokinetics of dienogest at steady state.

Evaluation of the effects of rifampicin, ketoconazole and erythromycin on the steady-state pharmacokinetics of the components of a novel oral contraceptive containing estradiol valerate and dienogest in healthy postmenopausal women.

BACKGROUND: We evaluated the effects of cytochrome P450 3A4 (CYP3A4) induction and inhibition on steady-state pharmacokinetics of the components of a novel oral contraceptive (OC) containing estradiol valerate (E2V) and dienogest (DNG). STUDY DESIGN: CYP3A4 induction was assessed in an open-label, one-arm study. Sixteen healthy postmenopausal women received E2V 2 mg/DNG 3 mg (days 1-17) and concomitant rifampicin (600 mg, days 12-16). Ratios of the area under the serum concentration-time curve between 0 and 24 h [AUC(0-24 h)] and maximum serum concentration (C(max)) of E2 and DNG on days 17 and 11 (after and before rifampicin intervention) are presented. CYP3A4 inhibition was investigated in an open-label, parallel-group study in 24 healthy postmenopausal women receiving E2V 2 mg/DNG 3 mg (days 1-14) and concomitant ketoconazole (400 mg, n=12) or erythromycin (500 mg three times daily, n=12) on days 8-14. Mean ratios of AUC(0-24 h) and C(max) of E2 and DNG on days 7 and 14 are presented. RESULTS: Concomitant administration of rifampicin decreased systemic drug exposure and yielded geometric mean ratios for E2C(max) and AUC(0-24 h) of 75% and 56%, respectively. Corresponding mean ratios for DNG were 48% and 17%, respectively. Ketoconazole coadministration increased systemic drug exposure and yielded ratios of E2 of 165% and 157%, respectively, and ratios of DNG of 194% and 286%, respectively. Erythromycin coadministration also resulted in increased mean C(max) and AUC(0-24 h) of both E2 and DNG. Geometric mean ratios of C(max) and AUC(0-24 h) for E2 were 151% and 133%, respectively. Corresponding ratios for DNG were 133% and 162%, respectively. CONCLUSIONS: Significant drug-drug interactions are apparent when CYP3A4 modulators are coadministered with the components of a novel OC containing E2V/DNG. Coadministration of CYP3A4 modulators should be avoided where possible, and another type of contraception should be used when coadministration of CYP3A4 inducers like rifampicin is unavoidable.

Plasma concentrations of testosterone and nandrolone in racing and nonracing intact male horses.

Pennsylvania (PA) State Racing Commissions regulate the endogenous androgenic steroid, testosterone (TES), in racing intact males (RIM) by quantification of TES in post-race samples. Post-race plasma samples (2209) collected between March 2008 and November 2010 were analyzed for TES, nandrolone (NAN), and other anabolic steroids (ABS). Of the 2209 plasma samples, 2098 had quantifiable TES ≥ 25 pg/mL. Plasma (mean ± SD) concentrations of TES and NAN in RIM were 329.2 ± 266.4 and 96.0 ± 67.8 pg/mL, respectively. Only 64.6% of RIM had quantifiable concentration of NAN, and there was no relationship between TES and NAN. Plasma TES concentrations were significantly (P < 0.0001) higher during the months of April, May, June, July, and August. A significantly higher (P < 0.006) plasma TES was observed in Thoroughbred (TB) (347.6 ± 288.5 pg/mL) vs. that in Standardbred (STB) (315.4 ± 247.7 pg/mL). Plasma concentrations of TES from breeding stallions (BS) were 601.6 ± 356.5 pg/mL. Statistically significant (P < 0.0001) lower plasma concentrations of the two steroids were observed in RIM horses. Based on quantile distribution of TES in the RIM and BS populations, 99.5% were at or below 1546.1 and 1778.0 pg/mL, respectively. Based on this population of RIM, the suggested upper threshold plasma concentration of endogenous TES in horses competing in PA should remain at 2000 pg/mL.

Ex vivo spontaneous generation of 19-norandrostenedione and nandrolone detected in equine plasma and urine.

19-Norandrostenedione (NAED) and nandrolone are anabolic-androgenic steroids (AASs). Nandrolone was regarded solely as a synthetic AAS until the 1980s when trace concentrations of apparently endogenous nandrolone were detected in urine samples obtained from intact male horses (stallions). Since then, its endogenous origin has been reported in boars and bulls; endogenous NAED and nandrolone have been identified in plasma and urine samples collected from stallions. More recently, however, it was suggested that NAED and nandrolone detected in urine samples from stallions are primarily artifacts due to the analytical procedure. The present study was undertaken to determine whether NAED and nandrolone detected in plasma and urine samples collected from stallions are truly endogenous or artifacts from sample processing. To answer this question, fresh plasma and urine samples from ≥8 stallions were analyzed for the two AASs, soon after collection, by liquid chromatography hyphenated to tandem mass spectrometry (LC-MS/MS). NAED and nandrolone were not detected in fresh plasma samples but detected in the same samples post storage. Concentrations of both AASs increased with storage time, and the increases were greater at a higher storage temperature (37°C versus 4°C, and ambient temperature versus 4°C). Although NAED was detected in some fresh stallion urine samples, its concentration (<407 pg/mL) was far lower (<0.4%) than that in the same samples post storage (at ambient temperature for 15 days). Nandrolone was not detected in most of fresh urine samples but detected in the same samples post storage. Based on these results, it is concluded that all NAED and nandrolone detected in stored plasma samples of stallions and most of them in the stored urine samples are not from endogenous origins but spontaneously generated during sample storage, most likely from spontaneous decarboxylation of androstenedione-19-oic acid and testosterone-19-oic acid. To our knowledge, it is the first time that all NAED and nandrolone detected in plasma of stallions and most of them detected in the urine have been shown to be spontaneously generated in vitro during sample storage. This finding would have significant implications with regard to the regulation of the two steroids in horse racing.

Simultaneous separation and determination of 16 testosterone and nandrolone esters in equine plasma using ultra high performance liquid chromatography-tandem mass spectrometry for doping control.

The potential for using testosterone and nandrolone esters in racehorses to boost the biological concentrations of these steroids and enhance athletic performance is very compelling and should be seriously considered in formulating regulatory policies for doping control. In order to regulate the use of these esters in racehorses, a sensitive and validated method is needed. In this paper, we report such a method for simultaneous separation, screening, quantification and confirmation of 16 testosterone and nandrolone esters in equine plasma by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Analytes were extracted from equine plasma by liquid-liquid extraction using a mixture of methyl tert-butyl ether and ethyl acetate (50:50, v/v) and separated on a sub-2 micron C(18) column. Detection of analytes was achieved on a triple-quadrupole mass spectrometer by positive electrospray ionization mode with selected reaction monitoring (SRM). Mobile phase comprised 2 mM ammonium formate and methanol. Deuterium-labeled testosterone enanthate and testosterone undecanoate were used as dual-internal standards for quantification. Limits of detection (LOD) and quantification (LOQ) were 25-100 pg/mL and 100-200 pg/mL, respectively. The linear dynamic range of quantification was 100-10,000 pg/mL. For confirmation of the presence of these analytes in equine plasma, matching of the retention time with mass spectrometric ion ratios from MS/MS product ions was used. The limit of confirmation (LOC) was 100-500 pg/mL. The method is sensitive, robust, selective and reliably reproducible.

Persistent primary hypogonadism associated with anabolic steroid abuse.

OBJECTIVE: To report a case of primary gonadal failure due to the chronic abuse of anabolic steroids used for bodybuilding. DESIGN: Case report. SETTING: Department of Diabetes and Endocrinology, Morriston Hospital, Swansea, Wales, United Kingdom. PATIENT(S): A 40-year-old man. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Clinical symptoms, levels of serum T, FSH, and LH. RESULT(S): Primary gonadal failure resulting from anabolic steroid use. CONCLUSION(S): We describe a case of initially secondary gonadal failure resulting from anabolic steroid use with subsequent primary gonadal failure and infertility. This case adds to the current literature and illustrates that the side effects of anabolic steroids can be prolonged and irreversible.

Subchronic nandrolone administration reduces cocaine-induced dopamine and 5-hydroxytryptamine outflow in the rat nucleus accumbens.

RATIONALE: The abuse of anabolic androgenic steroids (AASs) is not only a problem in the world of sports but is associated with the polydrug use of nonathletes. Investigations of the neurochemical effects of AAS have focused in part on the monoaminergic systems, involving, among other things, the development of dependence. We have previously shown that pretreatment with nandrolone decanoate attenuates dose-dependently the increase in extracellular dopamine (DA) concentration evoked by amphetamine and 3,4-methylenedioyxymethamphetamine in the nucleus accumbens (NAc). OBJECTIVES: The aim of this study was to investigate whether the nandrolone pre-exposure modulates the acute neurochemical and behavioral effects of cocaine in rats and whether the effects are long lasting. METHODS: DA, 5-hydroxytryptamine (5-HT), and their metabolites were measured from samples collected from the NAc by microdialysis. The behavior of the animals was recorded. RESULTS: The present study demonstrates that five injections of nandrolone (5 and 20 mg/kg) inhibited cocaine-evoked DA and 5-HT outflow in the NAc, locomotor activity (LMA), and stereotyped behavior in experimental animals, and that these effects are seen even after elimination of nandrolone from bloodstream. CONCLUSIONS: Given that accumbal outflow of DA and 5-HT, as well as LMA and stereotyped behavior, is related to gratification of stimulant drugs, this study suggests that nandrolone, at the doses tested, has a significant effect on the pleasurable properties of cocaine. Furthermore, because neurochemical and behavioral responses were still attenuated after a fairly long recovery period, it seems that nandrolone may induce long-lasting changes in the brains of rat.

Pharmacokinetics of an oral contraceptive containing oestradiol valerate and dienogest.

OBJECTIVE: To evaluate the pharmacokinetics of a combined oral contraceptive (OC) containing oestradiol valerate/dienogest (E2V/DNG) administered according to a four-phasic dosing regimen with an oestrogen step-down and a progestin step-up over 26 days of active treatment. METHODS: This Phase I, open-label study included healthy women aged 18-50 years. Treatment consisted of the administration of E2V 3 mg for 2 days, E2V 2 mg/DNG 2 mg for 5 days, E2V 2 mg/DNG 3 mg for 17 days, E2V 1 mg for 2 days, and placebo for 2 days. RESULTS: Pharmacokinetic data were analysed in 15 women. Stable E2 concentrations were maintained throughout the study. Minimum mean serum E2 levels were 33.6-64.7 pg/ml during E2V administration. The ratio of oestrone:E2 in serum was approximately 5:1. Minimum mean serum DNG levels were 6.8-15.1 ng/ml during DNG administration. Minimum concentrations of DNG increased only slightly during each phase of the regimen during which DNG was being administered. On day 24 the geometric mean C(max), C(ave) and t((1/2)) of DNG were 82.9 ng/ml, 33.7 ng/ml and 12.2 hours, respectively; the median t(max) was 1.5 hours. Serum sex hormone-binding globulin concentrations increased by 40% (within the normal range). Cortisol binding-globulin levels remained almost unchanged. Treatment was well tolerated. CONCLUSIONS: Treatment with an OC containing E2V and DNG was well tolerated and was associated with stable E2 concentrations over 28 days. The pharmacokinetics of DNG were consistent with previous findings. Minimum serum concentrations of DNG increased only slightly during phases of the regimen during which DNG was administered.