Being performance enhancing hormones, endogenous anabolic androgenic steroids (EAAS) are banned from most competitive sports by the World Anti-doping Agency (WADA). In anti-doping control laboratories, routine assays are mainly performed on urine samples of athletes in and out of competitions. Serum constitutes a promising alternative to urine as it is less subjected to manipulation or contamination that may influence the method sensitivity. The simultaneous determination of EAAS including conjugated metabolites using LC-MS is very challenging due to their contradicting chemical behaviors at the ionization interface of the mass spectrometer. This may prejudice their detection or limit the method sensitivity. Herein, we have addressed these challenges and developed a new method for the simultaneous determination of unconjugated, sulphate- and glucuronide-conjugated EAAS (Androsterone, Etiocholanolone, testosterone, epitestosterone, dihydrotestosterone, dehydroepiandrosterone, androstenedione and 17a-hydroxyprogesterone) in human serum using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The use of mass spectrometric detection in full scan mode facilitated the study of the most versatile adducts for detection and quantitation. A solid phase extraction method was developed for the sample preparation prior to analysis. The method limits of quantitation ranged from 0.006 to 7.904 ng/mL and the recoveries ranged from 70.2% to 96.5%. The method calibration was performed in untreated serum representing realistic matrix composition with correlation coeffecients ranged from 0.9859 to 0.9988. Finally, the serum-levels of the investigated steroids were determined in 4 male and 1 female human subjects to provide estimates of baseline levels based on individual values.

Concentrations of urinary steroids are measured in anti-doping test programs to detect doping with endogenous steroids. These concentrations are combined into ratios and followed over time in the steroidal module of the Athlete Biological Passport (ABP). The most important ratio in the ABP is the testosterone/epitestosterone (T/E) ratio but this ratio is subject to intra-individual variations, especially large in women, which complicates interpretation. In addition, there are other factors affecting T/E. Pregnancy, for example, is known to affect the urinary excretion rate of epitestosterone and hence the T/E ratio. However, the extent of this variation and how pregnancy affect other ratios has not been fully evaluated. Here we have studied the urinary steroid profile, including 19-norandrosterone (19-NA), in 67 pregnant women and compared to postpartum. Epitestosterone was higher and, consequently, the T/E and 5αAdiol/E ratios were lower in the pregnant women. Androsterone/etiocholanolone (A/Etio) and 5αAdiol/5βAdiol, on the other hand, were higher in the first trimester as compared to postpartum (p<0.0001 and p=0.0396, respectively). There was no difference in A/T during pregnancy or after. 19-NA was present in 90.5% of the urine samples collected from pregnant women. In this study, we have shown that the steroid profile of the ABP is affected by pregnancy, and hence can cause atypical passport findings. These atypical findings would lead to unnecessary confirmation procedures, if the patterns of pregnancy are not recognized by the ABP management units.

Androstenedione (4-androstene-3,17-dione) is banned by the World Anti-Doping Agency (WADA) as an endogenous steroid. The official method to confirm androstenedione abuse is isotope ratio mass spectrometry (IRMS). According to the guidance published by WADA, atypical steroid profiles are required to trigger IRMS analysis. However, in some situations, steroid profile parameters are not effective enough to suspect the misuse of endogenous steroids. The aim of this study was to investigate the atypical steroid profile induced by androstenedione administration and the detection of androstenedione doping using IRMS. Ingestion of androstenedione resulted in changes in urinary steroid profile, including increased concentrations of androsterone (An), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5α-diol), and 5β-androstane-3α,17β-diol (5β-diol) in all of the subjects. Nevertheless, the testosterone/epitestosterone (T/E) ratio was elevated only in some of the subjects. The rapid increases in the concentrations of An and Etio, as well as in T/E ratio for some subjects could provide indicators for initiating IRMS analysis only for a short time period, 2-22h post-administration. However, IRMS could provide positive determinations for up to 55h post-administration. This study demonstrated that, 5β-diol concentration or Etio/An ratio could be utilized as useful indicators for initiating IRMS analysis during 2-36h post-administration. Lastly, Etio, with slower clearance, could be more effectively used than An for the confirmation of androstenedione doping using IRMS.

The urinary steroid profile has been used in clinical endocrinology for the early detection of enzyme deficiencies. In the field of doping, its evaluation in urine samples is used to diagnose the abuse of substances prohibited in sport. This profile is influenced by sex, age, exercise, diet, and ethnicity, among others; laboratories own reference ranges might compensate for ethnic differences among population and inter-laboratory biases. This paper shows the reference ranges obtained in the Antidoping Laboratory of Havana for the following steroid profile parameters: ten androgens (testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstan-3α,17β-diol, 5β-androstan-3α,17β-diol, dehydroepiandrosterone, epiandrosterone, 11β-hydroxyandrosterone and 11β-hydroxyetiocholanolone), three estrogens (estradiol, estriol and estrone), two pregnanes (pregnanediol and pregnanetriol) and two corticosteroids (cortisol and tetrahydrocortisol). The urine samples (male: n = 2454 and female: n = 1181) and data obtained are representative of population from Latin-American countries like Cuba, Venezuela, Mexico, Dominican Republic, Guatemala and Chile. Urine samples were prepared by solid-phase extraction followed by enzymatic hydrolysis and liquid-liquid extraction with an organic solvent in basic conditions. Trimethylsilyl derivatives were analyzed by gas chromatography coupled to mass spectrometry. Reference ranges were established for each sex, allowing the determination of abnormal profiles as a first diagnostic tool for the detection of the abuse of androgenic anabolic steroids. The comparison with the Caucasian population confirms that the urinary steroid profile is influenced by ethnicity.

This study proposes a new analytical methodology for the determination of trace levels of testosterone (T) and epitestosterone (E) in urine matrices using bar adsorptive microextraction combined with liquid desorption followed by high-performance liquid chromatography with diode array detection (BAμE-LD/HPLC-DAD). The comparison of different sorbent coatings (five activated carbons, one styrene-divinylbenzene, two modified pyrrolidone, one ciano and one n-vinylpyrrolidone polymers) through BAμE showed that the latter phase presented much higher selectivity and capacity offering multiple mechanisms of interaction. Assays using this phase were performed on 25mL of water samples spiked at the 8.0μg/L level, yielded average recoveries of 92.1 and 93.4% for T and E, respectively, under optimized experimental conditions; BAμE (n-vinylpyrrolidone): 16h (1000rpm), pH 5.5; LD: acetonitrile, 30min under sonication treatment. From the developed analytical methodology, suitable detection limits were achieved (0.4μg/L) and good linear dynamic ranges (1.4-16.0μg/L) with remarkable determination coefficients (r(2)>0.9978). By using the standard addition methodology, the application of the present analytical approach on urine samples revealed good sensitivity. The proposed method, which operated under the floating sampling technology, proved to be a suitable sorption-based static microextraction alternative for screening T, E and the T/E ratio in urine samples for doping control purposes. The methodology showed to be easy to implement, demonstrating good reproducibility, sensitivity and robustness, allowing the possibility to choose the most selective sorbent coating according to the compounds of interest.

Previous studies carried out by the Authors had shown an increased urinary excretion of epitestosterone, a poorly active androgen mostly believed to be of adrenal origin, after HCG administration to presenescent and aging subjects. Purpose of this study was to ascertain whether or not this held in the young too. Testosterone excretion sharply decreased after the age of 60 (P less than 0,001); 17 KS on the other hand, decreased progressively in such a way that the aging group showed significantly lower (p less than 0,05) excretion rates than the only young-adult group. ET excretion decreased steadily but not significantly with age. ET showed a very good correlation to T (p less than 0,01) both under basal and poststimulatory conditions in each of the three age groups. ET percent increase showed a good correlation (p less than 0,01) to T percent increase only in young-adult and presenescent people, not in the elderly. Poor was the correlation between ET percent increase and 17 KS percent increase. Thus ET excretion pattern showed to be somewhat different from that of T and very different from that of 17 KS. As a conclusion (a) epitestosterone excretion rate seems to decrease steadily but not significantly with age; (b) epitestosterone probably is not mainly secreted by the adrenal gland. A major source may be the testis, but liver, prostate and blood red cells metabolism are not definitely ruled out.

Epitestosterone (17alpha-hydroxy-androst-4-en-3-one, EpiT) belongs to the list of prohibited substances of the World Anti-Doping Agency (WADA). Although it possesses no anabolic effect, it is presumed to be misused by athletes in order to mask administration of testosterone (T) by lowering the urinary T/EpiT ratio.To improve detection, an excretion study with 40 mg of orally administered EpiT was conducted focusing on the metabolites of EpiT: 5alpha- and 5beta-androstane-3alpha,17 alpha-diol (5aEpiD and 5bEpiD). A reference population of n = 74 volunteers was investigated to elucidate the urinary concentrations of these steroids.In order to prove whether an unusual finding in urinary concentrations or ratios is due to an illicit intake of steroids or due to physiological elevation, determination of carbon isotope ratios is advisable. A method for isotope ratio determination was developed to enable (13)C/(12)C ratios of EpiT, 5bEpiD, 5aEpiD, pregnanediol and androsterone and etiocholanolone to be measured from a single urine specimen. The method's validity was tested by applying linear mixing models and specificity was ensured by means of gas chromatography/mass spectrometry analysis. delta(13)C values at natural levels were determined with a reference population and both Delta values and corresponding reference limits were calculated.Considering the implemented EpiT-metabolites, a more than twofold extension of the detection time of EpiT administration was achieved with both the urinary concentration thresholds and the (13)C/(12)C ratios.

A sensitive and rapid liquid chromatographic (LC) method for the simultaneous determination of testosterone (T) and epitestosterone (E) in human urine samples has been developed and elaborated. The ratio of the both steroids (T/E) in human urine is a widely used as doping control indicator. A sample pretreatment by solid-phase extraction (SPE) after hydrolysis using 36% hydrochloric acid for determination of total level of T has been applied. Unconjugated (free) form of the both androgens were determined without hydrolysis steps, what makes novelty of the method, because simplifies the proposed procedure. In turn, the measurements of urinary free T and E provided the diagnostic information for excess adrenal production of steroids. The proposed LC assay was evaluated by analyzing a series of urine samples containing T, E and methyltestosterone (MT) as internal standard at the range of concentration 2-300 ng(-1)mL of both analyzed hormones. The proposed method was fully validated for specificity, linearity, limits of detection and quantitation, precision and trueness according to the current requirements concerning analytical methods. Interestingly, the developed LC method allows to obtain a sensitive enhancement with respect to UV detection with the quantitation limit for T and E equaled 2 ng mL(-1). The method was selective and reliable for identity and enable to detect changes of endogenous levels of T and E in urine independently of fluctuations characteristic for both analyzed endogenous hormone level in plasma.

BACKGROUND

In 2006 Floyd Landis won the world's most prestigious bicycle race, the Tour de France. However, not many days after the race's conclusion it was released to the press that the Laboratoire National de Dépistage du Dopage (LNDD) had found Landis' urine after stage 17 positive for synthetic testosterone.

METHODS

This review examines the instrumental data and methodology used by LNDD in the Landis case. The conclusions reached by LNDD were based on results of 2 separate instrumental methods. Subsequent to urine extraction and possibly derivatization, samples were initially screened via gas chromatography/mass spectrometry (GC/MS) using selected ion monitoring (SIM) to measure the ratio of testosterone to epitestosterone (T/E). Final confirmation of exogenous testosterone was determined by measuring the (13)C/(12)C stable isotope ratios in 4 metabolites of testosterone via gas chromatography combustion stable isotope ratio mass spectrometry (GC-C-IRMS).

CONCLUSIONS

T/E ratios determined by LNDD in Landis' stage 17 urine were unreliable due to the combined factors of an unsatisfactory extraction, high GC background, failure to obtain baseline peak separation for epitestosterone, unreliable quantization of the epitestosterone peak due to both peak overlap and because it was barely above background noise, and because only a single ion mass (432) rather than a minimum of 3 was used for SIM (in violation of both LNDD's SOP and WADA procedures). GC-C-IRMS methodology is less well known to the analytical chemistry community, but here too the results obtained by LNDD were unreliable. GC-C-IRMS errors can be briefly summarized as uncertain peak identification, unsuitable standards, and unreliable (and possibly biased) calculation of (13)C/(12)C ratios due to peak overlap as well as LNDD's usage of manual peak integration rather than use of the instrument system software.

Twenty-one healthy male volunteers aged 20-70 years were given transdermally 25 mg of 7-oxo-dehydroepiandrosterone daily in the form of an emulgel for 8 consecutive days. Morning blood was collected as follows: before application, and after the first, fourth and eighth doses (days 0, 2, 5 and 9), and then at different time intervals after termination of the treatment (days 16, 23, 37, 51, 72 and 100). Cortisol, testosterone, epitestosterone, estradiol, dehydroepiandrosterone and its sulfate, 7alpha- and 7beta-hydroxy-dehydroepiandrosterone, luteinizing hormone, follicle-stimulating hormone and sex hormone-binding globulin were measured in blood sera. In the course of treatment 7beta-hydroxy-dehydroepiandrosterone was significantly increased; testosterone and gonadotropins were lowered, but only after the first dose. All other significant changes were observed during the period after termination of the application:7beta-hydroxy-dehydroepiandrosterone remained increased for 28 days, 7alpha-hydroxy-dehydroepiandrosterone, testosterone, estradiol and sex hormone-binding globulin were decreased as late as day 63 and 91, respectively. On the other hand, epitestosterone was significantly increased between days 23 and 100. The levels of all other parameters studied were not significantly changed. The study points to an immediate as well as delayed effect of the short-term transdermal application of 7-oxo-dehydroepiandrosterone on relevant hormonal parameters.

The use of testosterone and its pro-drugs, such as dehydroepiandrosterone (DHEA), is currently regulated in horseracing by the application of international testosterone thresholds. However, additional steroidomic approaches, such as steroid ratios, to distinguish overall adrenal stimulation from drug administrations and an equine biological passport for longitudinal steroid profiling of individual animals could be advantageous in equine doping testing. Thus, DHEA concentrations and related ratios (testosterone [T] to DHEA and DHEA to epitestosterone [E]) were assessed in the reference population by quantitative analysis of 200 post-race gelding urine samples using liquid chromatography-tandem mass spectrometry. DHEA concentrations ranged between 0.9 and 136.6 ng/mL (mean 12.8 ng/mL), T:DHEA ratios between 0.06 and 1.85 (mean 0.43), and DHEA:E ratios between 0.21 and 13.56 (mean 2.20). Based on the reference population statistical upper limits of 5.4 for T:DHEA ratio and 48.1 for DHEA:E ratio are proposed with a risk of 1 in 10 000 for a normal outlier exceeding the value. Analysis of post-administration urine samples collected following administrations of DHEA, Equi-Bolic® (a mix of DHEA and pregnenolone) and testosterone propionate to geldings showed that the upper limit for T:DHEA ratio was exceeded following testosterone propionate administration and DHEA:E ratio following DHEA administrations and thus these ratios could be used as additional biomarkers when determining the cause of an atypical testosterone concentration. Additionally, DHEA concentrations and ratios can be used as a starting point to establish reference ranges for an equine biological passport.

Epitestosterone is a natural component of biological fluids of several mammals including man. For a long time it has been believed that it is a metabolite without any hormonal activity and without any marked relationship to the hormonal state in health and disease. The biosynthetic pathway as well as the site of its formation in man has not been unequivocally confirmed until now. It apparently parallels the formation of testosterone but, on the other hand, its concentration is not influenced by exogenously administered testosterone. This fact creates the basis of the present doping control of testosterone abuse. In 1989 an observation was presented in a dermatological study that epitestosterone has an effect counteracting the action of testosterone on flank organ of Syrian hamster. Further studies showed that a complex action consisting of competitive binding of epitestosterone to androgen receptor, of inhibition of testosterone biosynthesis and its reduction to dihydrotestosterone and of antigonadotropic activity could be demonstrated in rat, mice and human tissues. It can be presumed that epitestosterone as a natural hormone can contribute to the regulation of androgen dependent events as e.g. to the control of growth of prostate or body hair distribution.

To investigate the effect of gestagens on renal cell carcinoma 300-mg slices of normal, human kidney, renal cell carcinoma (RCC), and preoperatively irradiated RCC were subjected to a short term incubation with 200 pmoles of 3H-testosterone and 1 and 2 mug of gestonorone caproate. In the normal kidney 61.4 per cent of the testosterone added was metabolized, the oxidation products androstenedione and epitestosterone outweighing by 6:1 the reduction products 5alpha-dihydrotestosterone and androstanediol. In RCC only 22 per cent of the testosterone was metabolized, with 8.5 per cent being converted to 5alpha-androstanes. Gestonorone caproate essentially did not influence testosterone turnover. This can be explained by its action as an inhibitor of the reductive pathway of the testosterone metabolism only, which is insignificant in these tissues.

The drug test for exogenous administration of testosterone is based on the testosterone/epitestosterone ratio (T/E) in urine. Physiological and psychological stresses may alter plasma testosterone concentrations. The question is to know how much the psychological conditions of competition can modify the T/E ratio. In order to study this issue, 20 athletes practising modern pentathlon participated in a study designed to determine the effects of a pistol shooting trial on their hormonal response. Pistol shooting induces a high psychological stress without increasing energy expenditure. Venous blood samples were drawn before and after the trial according to the usual drug testing procedure. Athletes were separated into two groups: a group of young athletes (n = 10; mean age 19 +/- 0.3 years) and another group of aged subjects (n = 10; mean age 45 +/- 1.5 years). The rise in plasma testosterone concentrations reached 75% in older subjects versus 55% in younger ones. The plasma luteinizing hormone (LH) concentrations were not influenced by the trial. After shooting trial the elevation in cortisol concentrations was greater for older subjects than for younger ones (273 +/- 30 ng.ml-1 vs 173 +/- 7 ng.ml-1). The catecholamine response was identical in both groups. The urinary T/E ratio remained unchanged after the shooting trial and always remained lower than the International Olympic Committee limit of 6. These results indicate that the psychological stress associated with competition increases the production of steroid hormones (testosterone, cortisol), and that this phenomenon is more pronounced in older athletes. These hormonal changes do not influence the urinary excretion of steroid metabolites used as criterion for drug testing.

Sorbents were prepared by cross-linking β-cyclodextrin (β-CD) using two different types of cross-linker units at variable reactant mole ratios. The resulting polymers containing β-CD were evaluated as sorbents in micro-solid phase extraction (μ-SPE) format for the extraction of the endogenous steroids testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5αAdiol) and 5β-androstane-3α,17β-diol (5βAdiol). The best sorbent (C1; cyclodextrin polymer) showed superior extraction characteristics compared with commercial sorbents (C18 and Bond Elut Plexa). Parameters influencing the extraction efficiency of the C1 sorbent such as extraction and desorption times, desorption solvent and volume of sample were investigated. The extracts were separated using a Hypersil Gold column (50 × 2.1 mm, 1.9 μm) under gradient elution coupled to a LC-MS/MS. The compounds were successfully separated within 8 min. The method offers good repeatability (RSD < 10%) and linearity (r2>> 0.995) were within the range of 1-200 ng mL-1 for T and E, 250-4000 ng mL-1 for A and Etio and 25-500 ng mL-1 for 5αAdiol and 5βAdiol, respectively. The method was applied for the determination of steroid profile of urine from volunteers.

Dihydrotestosterone (DHT) can be used by an athlete as an anabolic steroid to evade the current International Olympic Committee approved drug tests. To investigate the possibility of a method for its detection, the heptanoate ester of DHT was administered to two male subjects (150 mg i.m.). Urine samples, collected before and after the injection, were subjected to enzymatic hydrolysis and the excretion rates of DHT, 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) and testosterone (T) were determined by radioimmunoassay. Relative changes in the excretion of DHT, 3 alpha-diol, 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol), 5 beta-androstane-3 alpha, 17 beta-diol (5 beta-diol), T and epitestosterone (17 alpha hydroxyandrost-4-en-3-one; Epi-T) were determined by gas chromatography-mass spectrometry (GC-MS). Following administration of DHT, the urinary excretion rates of DHT, 3 alpha-diol and 3 beta-diol increased when compared to those of T, Epi-T, 5 beta-diol and luteinizing hormone (LH). Concentrations of DHT in the plasma increased whereas those of T, LH and follicle stimulating hormone decreased. The changes following such modest doses of DHT suggest that these ratios of urinary hormones may be used for the detection of doping with DHT.

A high throughput method for simultaneous screening of anabolic steroids and their metabolites (4-esterendione, trenbolone, boldenone, oxandrolone, nandrolone, methandrostenolone, testosterone, 1-androstendione, ethisterone, normethandrolone, methyltestosterone, 16β-Hydroxystanozolol, epitestosterone, bolasterone, norethandrolone, danazol, stanozolol and androstadienone) in equine urine by online turbulent flow extraction coupled with liquid chromatography-tandem mass spectrometry was developed. The use of turbulent flow chromatography could simplify pretreatment of horse urine, which has complex matrices as well as high viscosity. The urine was extracted by mixed-mode cation exchange solid phase extraction, and hydrolyzed using β-glucuronidase/arylsulfatase. Then, the sample was automatically loaded on the TurboFlow Cyclone extraction column for removal of further matrix, followed by separation on a fused core C18 column before MS/MS detection. Optimization and validation of the method were discussed in detail. All analytes were rapidly detected within 10min with high sensitivity (picogram to nanogram per milliliter level), and no interference was observed. The linearity range was from 0.1-10ng/mL for nine steroids and 1.0-50ng/mL for the others, with correlation of coefficient values over 0.995. Precision and accuracy ranged from 0.1 to 14.5% and 1.7 to 12.4%, respectively. The developed method was successfully applied to the analysis of anabolic steroids in horse urine after administration of a model drug.

A common variant of the LHbeta subunit has a varying prevalence in various ethnic groups. The consequences of the presence of mutated luteinizing hormone (LH) concern borderline alterations in pituitary/gonadal function that could be mediated by an altered action of variant LH on gonadal steroidogenesis. A comparison of plasma concentrations of gonadal steroid sex hormones was completed in women heterozygous for variant LH and in women with the wild type of LH in three different age ranges. The sample was a randomly selected group of 177 normal women 16 to 72 years old. Variant LH was determined by immunofluorimetric methods using two combinations of monoclonal antibodies. The ratios of LH measured by the two assays indicated whether the subject was wild type homozygote, heterozygote or homozygote for the variant LHbeta allele. The carriers of the variant LH allele in the group of postmenopausal women showed higher serum testosterone levels than those with the wild type LH. This is in agreement with the clinical observations made previously showing a slightly higher androgenic action in the population with variant LH. No differences were detected in serum LH, FSH, epitestosterone and sex hormone binding globulin (SHBG).

Bacteria frequently found in equine urine samples may cause degradation of 17beta-OH steroids. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to evaluate the microbiological contamination of equine urine as a marker of poor storage conditions. Norethandrolone was used as the internal standard, and the linearity, sensitivity, precision and accuracy of the method were evaluated. 17beta-OH oxidation was demonstrated for testosterone, nandrolone, trenbolone and boldenone, but did not occur in alpha-epimers such as alpha-boldenone and epitestosterone, demonstrating the stereoselectivity of the reaction. A rapid test was performed by spiking one of the four 17beta-OH steroids in samples of diluted equine urine. The steroids were transformed into their respective ketones in the presence of bacterial activity. The test allows direct injection of diluted samples into the LC/MS system, without the need for prior extraction. Results show that the best method of storage is freezing at -18 degrees C. Urine specimens should be analyzed as soon as possible after thawing. This allows bacterial degradation of equine urine to be arrested temporarily, so that the urine can be used for qualitative or quantitative analysis of 17beta-OH steroids.

Although chemical derivatization for signal enhancement in drug testing is most often associated with gas chromatography, it also has the potential to improve the detection of analytes poorly ionized by atmospheric pressure ionization techniques, such as electrospray ionization used in liquid chromatography-mass spectrometry. A number of acidic compounds, namely drug glucuronides (e.g. conjugates of temazepam, oxazepam, lorazepam, morphine, testosterone, epitestosterone, 5-α-dihydrotestosterone, androsterone, p-nitrophenol, and paracetamol) were successfully derivatized with tris(trimethoxyphenyl) phosphoniumpropylamine to introduce a quaternary cation functionality to the analytes. Benzodiazepine glucuronides were more specifically investigated, and following positive mode electrospray ionization mass spectrometry, average improvements to peak areas as a result of derivatization were 67-, 6-, and 7- fold for temazepam, oxazepam, and lorazepam glucuronides. Average improvements to the signal-to-noise ratios for temazepam, oxazepam, and lorazepam glucuronides were 1336-, 371- and 217-fold, respectively. The values obtained for the derivatized conjugate were also typically higher than those for the underivatized parent drug. Urine containing benzodiazepine glucuronides was also successfully derivatized. The data indicates potential for the use of charge derivatization to improve the detection of molecules with acidic functionalities by liquid chromatography-mass spectrometry (LC-MS) techniques in certain scenarios.

Epitestosterone, a 17 alpha-epimer of testosterone is a normal constituent of body fluids in many species including man. It has long been believed that it is devoid of any biological significance. However, it is now demonstrated that in in vivo experiments on castrated male mice it counteracts the action of testosterone on androgen-dependent organs. In vitro experiments show that on the overall antiandrogenicity of epitestosterone participate true antiandrogenic action due to the binding to androgen receptors, strong 5 alpha-reductase inhibiting activity as well as a weak antigonadotropic activity. Epitestosterone is devoid of any embryotoxicity as checked by chick embryo-toxicity screening test.