Biomarkers identification in follicular fluid in relation to live birth in in vitro fertilization of women with polycystic ovary syndrome in different subtypes by using UPLC-MS method.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common infertility disorder which affects reproductive-aged women. However, metabolic change profiles of follicular fluid (FF) in lean and obese women diagnosed with and without PCOS remains unclear. METHODS: 95 infertile women were divided into four subgroups: LC (lean control), OC (overweight control), LP (lean PCOS), and OP (overweight PCOS). The FF samples were collected during oocyte retrieval and assayed by ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) metabolomics. RESULTS: A total of 236 metabolites were identified by metabolic analysis. The pathway enrichment analysis revealed that the glycerophospholipid metabolism (impact = 0.11182), ether lipid metabolism (impact = 0.14458), and primary bile acid biosynthesis (impact = 0.03267) were related to metabolic pathway between PCOS and control. Correlation analyses showed that epitestosterone sulfate was found positively correlated with fertilization rate in PCOS, while falcarindione, lucidone C. and notoginsenoside I was found to be negatively correlated. The combined four biomarkers including lucidone C, epitestosterone sulfate, falcarindione, and notoginsenoside I was better in predicting live birth rate, with AUC of 0.779. CONCLUSION: The follicular fluid of women with PCOS showed unique metabolic characteristics. Our study provides better identification of PCOS follicular fluid metabolic dynamics, which may serve as potential biomarkers of live birth.

False negative results in testosterone doping in forensic cases: Sensitivity of the urinary detection criteria T/E and T/LH.

At the Swedish national forensic toxicology laboratory, a measured testosterone/epitestosterone (T/E) ratio ≥ 12 together with testosterone/luteinizing hormone (T/LH) in urine > 400 nmol/IU is considered as a proof of exogenous testosterone administration. However, according to the rules of the World Anti-Doping Agency (WADA), samples with T/E ratio > 4 are considered suspicious and shall be further analysed by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) to confirm the origin of testosterone and its metabolites. The aim of this study was to investigate the possibility of false negative results and to estimate the frequency of negative results using the current criteria for detection of abuse of testosterone in forensic investigations. Urine and serum samples were collected by the police at suspected infringement of the doping law in Sweden. Fifty-eight male subjects were included in the study. Urinary testosterone was determined by gas chromatography-mass spectrometry (GC-MS), serum testosterone and LH-by immunoassay. The origin of testosterone and its metabolites was confirmed by means of GC-C-IRMS. Twenty-six of the 57 analysed subjects tested positive for exogenous testosterone using the criteria T/E ≥ 12 combined with T/LH > 400 nmol/IU. The IRMS analyses confirmed 47 positives; thus, 21 were considered false negatives. Negative predictive value was 32% (95% confidence interval [CI]: 16%-50%) and sensitivity 55%. No false positive subjects were found. The number of false negative cases using the current criteria for the detection of testosterone abuse and hence the low sensitivity indicates a need to discuss introduction of new strategies in forensic doping investigations.

Re-evaluation of combined ((ES/EG)/(TS/TG)) ratio as a marker of testosterone intake in men.

To detect doping with pseudo-endogenous anabolic steroids in sports, a urinary steroid profile with glucuronidated plus unconjugated androgens is used. In addition to analyze androgen glucuronide metabolites, it can be of interest to also include sulfate metabolites in the urinary steroid profile. The combined ratios of epitestosterone sulfate/epitestosterone glucuronide to the ratios of testosterone sulfate/testosterone glucuronide ((ES/EG)/(TS/TG)) have previously been investigated as a complementary biomarker for testosterone doping. In this restudy, the aim was to evaluate this biomarker in a larger study sample population. A single dose of 500-mg testosterone enanthate was administered to 54 healthy male volunteers. Urine was collected prior to (Day 0) administration and throughout 15 days and analyzed for the sulfate and glucuronide conjugates of testosterone and epitestosterone. The results show that the combined ratio increased to a larger extent than the traditional T/E ratio in all subjects. This increase was independent on UGT2B17 gene polymorphism. Moreover, a delayed peak of the combined ratio was observed in ~60% of the participants. The results confirm that complementary analyses of the sulfate metabolites may be a useful approach to detect testosterone doping in men.

Masking and manipulation.

The list of prohibited substances in sports includes a group of masking agents that are forbidden in both in- and out-of-competition doping tests. This group consists of a series of compounds that are misused in sports to mask the administration of other doping agents, and includes: diuretics, used to reduce the concentration in urine of other doping agents either by increasing the urine volume or by reducing the excretion of basic doping agents by increasing the urinary pH; probenecid, used to reduce the concentration in urine of acidic compounds, such as glucuronoconjugates of some doping agents; 5alpha-reductase inhibitors, used to reduce the formation of 5alpha-reduced metabolites of anabolic androgenic steroids; plasma expanders, used to maintain the plasma volume after misuse of erythropoietin or red blood cells concentrates; and epitestosterone, used to mask the detection of the administration of testosterone. Diuretics may be also misused to achieve acute weight loss before competition in sports with weight categories. In this chapter, pharmacological modes of action, intended pharmacological effects for doping purposes, main routes of biotransformation and analytical procedures used for anti-doping controls to screen and confirm these substances will be reviewed and discussed.

Changes in blood parameters after intramuscular testosterone ester injections - Implications for anti-doping.

Testosterone treatment stimulates the production of red blood cells and alters iron homeostasis. Thus, we investigated whether the 'haematological module' of the athlete biological passport (ABP) used by the World Anti-Doping Agency can be used to indicate misuse of testosterone. Nineteen eugonadal men received intramuscular injections of either 250 mg Sustanon®, a blend of four testosterone esters, or placebo on days 0 and 21 in a randomized, placebo-controlleddouble-blind design. Urine samples and blood samples were collected twice pre-treatment, at least 5 days apart, and on days 1, 3, 5, 10 and 14 post-injections to assess steroidal and haematological biomarkers of the ABP. The steroidal profile was flagged suspicious in all Sustanon®-treated subjects, whereas the haematological profile was flagged suspicious in six out of nine subjects. When both sensitivity and specificity were considered, reticulocyte percentage (RET%) appeared as the best marker of the haematological module for implying testosterone ester misuse. Atypical blood passport samples were used to select time points for further isotope-ratio mass spectrometry (IRMS) analysis of testosterone and its metabolites in simultaneously collected urine. In addition to the testosterone (T) to epitestosterone (E) ratio, the RET% and OFF-Score could help identify suspicious samples for more targeted IRMS testing. The results demonstrate that unexpected fluctuations in RET% can indicate testosterone doping if samples are collected 3-10 days after injection. From an anti-doping perspective, the haematological and steroidal modules of the ABP should complement each other when planning targeted follow-up testing and substantiating likely misuse of testosterone.

Characterizing the steroidal milieu in amniotic fluid of mid-gestation: A LC-MS/MS study.

Growth and development of an embryo or fetus during human pregnancy mainly depend on intact hormone biosynthesis and metabolism in maternal amniotic fluid (AF). We investigated the hormonal milieu in AF and developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of 14 sulfated and 6 unconjugated steroids in AF. 65 A F samples (male: female = 35: 30) of mid-gestation ranging from 16th week of gestation to 25th week of gestation were analyzed. Reference data of 20 steroid levels in AF of healthy women were provided. 13 sulfated and 3 unconjugated steroids were for the first time quantified in AF by LC-MS/MS. Highest concentrations were found for pregnenolone sulfate (PregS: mean ±â€¯SD, 8.6 ±â€¯3.7 ng/mL), 17α-hydroxypregnenolone sulfate (17OHPregS: 4.9 ±â€¯2.0 ng/mL), epitestosterone sulfate (eTS: 7.3 ±â€¯3.6 ng/mL), 16α-hydroxydehydroepiandrosterone sulfate (16OH-DHEAS: 21.5 ±â€¯10.7 ng/mL), androsterone sulfate (AnS: 9.2 ±â€¯7.4 ng/mL), estrone sulfate (E1S: 3.0 ±â€¯3.0 ng/mL), estriol 3-sulfate (E3S: 8.1 ±â€¯4.0 ng/mL) and estriol (E3: 1.2 ±â€¯0.4 ng/mL). Only testosterone (T) showed a significant sex difference (p < 0.0001). Correlations between AF steroids mirrored the steroid metabolism of the feto-placental unit, and not only confirmed the classical steroid pathway, but also pointed to a sulfated steroid pathway.

Isolation and quantification by high-performance liquid chromatography-ion-trap mass spectrometry of androgen sulfoconjugates in human urine.

Together with steroid glucuronides, sulfoconjugates may be used as markers of steroid administration as well as endogenous steroid production. A fast and sensitive analytical procedure has been developed for the simultaneous separation, determination and quantification of sulfate and glucuronide derivatives of testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio) and dehydroepiandrosterone (DHEA) in human urine. First, a weak anion-exchange solid-phase extraction support (SPE Oasis WAX) was used for complete and rapid separation of sulfates and glucuronides in two extracts after loading of urine sample (2 mL). Then sulfates were analyzed directly by high-performance liquid chromatography-ion-trap mass spectrometry (LC-MS/MS) with electrospray ionization in negative mode. Chromatographic separation of the targeted sulfoconjugates was achieved using a Waters XBridge C18 column (150 mm x 4.6 mm I.D., 5 microm) with gradient elution. Assay validation demonstrated good performance for instance for T sulfate (TS) and E sulfate (ES) in terms of trueness (89-107%), repeatability (3.4-22%) and intermediate precision (5.8-22%) over the range of 2-200 ng/mL (corresponding to 1.5-147 ng/mL as free steroids). Results obtained on biological samples demonstrated the suitability of this analytical strategy for direct measurement of androgen sulfoconjugates and glucuroconjugates in human urine.

Ion trap MS/MS of intact testosterone and epitestosterone conjugates--adducts, fragile ions and the advantages of derivatisation.

In ion trap mass spectrometry, fragile ions may fragment under the application of resonance ejection during precursor mass isolation, reducing MS/MS spectral intensity. In this study the steroidal epimers testosterone glucuronide (TG) and epitestosterone glucuronide (EG) have been chosen as a model for exploring whether compound structure is linked to ion trap fragility. Both compounds form multiple adducts by ESI-MS, namely protonation, ammonium and sodium, however, the mass spectrum of EG displays a more intense ammonium adduct peak than TG. [TG+NH(4)](+), [EG+NH(4)](+) and [EG+H](+) were found to be fragile ions. To explain the differences in adduct formation and fragility, molecular modelling was employed. Ammonium adduction was localised to the glucuronide ring oxygens and while EG has eight possible adduction sites, only seven were located for TG explaining the increased ammonium adduct abundance with EG. In EG the bond between the steroid and the glucuronide was slightly longer and the oxygen in this bond was more basic than TG. This shows that the EG bond is weaker which may contribute to the fact that [EG+H](+) but not [TG+H](+) is fragile. To investigate whether stability could be restored by chemical means, EG was derivatised with tris(trimethoxyphenyl)phosphonium chloride or methylated on the carboxylic acid and Girard P or methoxylamine on the 3-keto group. Derivatisation of the steroid rather than the glucuronide eliminated fragility and using a charged derivative eliminated adduct formation. This work demonstrates the importance of carefully considering the nature of the derivative and the site of derivatisation.

Comparison of partial filling MEKC analyses of steroids with use of ESI-MS and UV spectrophotometry.

Until now, partial filling micellar EKC-ESI-MS (PF-MEKC-ESI-MS) has seldom been applied for human biomolecules. In this study, determination of three electrically neutral endogenous steroid hormones, namely androstenedione, testosterone, and epitestosterone, by PF-MEKC-ESI-MS was investigated. Since ESI-MS and ESI-MS/MS behaviors of testosterone and epitestosterone proved to be nearly identical, efficient separation of the two compounds was required to obtain reliable identification. The chemical conditions as well as the instrumental parameters affecting the PF-MEKC-ESI-MS analysis were researched. In optimal conditions, ESI-MS showed excellent selectivity, and all the steroids could be identified using SIM. LODs were 0.75-5 microg/mL. The results obtained by PF-MEKC-ESI-MS were compared with those obtained by corresponding PF-MEKC-UV. PF-MEKC-UV provided better resolution, repeatability, and more than ten-fold higher sensitivity, in terms of LODs, than PF-MEKC-ESI-MS. The reasons for this were carefully examined. In comparison with PF-MEKC-UV, PF-MEKC-ESI-MS suffered from greater band broadening owing to the sheath-liquid interface. Resolution was also decreased owing to the elevated capillary temperature. Finally, we discovered that in the analysis of electrically neutral compounds, in-capillary sample concentration by micellar sweeping could be more efficiently utilized in PF-MEKC-UV than in PF-MEKC-ESI-MS.

Metabolic and isotopic signature of short-term DHEA administration in women: Comparison with findings in men.

The impact of dehydroepiandrosterone (DHEA) administration has been widely studied for anti-doping purposes in men, whereas only a few studies have been performed in women. In the present study, the impact of DHEA on the steroid profile parameters and their carbon isotopic ratios was explored. Eleven healthy young women and 10 healthy young men received two treatments: One with 100 mg/day of DHEA for 28 days and one with a placebo according to a double-blind crossover protocol. Urine and saliva (only in females) samples were collected before and for 72 hours after each short-term treatment. In all female subjects, concentrations of the urinary parameters of the steroid profile were highly impacted by short-term DHEA administration including epitestosterone (E). Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis was performed and positive results were observed for E in the four female subjects where E concentration was adequate for such analysis, whereas men results remained negative for E. Last, the ability of the Anti-Doping Administration and Management System (ADAMS) software used for the athlete biological passport to identify such doping was assessed. Of the 11 passports generated for female subjects, 10 were automatically classified as an atypical passport finding (ATPF). For the remaining passport with normal status in one woman, the variability of the concentrations prevented the ADAMS software from adjusting individual limits. The most impacted markers in women were T/E and 5αAdiol/E, with a detection window of 36 hours for 5αAdiol/E. In addition, good correlations were observed for DHEA and T concentrations in urine and saliva in females.

Application of testosterone to epitestosterone ratio to horse urine - a complementary approach to detect the administrations of testosterone and its pro-drugs in Thoroughbred geldings.

Detection of testosterone and/or its pro-drugs in the gelding is currently regulated by the application of an international threshold for urinary testosterone of 20 ng/mL. The use of steroid ratios may provide a useful supplementary approach to aid in differentiating between the administration of these steroids and unusual physiological conditions that may result in atypically high testosterone concentrations. In the current study, an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify testosterone (T) and epitestosterone (E). The method was used to analyze 200 post-race urine samples from geldings in order to generate the ratios for the reference population. Following statistical analysis of the data, an upper limit of 5 for T:E ratio in geldings is proposed. Samples collected from 15 geldings with atypical urinary testosterone concentrations (>15 ng/mL) but otherwise normal steroid profile, had T:E ratios within those observed for the reference population. The applicability of an upper T:E ratio to detect an administration was demonstrated by the analysis of a selection of incurred samples from testosterone propionate, dehydroepiandrosterone (DHEA), and a mixture of DHEA and pregnenolone (Equi-Bolic®) administrations. These produced testosterone concentrations above the threshold of 20 ng/mL, but also T:E ratios above the proposed limit of 5. In conclusion, consideration of the T:E ratio appears to be a valuable complementary aid to evaluate whether an atypical testosterone concentration could be caused by a natural biological outlier as opposed to the administration of these steroids. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluation of two glucuronides resistant to enzymatic hydrolysis as markers of testosterone oral administration.

Testosterone (T) has traditionally been the most commonly reported doping agent by doping control laboratories. The screening of T misuse is performed by the quantification of six endogenous androgenic steroids and the ratio T/E included in the Athlete Biological Passport (ABP). The inclusion of additional metabolites can improve the screening capabilities of ABP. In this study, the potential of 3α-glucuronide-6ß-hydroxyandrosterone (6OH-Andros3G) and 3α-glucuronide-6ß-hydroxyetiocholanolone (6OH-Etio3G) as markers of T oral administration was evaluated. These glucuronides have been shown to be resistant to enzymatic hydrolysis and their quantification by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was reported as the only way to obtain feasible results. Urine samples were collected from five volunteers before and after the oral administration of 40mg of T undecanoate and were analyzed by a LC-MS/MS method recently developed. Concentration of 6OH-Andros3G and 6OH-Etio3G compounds and those of the glucuronides of T (TG), epitestosterone (EG), androsterone and etiocholanolone were established and different concentration ratios were calculated. The detection windows (DWs) for the T administration obtained by each selected ratio were compared to the one of TG/EG. The results showed that four out of the nine tested markers presented DWs much larger for all volunteers than those obtained by the World Anti-Doping Agency established T/E marker or other alternative markers. The 6OH-Andros3G/EG, 6OH-Etio3G/EG, 6OH-Andros3G/TG and 6OH-Etio3G/TG markers were able to identify the T abuse up to 96h after the administration, extending our detection capability for the misuse up to 84h more than the classic marker. The importance of these markers was also highlighted by their prolonged capacity to detect the T misuse in the case of one volunteer whose TG/EG barely exceeded his individual threshold. As a consequence, the four markers presented in this study seem to have an exceptional potential as biomarkers of T oral administration.

Partial filling micellar electrokinetic chromatography analysis of androgens and testosterone derivatives using two sequential pseudostationary phases.

Separation of anabolic and androgenic steroids by micellar electrokinetic chromatography (MEKC) has been little studied. Simultaneous separation of the endogenous alpha-epimers testosterone and epitestosterone has not been achieved with any electroseparation technique. Here, a partial filling micellar electrokinetic chromatographic (PF-MEKC) method is described for the analysis of three endogenous steroid hormones (androstenedione, testosterone, epitestosterone) and two synthetic anabolic steroids (fluoxymesterone, methyltestosterone). The resolution efficiency of single-isomer sulphated gamma-cyclodextrins and the surfactants sodium dodecyl sulphate and sodium taurocholate was exploited. The method is based on the sequential introduction of short plugs of two different pseudostationary phases into the capillary. The separation was completed in less than 10 min. The method can be used in quantitative analysis. Linear correlation was obtained between concentration and peak area of 0.996 or better. The repeatability (RSD) of the compound peak areas ranged from 3.6% (methyltestosterone) to 6.2% (androstenedione). Limits of detection were between 73 microg/L (testosterone) and 160 microg/L (fluoxymesterone). As a demonstration of the method, androstenedione, testosterone and epitestosterone were determined in a spiked urine sample.

Development of a faster determination of 10 anabolic steroids residues in animal muscle tissues by liquid chromatography tandem mass spectrometry.

A method had been developed for determination of residues of 10 anabolic steroids (ASs) in animal muscle tissues by liquid chromatography tandem mass spectrometry (LC/MS/MS). After enzymolysis, the sample was extracted with tert-butyl methyl ether, cleaned up through reverse solid-phase extraction and further determined by LC/MS/MS under multiple reaction monitoring (MRM) mode. The limits of detection (LOD) of LC/MS/MS method used for testing epitestosterone (ETS), nandrolone (17 beta-NT), 17 alpha-methyl-testosterone (MTS), testosterone 17-propionate (PTS), medroxyprogesterone (MED), progesterone (PG), estrone (ESN), 17 beta-estradiol (17 beta-ES), 17alpha-ethynylestradiol (EES) and estriol (EST) in animal muscle ranged from 0.06 to 0.22 microg/kg, and the limits of quantification (LOQ) were from 0.12 to 0.54 microg/kg. Experiments on spiked samples of pork, beef, chicken and fish showed that at addition level of 1.0 microg/kg, the average recoveries of the ASs ranged from 64% to 77%, and coefficients of variation from 7.1% to 20.3%, while at addition level of 2.0 microg/kg, the average recoveries ranged from 70% to 89%, and coefficient of variation from 7.1% to 19.1%.

Alternative methodology for the analysis of progesterone, testosterone, and epi-testosterone in bovine liver and veal muscle.

Research has shown that traditional solvent extraction procedures used for the analysis of endogenous steroids often give inconsistent recoveries and results. However, a single-laboratory validation of a liquid chromatography/tandem mass specrometry method using 2 product ions per transition for progesterone, testosterone, and epi-testosterone in bovine liver and veal muscle showed accuracy and precision to within 23% at concentrations ranging from 0.5 to 2.0 microg/kg. Homogenized samples were pretreated with methanol to denature endogenous enzymes. Following removal of methanol, samples were treated overnight with Helix pomatia beta-glucuronidase to deconjugate glucuronide conjugates. Alkali digestion of the samples in KOH solutions was done under shaking at 37 degrees C for 30 min. The digestate was extracted with methyl tert-butyl ether, and the extracts were cleaned by partitioning between acetonitrile-hexane, followed by solid-phase extraction cleanup on silica cartridges. In bovine liver, average recoveries exceeded 54% for all analytes, and the within-run assay coefficients of variations were < 6 and 13% for high (2.0 microg/kg) and low (0.3 microg/kg) analyte concentrations, respectively. In veal muscle, average recoveries exceeded 60%, and the analysis of blind spikes gave accuracy estimates of over 85%, with coefficients of variation (CVs) < 15% for all analytes. The CVs for the multiple reaction monitoring ion ratios for all compounds were < 22% for all validation data. The method meets the requirements for confirmatory methods as outlined in 2002/657/EC. An analyst is capable of processing up to 20 samples within 5 days.

Intranasal delivery of Natesto® testosterone gel and its effects on doping markers.

The laboratory profile of intranasal testosterone gel has not been previously reported from an anti-doping perspective. Because intranasal testosterone gel is newly available as a commercial product, we sought to examine the laboratory parameters following administration of this formulation, with particular attention to anti-doping guidelines. Five healthy and active male subjects were administered testosterone intranasal gel three times daily for four weeks, using a pattern of five consecutive days on, two days off. Urine was collected after each five-day round of drug administration and analyzed using a full steroid screen and isotope ratio mass spectrometry (IRMS). Windows of detection for elevated testosterone/epitestosterone (T/E) and other steroid ratios, World Anti-Doping Agency (WADA) athlete biological passport (ABP) findings, and IRMS results were analyzed in this study. In the 0-24 h window post-administration, 70% of samples were flagged with a suspicious steroid profile and 85% were flagged as atypical passport findings according to the WADA ABP steroid module. In the 24-48 h window, 0% of samples displayed suspicious steroid profiles while 40% resulted in atypical passport findings. IRMS testing confirmed the presence of exogenous testosterone in 90% and 40% of samples in the 0-24 h and 24-48 h windows post-administration, respectively. Additionally, IRMS data were analyzed to determine commonalities in the population changes in δ13 C values of testosterone, androsterone, etiocholanolone, 5αAdiol, and 5ßAdiol. Though no discernible metabolic trend of the route of administration was identified, we discovered that intranasal gel testosterone is detectable using conventional anti-doping tests. Copyright © 2016 John Wiley & Sons, Ltd.

Facile synthesis of [16,16,17-2H3]-testosterone, -epitestosterone and their glucuronides and sulfates.

The analysis of trace components in complex biological matrices requires the use of reliable internal standards. For the gas chromatography/mass spectrometry (GC/MS) and high performance liquid chromatography/mass spectrometry (HPLC/MS) analyses, the stable isotope-labelled analogues of the analyte molecules are the most appropriate internal standards. In this work high-yield synthetic procedures for stably labelled and isotopically pure [16,16,17-2H3]-testosterone and- epitestosterone are reported. Synthetic methodologies for the glucuronidation and sulfation were established with the commercially available epitestosterone. Structure characterization of 4-androsten-17 alpha-ol-3-one methyl-2',3',4'-tri-O-acetyl-beta-D-glucuronate was made by two-dimensional nuclear magnetic resonance (COSY). Subsequently glucuronidation of [16,16,17-2H3]-testosterone and sulfation of [16,16,17-2H3]-epitestosterone were carried out at greater than 60% yield. However, the yield from the glucuronidation of epitestosterone was not as high. Electrospray mass spectrometry of four conjugates: testosterone sulfate, epitestosterone sulfate, testosterone glucuronide and epitestosterone glucuronide was carried out in the positive ion mode at a number of orifice voltages (50-95 V). Studies of the collisionally induced dissociation at both the interface and in the collision cell (MSMS) confirmed that the glycosidic bond of epitestosterone glucuronide was more labile than that of testosterone glucuronide. Use of the deuterated internal standards is reported to demonstrate the direct analysis of the steroid conjugates by HPLC/MS.

Differences in natural steroid hormone patterns of beef from bulls and steers.

This investigation gives an overview of the concentrations of naturally occurring androgens, progestogens, corticosteroids, and their precursors and metabolites in meat from bulls and steers. A recently developed gas chromatography-mass spectrometry IGC-MS) method with improved sensitivity for steroid analysis was used. Eighty-two beef samples were analyzed using the GC-MS method. Beef from bulls contained higher concentrations of testosterone, which is an anabolic androgen, and its metabolite epitestosterone (P < .01) and the androgen precursor dehydroepiandrosterone (P < .05) than beef from steers. Beef from steers contained higher (P < .05) concentrations of the basic hormone precursor pregnenolone and cortisol, which is a catabolic corticosteroid, than beef from bulls. A classification of an unknown beef sample to one of the categories (bull or steer) was possible in most cases (>90%) using a masculinity index (MI) that was calculated using the concentrations of testosterone, epitestosterone, and pregnenolone. Because the hormonal status of beef cattle is related to meat quality characteristics, such as tenderness or fat and protein distribution, the MI may contribute to meat quality assessment and meat quality control.

Feasibility of on-line supercritical fluid extraction of steroids from aqueous-based matrices with analysis via gas chromatography-mass spectrometry.

The solubility of testosterone, boldenone, androstenone, etiocholanolone, and epitestosterone are measured in pure supercritical CO2. Testosterone exhibited the highest solubility in supercritical CO2. The solubility of all steroids except epitestosterone increased by one order of magnitude with increasing pressure from 100 to 400 atm. Epitestosterone had the lowest solubility in supercritical CO2 and its solubility was not affected by pressure. The extraction efficiency of steroids from an aqueous saline environment exceeded 95%. Because of the partial solubility of water in supercritical CO2, the addition of a moisture trap after the aqueous vessel is necessary to prevent the plugging and deterioration of the gas chromatographic (GC) column. It is demonstrated that on-line supercritical fluid extraction-GC-mass spectrometry is feasible for the quantitative extraction and analysis of steroids from both saline and urine solutions. However, it is determined that the adsorbent vessel filled with Hydromatrix is not sufficient to trap all the moisture, and after 3 to 4 extractions, the GC column efficiency lowered.

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.