Determination of testosterone propionate in human plasma by gas chromatography-mass spectrometry

A specific, sensitive and accurate quantitative analysis of testosterone propionate in human plasma was developed using gas chromatography-mass spectrometry-selected-ion monitoring. For the calculation of testosterone propionate in plasma, peak height ratios were measured by selected-ion monitoring performed on the molecular ions of the trifluoroacetyl derivative of testosterone propionate (m/z 440) and testosterone propionate-19,19,19-d3 (m/z 443). The sensitivity of the method was judged from the lower limit of the detection of the mass spectrometer which was at 20 pg. The inter-assay coefficients of variation and relative error at a concentration of 1.31 ng/ml of plasma were 5.47% and -2.3%, respectively. The method described was applied to the determination of plasma concentrations of testosterone propionate-19,19,19-d3 following an intramuscular dose of testosterone propionate-19,19,19-d3 in a healthy male volunteer.     

Determination of plasma testosterone by mass fragmentography using testosterone-19-d3 as an internal standard. Comparison with radioimmunoassay

Analytical procedures for the measurement of testosterone by mass fragmentography (MF) using trideuterated testosterone (testosterone-19,19,19-d3) are described. For the calculation of plasma testosterone, peak height ratios were measured by MF performed on the molecular ions of the TFA derivative of testosterone (m/e 480) and testosterone-19,19,19-d3 (m/e 483). The sensitivity of the method was judged from the lower limit of detection of the mass spectrometer which was at 10 pg. For the measurement of the precision, the inter- and intra-assay coefficients of variation (C.V.) were calculated by using a pooled plasma sample; they were 3.15% and 1.79%, respectively. The specificity was investigated by the use of 5 alpha-dihydrotestosterone and the MF method was found to afford a highly selective technique. These results obtained by MF have been compared with the results obtained by a radioimmunoassay method.     

Simultaneous measurements of endogenous and deuterium-labelled tracer variants of androstenedione and testosterone by capillary gas chromatography-mass spectrometry

A capillary gas chromatographic-mass spectrometric method for the simultaneous determination of androstenedione and testosterone in human plasma using [19,19,19-2H3]androstenedione and [19,19,19-2H3]testosterone as internal standards is described. For calculation of plasma androstenedione and testosterone, peak heights were measured by selected-ion monitoring of the molecular ions of the heptafluorobutyryl derivatives of androstenedione and [2H3]androstenedione (m/z 482 and 485) and of testosterone and [2H3]testosterone (m/z 680 and 683). The isotope dilution method needed no complex corrections for contributions and provides a sensitive and reliable technique with good accuracy, precision and reproducibility.     

Electrospray collision-induced dissociation of testosterone and testosterone hydroxy analogs

Complications with the gas chromatographic analysis of steroids prompted the use of alternative techniques for their identification. High-performance liquid chromatography/mass spectrometry with atmospheric pressure ionization allowed the collection of data for structural identification of these compounds. The objective of this study was to investigate the up-front collision-induced dissociation (UFCID) electrospray ionization (ESI) mass spectra of testosterone and monohydroxylated testosterones. The positive ion UFCID ESI mass spectrum of testosterone showed three significant ions at m/z 97, 109 and 123. The relative abundance of these ions in the UFCID ESI mass spectra of monohydroxylated testosterones varied with the position of the hydroxy group. Statistical data allowed the prediction of hydroxy group position on testosterone by evaluation of the relative abundance of the m/z 97, 109, 121 and 123 ions. Data from the ESI mass spectral analysis of testosterone in a deuterated solvent and from the analysis of cholestenone and 4-androstene-3 beta, 17 beta-diol indicated that the initial ionization of testosterone occurred at the 3-one position. CID parent ion monitoring analyses of the m/z 97, 109 and 123 ions indicated that each resulted from different fragmentation mechanisms and originated directly from the [M + H]+ parent ion. The elemental composition of these fragment ions is proposed based on evidence gathered from the CID analysis of the pseudo-molecular ions of [1,2-2H2]-, [2,2,4,6,6-2H5]-, [6,7-2H2]-, [7-2H]-, [19,19,19-2H3]- and [3,4-13C2]testosterone. The structure and a possible mechanism of formation of the m/z 109 and 123 ions is presented. The results of this study advance the understanding of the mechanisms of collision-induced fragmentation of ions.     

Study of the metabolism of testosterone, nandrolone and estradiol in cattle.

The current metabolism study was undertaken to identify key analytes in urine, plasma and bile following testosterone, nandrolone and estradiol administrations to cull cows, heifers and steers. This information will be used to develop confirmatory analysis procedures. In the present study, mixtures (1:1) of testosterone, nandrolone or estradiol and their deuterium labelled analogues were administered to cull cows, heifers and steers. Two analogues of deuterium labelled testosterone were synthesised and administered, to facilitate identification of metabolites. Following administration, urine, plasma and bile samples were collected and subjected to solid phase extraction. The extracts were derivatised and analysed by GC-MS. The major analytes derived from the administered steroids were identified on the basis of the twin ion peaks produced for their non-labelled and deuterium labelled analogues and their stereochemistries determined by comparison of retention times with appropriate reference standards. Using suitable internal markers, excretion profiles for the major analytes in urine and plasma have been determined and levels in isolated bile samples estimated. This work is on-going, and this paper is a summary of some of the studies completed to date.     

Studies on neurosteroids XX. Liquid chromatography-tandem mass spectrometric method for simultaneous determination of testosterone and 5alpha-dihydrotestosterone in rat brain and serum

Testosterone (T) and 5alpha-dihydrotestosterone (DHT) are now referred to not only as androgenic steroid hormones, but also as neuroactive steroids, because they elicit anesthetic and anxiolytic effects. Methods using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) coupled with derivatization are developed and validated to examine rat brain and serum levels of T and DHT and their stress-induced changes. The steroids are extracted with methanol-acetic acid from the brain tissue or serum, purified using solid-phase extraction cartridges, derivatized with a permanently charged reagent, 2-hydrazino-1-methylpyridine, and subjected to LC-MS-MS. [19,19,19-(2)H3]-T is used as the internal standard. The intra- and inter-assay coefficients of variation are below 10.0%, and the analytical recoveries are 98.1-103.0%. The developed methods are applied to the animal study and it was found that a fair amount of DHT is continuously and locally synthesized in the brain, and its level is not changed by the immobilization stress and depends on the brain T level.     

Simultaneous determination of testosterone and androstadienone (sex attractant) in human plasma by gas chromatography-mass spectrometry with high-resolution selected-ion monitoring

Androsta-4,16-dien-3-one (androstadienone) and androst-4-en-3-one-17 beta-ol (testosterone) in healthy human plasma were simultaneously determined under several experimental conditions by gas chromatography-mass spectrometry with high-resolution selected-ion monitoring. Internal standards were [2,2,4,6,6-2H5]androstadienone and [2,2,4,6,6-2H5]testosterone. Samples were extracted with an Extrelut column, purified using Lipidex 5000 and converted into hydroxime-trimethylsilyl derivatives for determination. Physiological concentrations of androstadienone and testosterone found in eleven healthy men were 2.05 +/- 0.74 and 18.6 +/- 4.9 pmol/ml in plasma (mean +/- S.D.), respectively. No correlation was observed between these steroid concentrations.     

Determination of plasma testosterone by mass fragmentography using [3,4-13C]testosterone as an internal standard

The combination of glass capillary gas chromatography--mass spectrometry is especially suitable for the recognition of compounds. The use of [3,4-13C]testosterone as internal standard, mass fragmentography and isotope ratio measurement have been applied to the quantitative determination of testosterone in plasma. This paper describes the method, using tert.-butyldimethylsilylmethoxime and di-heptafluorobutyrate derivatives. The calibration graph in isotopic dilution is examined. The results obtained are compared with the results obtained by radioimmunoassay. The sensitivity of the method is judged from the lower limit of detection: 4.5 pg. The precision, and inter- and intra-assay are calculated.     

Detectability of testosterone esters and estradiol benzoate in bovine hair and plasma following pour-on treatment

The abuse of synthetic esters of natural steroids such as testosterone and estradiol in cattle fattening and sports is hard to detect via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. An interesting alternative can be provided by the analysis of the administered synthetic steroids themselves, i.e., the analysis of intact steroid esters in hair by liquid chromatography tandem mass spectrometry (LC/MS/MS). However, retrospective estimation of the application date following a non-compliant finding is hindered by the complexity of the kinetics of the incorporation of steroid esters in hair. In this study, the incorporation of intact steroid esters in hair following pour-on treatment has been studied and critically compared with results from intramuscular treatment. To this end animals were pour-on treated with a hormone cocktail containing testosterone cypionate, testosterone decanoate and estradiol benzoate in different carriers. The animals were either treated using injection and pour-on application once or three times having 1 week between treatments using injection and pour-on application. Animals were slaughtered from 10–12 weeks after the last treatment. Both hair and blood plasma samples were collected and analysed by LC/MS/MS. From the results, it is concluded that after single treatment the levels of steroid esters in hair drop to CCβ levels (5–20 μg/kg) after 5–7 weeks. When treatment is repeated two times, the CCβ levels are reached after 9–11 weeks. Furthermore, in plasma, no steroid esters were detected; not even at the low microgramme per litre level but—in contrast with the pour-on application—after i.m. injection, significant increase of 17β-testosterone and 17β-estradiol were observed. These observations suggest that transport of steroid esters after pour-on application is not only performed by blood but also by alternative fluids in the animal so probably the steroid esters are already hydrolysed and epimerized before entering the blood.     

Preparation of 19,19,19-trifluororetinal(9-trifluoromethylretinal)

Synthetic work of all-trans-19,19,19-trifluororetinal (7E,9Z,11E,13E) is described.     

Antibody development to testosterone and its application in capillary electrophoresis-based immunoassay

The present report describes a rapid, simple, and highly selective approach to perform testosterone competitive immunoassay by CE and LIF detection. The method involves using synthesized fluorescence-labeled testosterone as a tracer to compete with testosterone. Two polyclonal antibodies (antibody (Ab) arised from T-3-BSA (Ab(3)) and Ab arised from T-17-BSA (Ab(17))) and their respective tracers have been optimized and Ab(3) system is used for the quantification of testosterone by CE-based immunoassay. The method is developed with a wide working range of 3.70-2000 ng/mL and an LOD at 1.11 ng/mL. Tests for normal and positive urine samples show that this method has the potential to be applied in testosterone doping control.     

Inhibitors of testosterone biosynthetic and metabolic activation enzymes

The Leydig cells of the testis have the capacity to biosynthesize testosterone from cholesterol. Testosterone and its metabolically activated product dihydrotestosterone are critical for the development of male reproductive system and spermatogenesis. At least four steroidogenic enzymes are involved in testosterone biosynthesis: Cholesterol side chain cleavage enzyme (CYP11A1) for the conversion of cholesterol into pregnenolone within the mitochondria, 3β-hydroxysteroid dehydrogenase (HSD3B), for the conversion of pregnenolone into progesterone, 17α-hydroxylase/17,20-lyase (CYP17A1) for the conversion of progesterone into androstenedione and 17β-hydroxysteroid dehydrogenase (HSD17B3) for the formation of testosterone from androstenedione. Testosterone is also metabolically activated into more potent androgen dihydrotestosterone by two isoforms 5α-reductase 1 (SRD5A1) and 2 (SRD5A2) in Leydig cells and peripheral tissues. Many endocrine disruptors act as antiandrogens via directly inhibiting one or more enzymes for testosterone biosynthesis and metabolic activation. These chemicals include industrial materials (perfluoroalkyl compounds, phthalates, bisphenol A and benzophenone) and pesticides/biocides (methoxychlor, organotins, 1,2-dibromo-3-chloropropane and prochloraz) and plant constituents (genistein and gossypol). This paper reviews these endocrine disruptors targeting steroidogenic enzymes.     

Trace level quantification of deuterated 17beta-estradiol and estrone in ovariectomized mouse plasma and brain using liquid chromatography/tandem mass spectrometry following dansylation reaction

A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method coupled with dansylation was developed for the simultaneous quantification of exogenously administered deuterated 17beta-estradiol-d4 (E2-d4) and its metabolite, estrone-d4 (E1-d4), in mouse plasma and brain homogenates. The dansylation reaction was simple, fast, and sensitive, and a lower limit of quantification of 50 pg/mL was achieved by using 50 microL of mouse plasma. Interference from endogenous 17beta-estradiol and estrone in plasma and brain samples was minimized by the use of deuterated-E2 as well by utilizing ovariectomized (OVX) mice. The recovery of dansylated derivative exceeded 83% and the reaction was completed within approximately 3 min. The intra- and inter-day assay precision were better than 12.9% and assay accuracy ranged between 92-104% for E1-d4 and E2-d4 in plasma, respectively. The absorption of E2-d4 at both 1 and 3 mg/kg P.O. was rapid, reaching peak plasma concentrations (Cmax) at 5 min post-dose that was the earliest time point obtained, and were 1.1 and 13.8 ng/mL, respectively; the Cmax values for the estrone metabolite, E1-d4, were 1.1 and 43.2 ng/mL, respectively. The area-under-the-plasma-time curve (AUC(0-2 h)) values were determined to be 0.65 and 2.90 ng. h/mL for E2-d4 and 0.77 and 6.74 ng. h/mL for E1-d4, respectively, at 1 and 3 mg/kg. The mean brain-to-plasma ratio for E1-d4 and E2-d4 after P.O. administration of E2-d4 to the OVX mice at 1 and 3 mg/kg indicated that both E1-d4 and E2-d4 were present in the brain as well as in the circulation.     

Radioimmunoassay of Testosterone in Murine Plasma

Abstract

This report describes a radioimmunoassay method for measuring unconjugated plasma testosterone (T) and its application to the measurement of T in murine plasma. The parameters of assay reliability, sensitivity, and accuracy were established. Di-hydrotestosterone, the only steroid which showed significant cross-reaction with the testosterone antibody used, was not found in significant quantities in male mice of 19 inbred strains examined. This suggested that chromatographic separation of T from other plasma steroids was unnecessary for the measurement of T in males of this species. Biological levels of T in normal adult male mice of 19 inbred strains showed a range of 0.64 to 21.9 ng/ml plasma and a mean of 5.10 ng/ml. Castration reduced testosterone levels to about 4% of the normal level; castration plus adrenalectomy reduced testosterone to levels below the sensitivity of this method. These investigations demonstrated the validity and reliability of this method for measuring uncanjugated plasma T in male mice.     

Direct radioimmunoassay of testosterone in human saliva

A simple direct radioimmunoassay for testosterone in 400 μl of whole human saliva is reported. Neither extraction nor chromatography is needed. The direct assay involves a commercially available, highly selective antiserum raised against testosterone-19-(O-carboxymethyl)-ether-BSA, an iodinated tracer, a serum added to give parallel inhibition of analyte and tracer binding to serum proteins and a double antibody/polyethylene glycol separation technique. The lower limit of determination is about 3 pM salivary testosterone and the calibration curve covers the range of clinical interest both for males and females (0–868 pM). The assay is specific as judged from recovery and dilution tests, from the cross-reactivity of the antiserum used and from comparisons with an extraction procedure. Within-assay and between-assay relative standard deviations are 4.1, 1.9, 10.5% and 6.3, 2.8, 15.3% for saliva samples with testosterone concentrations of 310, 117 and 52.7 pM, respectively.     

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.     

Determination of testosterone:epitestosterone ratio after pentafluorophenyldimethylsilyl-trimethylsilyl derivatisation using gas chromatography-mass spectrometry in equine urine

A highly specific method is described for measuring the testosterone:epitestosterone ratio in equine urine by gas chromatography-mass spectrometry (GC-MS) with stable isotope internal standards. The procedure was based on Serdolit Pad-1 resin extraction, enzymatic hydrolysis, and chemical derivatisation prior to instrumental analysis. The mixed derivatives, 3-trimethylsilyl-17-pentafluorophenyldimethylsilyl ether (3-TMS-17-flophemesyl) testosterone and epitestosterone, were found to have excellent analytical properties. The specificity of the derivatisation method exploits a unique feature of steroids: the selective exchange of the alcoholic flophemesyl ether for the trimethylsilyl ether. The sensitivity and specificity of the mixed 3-TMS-17-flophemesyl derivatives allow adequate determinations of testosterone and epitestosterone, even in urine from mares, in 5 ml samples. The repeatability of testosterone and epitestosterone was 6.2 and 5.7%, respectively, and their reproducibility was in the range of 6.4-8.7%.     

Solid phase radioimmunoassay for testosterone in human serum using antibodies coupled to magnetizable cellulose

Summary A simple user-friendly, solid phase radioimmunoassay for testosterone in human serum based on magnetic particles is described. IgG fractions precipitated from polyclonal testosterone antiserum were coupled to magnetizable cellulose particles using carbonyl diimidazole. The prepared antibody solid phase was stable for one year when stored at 4 °C. The optimized assay involves the incubation of 50 ml of testosterone standards (0.3-10 ng/ml), 100 ml of magnetizable cellulose particle coupled antibody suspension and 100 ml of ¹²⁵I-testosterone derivative for 4 hours at 37 °C. At the end of the incubation, the tubes were placed on a magnetic rack for 10 minutes after the addition of wash buffer and decanted. The sensitivity of the assay is 0.2 ng/ml. The intra-assay variation was <15% throughout the assay range. The recovery varied from 88 to 115%. On dilution of samples having high levels of testosterone, linearity ranged between 87 and 125%. Correlation coefficient of 0.978 was obtained when the developed solid phase assay was compared to the earlier established liquid phase assay.     

Simultaneous quantitation of testosterone,estradiol, ethinyl estradiol,and 11‐ketotestosterone in fathead minnow fish plasma by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry

In the present work, for the first time, a rapid and sensitive liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry (LC/APPI‐MS/MS) method has been developed and validated for the simultaneous quantitation of testosterone, estradiol, ethinyl estradiol, and 11‐ketotestosterone in fathead minnow fish plasma using no more than 10 µL of plasma. Compounds present in plasma were directly derivatized with dansyl chloride and 25 µL of the derivatized mixture was injected into the LC/APPI‐MS/MS system. The gradient chromatographic elution was achieved on an Agilent Zorbax SB‐C18 analytical column (2.1 mm × 50 mm, 1.8 µm particle size) with mobile phases consisting of acetonitrile, water and acetic acid. The flow rate was 0.5 to 0.7 mL/min and the total run time was 11.5 min. The lower limits of quantitation for testosterone, estradiol, ethinyl estradiol, and 11‐ketotestosterone and were 1, 1, 1, and 2.5 ng/mL, respectively. Intra‐batch precision was less than 19.4% and inter‐batch precision was less than 11.7% for all four analytes. Accuracy was within 83.5–115.4% of nominal concentrations. This method is used for quantitation of sex steroid levels in fathead minnow tested in endocrine disruptor screening experiments. Copyright © 2009 John Wiley & Sons, Ltd.     

Application of chemiluminescence immunoassays for steroid hormones in clinical endocrinological investigations in women

Immunoassays based on chemiluminescence for the measurement of serum and plasma steroids (estradiol, estriol, progesterone, testosterone, and cortisol), urinary steroid conjugates (estrone-3-glucuronide, estriol-16α-glucuronide and pregnanediol-3α-glucuronide) and peptide hormones (choriogonadotropin and luteinizing hormone) are surveyed briefly. These immunoassays are simple, robust and valid alternatives to radioimmunoassay. Homogeneous procedures and recent solid-phase assays based on purified specific antibodies, covalently coupled to polymer beads are discussed. Some new results are presented for solid-phase chemiluminescence immunoassays: estradiol is quantified in extracts of serum by using a monoclonal antibody to estradiol with estradiol-6-carboxymethyloxime-aminobutylethyl isoluminol as the marker ligand, and progesterone is quantified in unextracted serum by using a polyclonal antibody to progesterone, progesterone-11-hemisuccinyl-aminobutylethyl isoluminol as the marker ligand, and danazol (17α-pregna-2,4-dien-20-yno[2,3-d]-isoxazol-17-ol) to displace progesterone from serum binding-proteins. Their clinical utility is demonstrated.