Steroids In Human Urine Research Articles

A screening method for the simultaneous detection of glucocorticoids, diuretics, stimulants, anti-oestrogens, beta-adrenergic drugs and anabolic steroids in human urine by LC-ESI-MS/MS

This paper presents a general screening method, based on liquid chromatography/mass spectrometry (LC/MS), for the simultaneous detection in human urine of 72 xenobiotics (21 diuretics, 16 synthetic glucocorticoids, 17 beta-adrenergic drugs, 10 stimulants, 5 anti-oestrogens and 3 anabolic steroids), excreted free or as glucuro-conjugates in urine. Although the method has been specifically designed and evaluated in view of its potential application to anti-doping analyses, it can also be effective in other areas of analytical toxicology. Sample preparation was based on two liquid/liquid separation steps (performed at alkaline and at acid pH, respectively) of hydrolyzed human urine, and then an assay by LC/MS-MS in positive and negative ionization mode using an electrospray ionization source (ESI) and multiple reaction monitoring (MRM) as the acquisition mode. The overall time needed for an LC run was less than 15 minutes. All compounds showed good reproducibility in terms of both the retention times (CV%<1) and the relative abundances of the diagnostic transitions (CV%<10). The limits of detection (LOD) were in the range of 1-50 ng/mL for glucocorticoids, anti-oestrogens and steroids, and 50-500 ng/mL for diuretics, beta-adrenergic drugs and stimulants, thus satisfying the minimum required performance limits (MRPL) set by the World Anti-Doping Agency (WADA) for the accredited anti-doping laboratories.

Preventive doping control analysis: liquid and gas chromatography time‐of‐flight mass spectrometry for detection of designer steroids

A new combined doping control screening method for the analysis of anabolic steroids in human urine using liquid chromatography/electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (LCoaTOFMS) and gas chromatography/electron ionization orthogonal acceleration time-of-flight mass spectrometry (GCoaTOFMS) has been developed in order to acquire accurate full scan MS data to be used to detect designer steroids. The developed method allowed the detection of representative prohibited substances, in addition to steroids, at concentrations of 10 ng/mL for anabolic agents and metabolites, 30 ng/mL for corticosteroids, 500 ng/mL for stimulants and beta-blockers, 250 ng/mL for diuretics, and 200 ng/mL for narcotics. Sample preparation was based on liquid-liquid extraction of hydrolyzed human urine, and the final extract was analyzed as trimethylsilylated derivatives in GCoaTOFMS and underivatized in LCoaTOFMS in positive ion mode. The sensitivity, mass accuracy, advantages and limitations of the developed method are presented.

Screening for anabolic steroids in doping analysis by liquid chromatography/electrospray ion trap mass spectrometry

A fast and selective LC/MS/MS method for the screening of four anabolic steroids in human urine has been developed and validated. Liquid-liquid extraction with diethyl ether was applied after enzymatic hydrolysis. Analyses were performed on an ion trap mass spectrometer equipped with electrospray ionisation. MS/MS was applied for all compounds. The analytical run time was 11 min. The LOD for all compounds varied between 1 and 10 ng/mL. Left-over A samples, which were declared positive by GC/MS for the presence of 3'-hydroxystanozolol, were assessed using the described method.

Screening for unknown synthetic steroids in human urine by liquid chromatography‐tandem mass spectrometry

Chemically modified steroids (designer steroids), including tetrahydrogestrinone and norbolethone, pose a threat to the integrity of the sport community. These compounds have recently been detected in urine specimens from athletes, resulting in temporary or permanent suspension from amateur and/or professional competition. Triple quadrupole mass spectrometers enable doping control laboratories to screen for unknown, anabolic, androgenic steroids utilizing precursor ion scans. On the basis of common dissociation patterns of steroids with common structural features, characteristic product ions were selected to serve as diagnostic markers for previously unidentified drugs or drug metabolites in human urine samples. An assay was established to complement standard screening procedures. Urine specimens were enzymically hydrolyzed, partitioned into ether, concentrated, and analyzed by precursor ion scanning. Spectra from samples fortified with eight standard compounds (methyltestosterone, ethyltestosterone, 1-testosterone, gestrinone, dihydrogestrinone, tetrahydrogestrinone, norbolethone, and propyltrenbolone) and one deuterium-labeled analog (d(4)-tetrahydrogestrinone) at 50 ng/ml of urine, had precursor ion peaks other than those from common endogenous steroids. Subsequent product ion scan experiments on precursor ions of peaks of unknown origin provided structural identification of the unknown compounds.

Screening of free 17-alkyl-substituted anabolic steroids in human urine by liquid chromatography–electrospray ionization tandem mass spectrometry

A qualitative liquid chromatography–electrospray ionization tandem mass spectrometry method was developed for screening of the abuse of 4-chlorodehydromethyltestosterone, danazol, fluoxymesterone, formebolone, metandienone, oxandrolone, and stanozolol. The introduced method measures simultaneously nine different 17-alkyl-substituted anabolic androgenic steroids or their unconjugated metabolites in human urine, using methyltestosterone as an internal standard. Sample preparation involved one-step liquid extraction. Liquid chromatographic separation was achieved on a reversed-phase column with methanol–water gradient containing 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. Compounds were ionized in the positive mode and detected by multiple reaction monitoring. All steroids within the study could be selectively detected in urine with detection limits of 0.1–2.0 ng/ml. The method showed good linearity up to 250 ng/ml with correlation coefficients higher than 0.9947. With simple and fast sample preparation, low limits of detection, and high selectivity and precision, the developed method provides advantages over the present testing methods and has the potential for routine qualitative screening method of unconjugated 17-alkyl-substituted anabolic steroids in human urine.

Solvent and solid-phase extraction of natural and synthetic anabolic steroids in human urine

Liquid–liquid (using dichloromethane) and liquid–solid extraction processes (using disposable C 18 cartridges) were applied to human urine samples spiked with 15 androgenic anabolic steroids (natural and synthetic). The extraction recoveries were assessed from different HPLC separations of anabolic steroids using water–acetonitrile mobile phase, and using calibration graphs obtained by injection into HPLC of standard samples of these compounds before and after extraction. The procedures, including sample preconcentration, showed extraction efficiencies over 90% which were independent on a wide range of concentrations tested. Solid phase extraction yielded poor results for oximetolone, danazol and dehydroepiandrosterone. For real urine samples, hydrolysis using β-glucuronidase and washing using sodium hydroxide before and after solvent extraction, respectively, is recommended.

High-throughput sample preparation and analysis using 96-well membrane solid-phase extraction and liquid chromatography-tandem mass spectrometry for the determination of steroids in human urine

A 96-well solid-phase extraction (SPE) system is used to rapidly prepare human urine samples for high-throughput quantitative analysis of two steroids, equilenin and progesterone, by liquid chromatography-tandem mass spectrometry using deuterated estrone as the internal standard. We define high-throughput here as analysis of 384 samples in a 24 h period. A total of 384 samples and standards were extracted by an individual in one day and subsequently analyzed within a 24 h period. The inter- and intratray accuracy and precision obtained over the course of these injections was within 8% coefficient of variation when analyzed by atmospheric pressure chemical ionization mass spectrometry using positive ion detection. A semiautomated sample processing workstation was used to add internal standard and then process 96 samples at a time. The recovery of the analytes from the SPE was approximately 85%. The accuracy and precision obtained was comparable to that ordinarily obtained using manual sample preparation techniques.

Determination of anabolic steroids by gas chromatography/negative-ion chemical ionization mass spectrometry and gas chromatography/negative-ion chemical ionization tandem mass spectrometry with heptafluorobutyric anhydride derivatization.

A gas chromatography/mass spectrometry (GC/MS) method is described which uses negative ion chemical ionization (NCI) and tandem mass spectrometry (MS/MS) for the determination of eight anabolic steroids in human urine. Eight anabolic steroids were derivatized by heptafluorobutyric anhydride (HFBA), and were determined using GC/NCI-MS and GC/NCI-MS/MS. The linear correlation coefficients for calibration in NCI-MS/MS were in the range 0.9880-0.9988. This method of derivatization with HFBA for use with GC/NCI was useful in determinations of 19-norandrosterone, boldenone, 19-noretiocholanolone, 2-methylandrosterone, nandrolone, 1-methyleneandrosterone, 1-methylandrosterone, 4-dihydroboldenone and mesterolone. The detection limits of this procedure were 5-20 ppb at a signal-to-noise (S/N) ratio of 3.

Rapid determination of corticosteroids in urine by combined solid phase microextraction/liquid chromatography/mass spectrometry.

Solid-phase microextraction combined with fast short-column liquid chromatography/mass spectrometry (SPME/LC/MS) was used for isolating and analysing 11 corticosteroids and 2 steroid conjugates from urine samples. Several SPME parameters such as polarity of fibres, extraction time and effect of ionic strength, were investigated, and their impact on the SPME/LC/MS technique was studied. To demonstrate the potential of the SPME/LC/MS method, its application to the determination of steroids in human urine was investigated. The method was shown to be sensitive with detection limits between 4 and 30 ng/mL and precision between 4.9 and 11.1% RSD through the use of an internal standard technique. The versatility of the method was also exhibited by its excellent linearity in the concentration range of 20 to 20,000 ng/mL in urine for all investigated compounds. A comparison of the SPME/LC/MS method with LC/MS methods utilizing traditional sample preparation techniques, shows that the former offers similar performance in terms of precision, linearity and detection limits, but is clearly easier to use and faster to perform.

Determination of Steroids in Human Urine by Micellar Liquid Chromatography

A simple and sensitive method for the determination of steroids using micellar liquid chromatography is described. The steroids, including hydroxycorticosterone. corticosterone, northisterone, testosterone, mexdroprogesterone acetate and progesterone, were separated by reversed-phase using a micelles mobile phase following UV detection at 245 nm. The parameters affecting retention of the test solutes such as the concentration of sodium dodecyl sulfate (SDS) and n-butanol-1 in the mobile phase were investigated. It was found that the retention of the solutes was dependent on the composition of mobile phase. The linear calibration plots range from 0.1 to 10 μg ml−1 in mobile phase containing 5.0 × 10−2 mol l−1 SDS/9 % n-butanol-1 at pH 6.0, and the detection limit in order of 0.1 μg ml−1 was obtained. The proposed method was used for the determination of steroids in urine using direct injection of samples without previous treatment.

Direct determination of anabolic steroids in human urine by on-line solid-phase extraction/liquid chromatography/mass spectrometry.

A fully automated method is presented for the determination of anabolic steroids in human urine. On line solid-phase extraction with automatic cartridge exchange is used for sample clean-up followed by an high-performance liquid chromatographic isocratic separation. A narrow-bore 5 microns reversed-phase column (250 x 2.1 mm i.d.) at a flow rate of 0.2 ml min-1 is connected to a particle beam interface linked to a single-quadrupole mass spectrometer. No derivatization is needed and normal electron impact mass spectra are obtained for a series of up to 28 steroids and metabolites. Urine samples collected following the ingestion of therapeutic doses of some of the steroids were analysed and the detection and confirmation of excreted compounds accomplished. A dramatic reduction in human intervention and overall analysis time was achieved compared with other common approaches.

Application of β-cyclodextrin for the analysis of estrogenic steroids in human urine by high-performance liquid chromatography

A procedure is described for simultaneous determination of estriol, estrone and 17β-estradiol in human urine. After acid hydrolysis of the sulphate conjugates, the estrogens were extracted into diethyl ether. The ether extracts were concentrated and applied directly to an HPLC column. Using a 25 cm C-18 column and acetonitrile-water modified by the addition of β-cyclodextrin as mobile phase, the separation of estriol, estradiol and estrone was achieved within 20 minutes. The extraction of estrogens from the biological matrix is excellent. Estrogens were detected using a UV-detector (280 nm) or a spectrofluorimetric detector (λexc=280nm, λem=312 nm). The latter detection system has been used for the determination of estrogens in the urine of non-pregnant women. The procedure is simple and can be used in clinical practice.

Monosulfates of 16-oxygenated ketonic C 19 steroids in adult human urine

A monosulfate fraction of steroids in human urine was obtained by chromatography on Sephadex LH-20. After solvolysis, fractionation on silicic acid and thin-layer chromatography, the steroids were subjected to gas-liquid Chromatographic and gas Chromatographic-mass spectrometric analysis. The following 16-hydroxylated ketonic c 19 steroids were identified in the monosulfate fraction of normal female and male urine: 16a-hydroxydehydroepiandrosterone, 16a-hydroxyandrosterone, 16a-hydroxyepiandrosterone, 16a-hydroxyetiocholanolone, 3a, 16a-dihydroxyandrost-5-en-17-one, and 16β-hydroxydehydroepiandrosterone. In addition, 3β, 17β-dihydroxyandrost-5-en-16-one was shown to be present in the same fraction of urine. Some quantitative values of the urinary excretion of these compounds are given.

Biosynthesis of 18-hydroxytestosterone in the human foetal liver.

After incubation of testosterone with 105 000 X g microsomes of human foetal liver, a C19 steroid with a 3‐oxo‐Δ4‐structure was isolated by ascending one‐dimensional thin‐layer chromatography. Gas chromatography‐mass spectrometry revealed that the compound was identical with 18‐hydroxytestosterone. The significance of this finding in relation to earlier reports of 18‐hydroxylated steroids in human urine is discussed.

Identification of 3α-hydroxyandrost-5-en-17-one in human plasma, urine and bile

In the monosulfate fraction of the steroids in human urine and bile from normal subjects as well as in blood plasma from uremic and some normal subjects 3α-hydroxyandrost-5-en-17-one has been identified. The identification was based on gas-liquid chromatographic and gas chromatographic-mass spectrometric analysis of the compound and several of its derivatives.

STUDIES ON THE »DIFFERENTIAL« HYDROLYSIS OF STEROID CONJUGATES IN URINE

ABSTRACT The conditions for optimal yields from human urine of corticosteroids and 17-ketosteroids were investigated by means of different methods of hydrolysis and extraction. With β-glucuronidase hydrolysis and simultaneous dialysis, almost all tetrahydro derivatives of cortisone, hydrocortisone, corticosterone, as well as etiocholanolone, androsterone and 11-oxygenated 17-ketosteroids are liberated. The dialysis does not proceed quantitatively, particularly with regard to the isolation of 17-ketosteroids. Hydrocortisone and cortisone seem to occur as free steroids in human urine. They can be extracted at pH 7.0 without preceding hydrolysis. The continuous extraction with ether or ethyl acetate at pH 1.0 hydrolyses the sulphuric acid esters of dehydroepiandrosterone and androsterone, but no measurable amounts of corticosteroid conjugates. Extraction at an acid pH leads to considerable spoiling of the extracts by unspecific tetrazolium blue positive chromogens. For the simultaneous hydrolysis and extraction of corticosteroids and 17-ketosteroids from one urine sample the following procedure is proposed: β Glucuronidase hydrolysis and dialysis for three days; extraction at pH 7.0 with ether of the watery contents of the dialyzing tube; continuous extraction with ether at pH 0.5 for 48 hours.

Urinary excretion of 17-ketogenic steroids in normal subjects before and after stimulation with intramuscular zinc-ACTH.

The estimation of 17-ketogenic steroids in human urine has proved a useful means of indirectly measuring the adrenocortical production of cortisone and hydrocortisone. Studies of precision and accuracy indicate that this procedure is practical for routine clinical purposes. The excretion of 17-ketogenic steroids in day and night 12-hour urine samples was determined in a group of 31 normal subjects. The basal 24-hour excretion was 13.1 mg. (S.D.±5.0) for males and 10.8 mg. (S.D.±2.9) for females. A diurnal variation was noted. The output of 17-ketogenic steroids was also studied during two consecutive 12-hour periods following single intramuscular injections of Cortrophin-Zinc. The mean response was approximately proportional to the basal output. A maximal response in terms of both magnitude and duration was obtained with doses of 30 to 40 (USP) units. Larger doses produced no significant increase in steroid output nor prolongation of action. Repeated responses of 17-KGS to intramuscular ACTH may vary considerably. Individual differences between subjects or within the same subject on different occasions largely account for these variations, which appear to be correlated with variations in basal excretion from day to day.

Enzymatic estimation of steroids in human urine.

The enzymatic microestimation of urinary steroid alcohols by means of ±- and ²-hydroxysteroid dehydrogenases is described. These highly purified enzymes catalyze selective and stereospecific oxidations of certain hydroxy-steroids to ketosteroids. Diphosphopyridine nucleotide functions as hydrogen acceptor and the assays are based on the spectrophotometric measurement of the reduced pyridine nucleotide. Methods are described for the preparation of extracts of human urine suitable for enzymatic assay. The reliability of the procedures has been established by a variety of criteria including the recovery of physiological amounts of steroids added to urineValues for the excretion of ±- and ²-hydroxysteroids have been determined in normal adult subjects of both sexes and in several patients with abnormalities of the endocrine system. The estimation of individual urinary steroids separated by paper chromatography is demonstrated. THE estimation of urinary steroids in man has been of interest for approximately 20...

THE DETERMINATION OF ADRENOCORTICAL STEROIDS IN HUMAN URINE1

introduction Various methods for the determination of adrenocortical steroids in human urine have been proposed in recent years. Hormonally active corticosteroids have been measured by bioassay (e.g., Selye and Schenker 1938, Eggleston et al. 1946, Venning et al. 1946), but the relatively small concentrations of active material, particularly in normal human urine (Venning and Kazmin 1946, Heard 1948) has required the processing of large amounts of urine. The probability of the presence in urine of typical adrenocortical substances lacking biological activity has led to attempts at their measurement. Advantage has been taken of the fact that certain typical adreno-corticosteroids possess significant reducing activity by virtue of an α-ketol side chain at C-17 (Talbot et al. 1945) as well as the α, β unsaturation in ring A (Heard and Sobel 1946). In addition fairly specific determination of certain types may be had by periodic acid oxidation which generates formaldehyde from either α-ketol or glycol side chains (Lowenstein et al. 1946, Daughaday et al. 1948). In this paper we will compare the results of assay of reducing with formaldehydogenic activity.

ADRENAL CORTICAL HORMONE EXCRETION IN ENDOCRINE AND NONENDOCRINE DISEASE AS MEASURED BY CHEMICAL ASSAY

IN A previous paper (1) a method was described for the estimation of adrenal cortical steroids in human urine by the quantitative measurement of the formaldehyde liberated by oxidation with periodic acid from urinary extracts. This method has been used in the study of normal subjects and in patients with endocrine and nonendocrine diseases; the results are presented in this paper. The technique which we have employed measures a chemical property of the urinary steroids possessing a C-17 side chain. Presumably, not only active compounds but also inactive substances are measured. This method, therefore, does not possess the same specificity possessed by bioassay (2, 3) and its validity must rest on how well the results conform to the known pathologic physiology of the adrenal and the results obtained using bioassays. The possibility exists that, because chemical methods measure both active and inactive substances, information may be obtained which is of different significance and would supplement information obtained by bioassay.