Urinary excretion of 5beta-pregnane-3alpha,20alpha,21-triol in human gestation

The steroids in urine from normal pregnant women have been studied. After extraction of conjugate steroids, solvolysis and enzymatic hydrolysis, the liberated steroids were separated by chromatography on Sephadex LH-20, and were analysed by gas-liquid chromatography and gas chromatography mass spectrometry. The following steroids were isolated and completely identified in the LH-20 fraction 7: 5beta-pregnane-3alpha,20alpha-diol, 5beta-pregnane-3alpha,17,20alpha-triol, 5beta-pregnane-3alpha,20alpha,21-triol and 5alpha-pregnane-3beta,16alpha,20alpha-triol. In addition, two metabolites tentatively identified as 5xi-pregnane-2xi,3xi,20xi-triol and 2xi,3xi,16xi-trihydroxy-5xi-pregnan-20-one, have not been reported as occcurring in urine from pregnant women. The 5beta-pregnane-3alpha,20alpha,21-triol was detected only in the third trimester of pregnancy and the urinary excretion values are between 320 and 650 microgram per 24 h. With the present data, it is not possible to establish the precursor(s) of this steroid. However, these results tentatively suggest that 5beta-pregnane-3alpha,20alpha,21-triol arises from foeto-placental unit.     

Steroids in porcine follicular fluid: analysis by HPLC, capillary CG and capillary CG/MS after purification on SEP-PAK C18 and ion exchange chromatography

Steroids in porcine follicular fluid have been concentrated by reverse phase chromatography in SEP-PAK C18 and purified further on the cation exchanger SP-Sephadex C-25. Fractionation into unconjugated neutral and phenolic steroids, glucuronides and sulfates was carried out on triethylaminohydroxypropyl Sephadex LH-20 (TEAP-LH-20). The unconjugated neutral fraction was analysed by high pressure liquid chromatography (HPLC) on a C18 radial cartridge 5 mm I.D.; 10 mu, or on a C18 5 mu RESOLVE column, and by capillary gas chromatography (GC) on a 12 M OV-1 cross linked fused silica column. Testosterone, progesterone and androstenedione were the major steroids detected by HPLC monitored at 254 nm, although 17- hydroxy-, 20 alpha-dihydro- and 20 beta-dihydroprogesterone were also present. Pregnenolone, pregnanediol, dehydroepiandrosterone, 17-hydroxypregnenolone and androsterone were detected by capillary CG as their 0-methyloxime trimethylsilyether derivatives. Further confirmation of structure was provided by complete mass spectral data or by selective ion monitoring (SIM).     

Analyses of steroids in saliva using highly selective mass spectrometric techniques

Highly selective techniques of gas chromatography mass spectrometry have been used in the unequivocal identification of salivary steroids at concentrations ranging from 20 pg ml-1 to 20 ng ml-1. Oestradiol-17 beta, for example, has been identified in pregnancy saliva by gas chromatography high resolution mass spectrometry selected ion monitoring of the bis-TMS ether and by gas chromatography mass spectrometry metastable peak monitoring of the bis-tert-butyldimethylsilyl ether. Dehydroepiandrosterone sulphate has been identified in saliva, following enzymic hydrolysis, by gas chromatography high resolution mass spectrometry selected ion monitoring of the tert-butyldimethylsilyl ether and methyloxime tert-butyldimethylsilyl ether. These initial analyses have been designed to guide the development of routine immunoassay procedures which may subsequently be validated by comparison with reference gas chromatographic mass spectrometric methods.     

The identification and quantification of three new 6alpha-hydroxylated corticosteroids in human neonatal urine

The 24 hours urines of six two days old fullterm newborn infants were investigated for polar corticosteroids. 6alpha-hydroxy-tetrahydrocortisone, 6alpha-hydroxy-20alpha-cortolone and 6alpha-hydroxy-20beta-cortolone were identified by gas chromatographic-mass spectrometric comparison of the urinary steroids to compounds synthesized previously. These 6alpha-hydroxylated corticosteroids as well as seven other polar corticosteroids were quantified by gas chromatography or mass fragmentography. It was shown that the newly identified steroids constituted a quantitatively important part of the neonatal urinary corticosteroids. The unconjugated- and glucuronic acid conjugated steroids were quantified separately. It was found that the extent of glucuronoconjugation decreased with increasing polarity of the steroid moiety.     

Identification and quantitation of free and conjugated steroids in milk from lactating women

A method for analysis of metabolic profiles of free and conjugated steroids in milk has been developed. Milk is diluted with aqueous triethylamine sulphate and liquid-solid extraction is achieved on a Sep-Pak C18 cartridge at 60-64 degrees C. Steroids are purified by chromatography on small columns of Lipidex 5000 and sulphohydroxypropyl Sephadex LH-20 [H+] prior to separation into neutral and phenolic compounds, glucuronide, mono- and disulphate conjugate groups on the lipophilic strong anion exchanger triethylaminohydroxypropyl Sephadex LH-20 (TEAP-LH-20). Conjugated steroids are released by enzymatic or solvolytic procedures and separated into a neutral and a phenolic fraction on TEAP-LH-20. The O-methyloxime and trimethylsilyl ether derivatives of the steroids are analyzed by capillary column gas chromatography-mass spectrometry. Fifty steroids were identified in milk collected from women a few days after delivery. Quantitatively about 80% were present as sulphates, 15% as glucuronides and only 5% were unconjugated steroids. The steroid pattern was similar to that in late pregnancy plasma with pregnanolone, pregnanediol and pregnanetriol isomers and dehydroepiandrosterone being predominant. About 10% of the steroid content consisted of estrogens. The total concentration of steroids 2 days after delivery was 20-116 ng/ml, i.e. about 1-5% of the concentration was about 10 ng/ml 1 month after delivery. In one milk sample, collected 2 days after delivery, the steroid concentration (3.6 micrograms/ml) was similar to that in plasma.     

Sample purification using a C18-bonded reversed-phase cartridge for the quantitative analysis of corticosteroids in adrenal cell cultures by high-performance liquid chromatography or gas chromatography—mass spectrometry

Quantitative extraction and subsequent purification of small biological samples often involve cumbersome procedures. We have devised a short and efficient method for the quantitative extraction of the corticosteroid and the 20α reduced steroid series from culture medium containing 20% sera in a single, pure fraction with separation from cholesterol. Passage through a C₁₈-bonded reversed-phase Sep-Pak^® cartridge of the acidified culture medium and subsequent extraction of the steroid fraction with methanol yields a single fraction containing all steroids in 90% recovery and reduced quantities of cholesterol down to 30%. The extract can then be used without further purification for quantitative analysis by high-performance liquid chromatography or derivatized and analyzed by gas chromatography and gas chromatography—mass spectrometry.     

Adrenal tissue in the male African catfish,Clarias gariepinus: localization and steroid hormone secretion

Interrenal-like tissue in male African catfish is localized in paired organs, situated retroperitoneally anterior of the kidney. Histological and enzyme-histochemical (3-hydroxysteroid dehydrogenase) reactions were carried out on sections of these organs in order to localize steroid-producing cells. In vitro incubations were carried out to determine the steroidogenic capacity of the interrenal-like tissue. Twenty-one steroids could be identified and quantified in the incubation medium, by means of gas chromatography followed by mass spectrometry. Cortisol, 5-and 5-androstanedione and androstenedione together comprised about 80% of the total steroid content. In a previous study, twenty testicular steroids were detected in plasma after castration. In the present investigation we have shown that most of these steroids can be produced by the interrenal-like tissue, which thus can be considered as an extra-testicular source of gonadal steroids.     

Gas chromatography profile of estrogens: Application to pregnancy urine

A method for the simultaneous quantitation of 7 estrogens in pregnancy urine is described. It involves enzymatic hydrolysis, extraction of free steroids, ion-exchange column chromatography and quantitation of the trimethylsilyl derivatives by gas chromatography on OV 1. Data obtained from normal and twin pregnancies and from women with anencephalic fetus or intra uterine fetal death are analysed. The sensibility of the method is about 40 μg of each estrogen by liter of urine.     

Analysis of profiles of conjugated steroids in urine by ion-exchange separation and gas chromatography—mass spectrometry

A simplified, flexible method for the analysis of metabolic profiles of steroids in urine is described. Solid extraction with Amberlite XAD-2 or Sep-Pak C_{1 g} cartridges is followed by group fractionation of unconjugated neutral and phenolic steroids, monoglucuronides, monosulphates and disulphates on the lipophilic strong anion exchanger triethylaminohydroxyproply Sephadex LH-20 (TEAP-LH-20). Following brief enzymatic hydrolysis or solvolysis the steroids are purified on TEAP-LH-20. O-Methyloxime and trimethylsilyl ether derivatives are prepared and purified by filtration through Lipidex 5000, and are then analyzed by glass capillary column gas—liquid chromatography and gas chromatography—mass spectrometry.Between 2 and 5 ml of urine are used for a comprehensive analysis. Unconjugated neutral and phenolic steroids are isolated in half a day, corresponding steroids in the conjugate fractions in two days. No fraction containing steroids is discarded, but the analysis can be limited to a selected fraction.     

Testosterone and estradiol assays: current and future trends

Sex steroid measurements for the investigation of endocrine disorders have been fraught with accuracy and imprecision problems since the advent of high throughput, direct assays almost 10 years ago on automated analyzers. Results from testosterone and estradiol measurements at the low end of detectability have suffered the most and there are few automated systems that can accurately measure these steroids in women, children and hypogonadal males on a routine basis. With the advent of mass spectrometry coupled to either gas chromatography or liquid chromatography, an improved approach to the measurement of these steroids has developed that shows promise for accurately and precisely measuring testosterone and estradiol in all patient populations including women and children. These mass spectrometry based methods for the sex steroids have been established as higher order reference method procedures that will resolve the issues of low end sensitivity measurements for these steroids, provide for appropriate standardization and reference materials and align most laboratories in hospital and reference laboratories to generate results that are inter-changeable between laboratories and methods.     

Disulphates of 16-oxygenated ketonic c19 steroids as biliary metabolites of androsterone sulphate in female rats

The chemical synthesis of 16β-hydroxyandrosterone was described preparatory to studies of the disulphates of the 16-oxygenated ketonic C₁₉ steroids present in the bile of female rats dosed with [³H]androsterone sulphate. The biliary metabolites were separated by chromatography on Sephadex LH-20 to afford monosulphate and dicon jugate fractions. After solvolysis of the diconjugate fraction, the liberated steroids were separated by partition chromatography on Celite 545 and analyzed by gas chromatography-mass spectrometry. In addition to 3α, 17β-dihydroxy-5α-androstan-16-one isolated previously, 16β-hydroxyandrosterone was identified as a disulphate.     

Method for combined analysis of profiles of conjugated progesterone metabolites and bile acids in serum and urine of pregnant women

A method for analysis of profiles of conjugated progesterone metabolites and bile acids in 10 ml of urine and 1–4 ml of serum from pregnant women is described. Total bile acids and neutral steroids from serum and urine were extracted with octadecylsilane-bonded silica. Groups of conjugates were separated on the lipophilic ion-exchanger triethylaminohydroxypropyl Sephadex LH-20 (TEAP-LH-20). Fractions were divided for steroid or bile acid analyses. Sequences of hydrolysis/ solvolysis and separations on TEAP-LH-20 permitted separate analyses of steroid glucuronides, monosulfates and disulfates and bile acid aminoacyl amidates, sulfates, glucuronides and sulfate-glucuronides. Radiolabelled compounds were added at different steps to monitor recoveries and completeness of separation, and hydrolysis/solvolysis of conjugates was monitored by fast-atom bombardment mass spectrometry. The extraction and solvolysis of steroid disulfates in urine were studied in detail, and extraction recoveries were found to be pH-dependent. Following methylation of bile acids, all compounds were analysed by capillary gas chromatography and gas chromatography—mass spectrometry of their trimethylsilyl ether derivatives. Semiquantification of individual compounds in each profile by gas—liquid chromatography had a coefficient of variation of less than 30%. The total analysis required 3 days for serum and 4 days for urine.     

Quantitative analysis of steroid profiles from urine by capillary gas chromatography : I. Accuracy and reproducibility of the sample preparation

A method is described for the determination of steroid profiles from urine by means of gas chromatography using high-efficiency glass capillary columns. The accuracy and reproducibility of essential steps in the sample preparation (extraction of steroids and steroid conjugates by means of XAD-2, enzymatic hydrolysis with Helix pomatia juice, solvolysis in acidified ethyl acetate and alkali wash) are established using different endogenously labelled urine samples, obtained from normal subjects to whom labelled steroids had been administered. Preliminary results are given on the reproducibility of the derivatization procedure (formation of methoxime-trimethylsilyl (MO-TMS) ethers), the gas chromatographic analysis and the whole method. Two procedures for the purification of MO-TMS steroid derivatives are compared. Application of the method to urine samples of patients with various endocrine disorders is included.     

Excretion of estriol, estetrol, 16-epi-estriol, 16-keto-estradiol and 16-hydroxyestrone in the 24-hour urine of pregnant women in the last trimester]

In this publication a method is given which allows simultaneous estimation of estriol, estetrol, 16-epiestriol, 16-ketoestradiol and 16-hydroxyestrone in urine of pregnant women. First conjugates are precipitated with ammoniumsulfate and hydrolyzed. Then the steroids are extracted and converted to azodyes by reacting with the diazonium salt dark blue r. After separation by thin layer chromatography the azodyes are measured by remission analysis with a chromatogramm spectrophotometer. From the data obtained from 66 cases norm groups were set up for the excretion of the steroids in the 3rd trimester of pregnancy. In the last month of pregnancy the average excretion, expressed in % of excreted estriol, is as follows: estetrol 5,7%, 16-epiestriol 3,1%, 16-ketoestradiol 7,0%, 16-hydroxyestrone 5,3%.     

Gas chromatographic and mass spectrometric analysis of conjugated steroids in urine

This study was carried out qualitatively and quantitatively to investigate the presence and the concentrations of anabolic steroids in urine collected from orally administered humans. Microanalysis of conjugated steroids by gas chromatography and mass spectrometry (GC/MS) has been carried out. Following oral administration three major metabolites of anabolic steroid drugs have been detected and partially characterized. The six steroids can be analysed at the same time in 17 min. The lower detection limit was 10 ng/ml in 5 ml of urine. The conjugated steroids from urine were centrifuged to 2,430g for 10 min, the supernatant solution passed through Amberlite XAD-2 column and the steroids eluted fraction esterified by using MSTFA and TMSI. The rate of metabolism and urinary excretion seem to be reasonably fast.     

Fractionation of fecal neutral steroids by high performance liquid chromatography

Fecal neutral steroids were fractionated by high performance liquid chromatography (HPLC) into three major fractions: 5 beta-H, 3-keto steroids; 5 beta-H, 3 beta-hydroxy steroids; and 5 alpha-H and delta 5-3 beta-hydroxy steroids. This separation was achieved in about 10 minutes, with greater than 97% recovery of standards in each fraction. Gas-liquid chromatographic quantitation of fecal steroids fractionated by either HPLC or thin-layer chromatography gave nearly identical results. A method using both C18 reverse phase and silica HPLC to purify radiolabeled sterols is also described.     

Formation of C19 11-hydroxysteroids by porcine Leydig cells.

In a previous study, we reported the presence of 11 beta-hydroxyandrostenedione and 11 beta-hydroxytestosterone in testicular vein blood from mature male pigs. Since C19 steroids with an oxygen function at C11 have not been recorded as products of steroid biosynthesis in normal mammalian testes, we have examined their possible production in purified preparations of porcine Leydig cells. Both androstenedione and cortisol were added as substrates in studies using cell incubations of Leydig cells from mature boars (greater than 8 months old). Steroids were recovered from media by solid-phase extraction and separated by reversed-phase high performance liquid chromatography. Peaks corresponding to retention times of authentic standard steroids were seen for both 11 beta-hydroxyandrostenedione and 11 beta-hydroxytestosterone from each substrate. Generally, lesser amounts of C19 11-oxosteroids were noted also. Definitive confirmation was made by gas chromatography - mass spectrometry for 11 beta-hydroxyandrostenedione in the media.     

A rapid gas-liquid chromatographic method for the quantitation of urinary estrone, estradiol-17 , estriol, and pregnanediol throughout pregnancy

A rapid, sensitive and inexpensive method is presented for the determination of urinary estrone (E₁), estradiol-17β (E₂), estriol (E₃), and pregnanediol (P₂) in pregnancy urine. The assay involves enzymatic hydrolysis, ion-exchange chromstography, and final quantitation of the steroids as heptafluorobutyric anhydride derivatives in a gas Chromatograph. Twenty to 30 samples may be prepared for gas-liquid chromatography in one working day. For six samples, the results including gas-liquid chromatography and calculation can be obtained eight hours after receiving the urine specimens thus competing in speed with radioimmunoassays. The method is sensitive enough to allow urines from less than six weeks after the last menstrual period to be quantitated.     

Production and secretion of C-19 steroids by rat and guinea pig adrenals

The concentrations of C-19 steroids were measured in guinea pig and rat adrenals before and after castration as well as after stimulation with adrenocorticotropin hormone (ACTH). Characterization of adrenal C-19 steroids was also carried out by isolation with high-performance liquid chromatography and gas chromatography/mass spectrometry (GC/MS). From radioimmunoassay (RIA) data, androstenedione (4-DIONE) and 11 beta hydroxyandrostenedione (11 beta-DIONE) were the major C-19 steroids found in guinea pig adrenals, and castration induced a decrease of 4-DIONE levels only while all other C-19 steroids remained unchanged. In rat adrenals, the major C-19 steroids were 4-DIONE and testosterone, and they were also markedly inhibited after castration. With the exception of 11 beta-DIONE, all other C-19 steroids in circulation were eliminated after castration in both animals species. After ACTH administration in the guinea pig, adrenal 4-DIONE and 11 beta-DIONE levels were markedly stimulated, while an increase of only 11 beta-DIONE was observed in plasma. In the rat, ACTH had a small stimulatory effect on adrenal 52-androstane-3 alpha, 17 beta-diol (3 alpha-DIOL) and plasma 11 beta-DIONE levels. Analysis of guinea pig adrenal steroids by GC/MS confirmed the presence of C-19 steroids in adrenals (namely, 4-DIONE and 11 beta-DIONE) while, in the rat, this could not be confirmed. Our data indicate that production of C-19 steroids occurs in guinea pig adrenals, and 11 beta-DIONE is the major C-19 steroid as well as the only C-19 steroid secreted into the circulation. In the rat, the production of C-19 steroids detected by RIA is not supported by GC/MS data.     

Study of a virilizing adrenal cortical tumor. Analysis of urinary steroids by gas-liquid chromatography and mass spectrometry]

The C19 and C21 urinary steroids from a virilizing adrenal tumour with high levels of plasma 17alpha-progesterone and its urinary metabolites have been identified and quantitated by gas chromatography and mass spectrometry of sephadex fractions of the total urinary extract. Of the fifty five identified steroids thirteen were new compounds or known compounds not found before in such a case. The actiology of the apparent 21-steroid hydroxylase deficiency is discussed at the light of these analytical results and of the hormonogenesis enzymatic induction of the tumour biopsy.