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A highly sensitive derivatization method for liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry of dehydroepiandrosterone (DHEA), testosterone (T), pregnenolone (P5), and 17alpha-OH-pregnenolone (17-OHP5) was developed based on the use of fusaric acid as a reagent. DHEA, P5, and 17-OHP5 were rapidly and quantitatively converted to the 3-fusarate esters by treatment with fusaric acid and 2-methyl-6-nitrobenzoic anhydride. The positive ESI-mass spectra of the fusarate esters of each steroid were dominated by the appearance of [M + H](+) as base peaks. The fusarate derivatization of these steroids showed 17.6-fold (DHEA), 11.9-fold (P5), 3.3-fold (17-OHP5), and 1.8-fold (T) higher sensitivity to those of the corresponding picolinate derivatives in LC-selected reaction monitoring., (2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.)

New derivatization reagents, 1-(2,4-dinitro-5-fluorophenyl)-4-methylpiperazine (PPZ) and 4-(4-methyl-1-piperazyl)-3-nitrobenzoyl azide (APZ), were developed for the liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) determination of steroids having a hydroxy group. PPZ reacted with a phenolic hydroxy group in estrogens. After quaternarization of the PPZ-estrogens with methyl iodide, the resulting derivatives provided more than a 2000-fold higher sensitivity compared to the intact estrogens. After derivatization of steroids having an alcoholic hydroxy group (5-ene-steroids or 5alpha-reduced steroids) with APZ followed by methylation, their detection responses increased more than 500 times. These derivatization procedures coupled with LC-ESI-MS/MS were successfully used for the determination of estrogens in the serum and prostatic 5alpha-dihydrotestosterone.

Synthesis and liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) behaviors of the picolinoyl, 6-methylpicolinoyl, nicotinoyl, 2-methoxynicotinoyl and isonicotinoyl derivatives of the hydroxysteroids estrone, estradiol, 3beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) and testosterone in positive mode were investigated. Each steroid was converted to the corresponding pyridine-carboxylate derivative by the acyl chloride method or the mixed anhydride method using the corresponding free acids and 2-methyl-6-nitrobenzoic anhydride; in each case, the latter method principally gave a better yield. The pyridine-carboxylate derivative of each steroid exhibited a clear single peak in liquid chromatography with a reversed phase column and CH(3)CN-0.1% CH(3)COOH as a mobile phase. The positive-ESI-mass spectra of the picolinoyl, 6-methylpicolinoyl and 2-methoxynicotinoyl derivatives showed a predominance of [M+H](+), whereas [M+H+CH(3)CN](+) was observed with high intensity in the nicotinoyl and isonicotinoyl derivatives. Even in the case of estradiol, with its two hydroxyl groups, a single charged ion of [M+H](+) or [M+H+CH(3)CN](+) was observed in the positive-ESI-mass spectrum of each derivative. The results revealed that picolinoyl derivatization is a simple and versatile method suitable for the sensitive and specific determination of hydroxysteroids by LC-ESI-MS (selected reaction monitoring).

Chemical investigation of the dichloromethane extract of the Red Sea marine sponge Lamellodysidea herbacea led to the isolation of four novel polyhydroxysteroids: cholesta-8-en-3beta,5alpha,6alpha,25-tetrol (1), cholesta-8(14)-en-3beta,5alpha,6alpha,25-tetrol (2), cholesta-8,24-dien-3beta,5alpha,6alpha-triol (3), and cholesta-8(14),24-dien-3beta,5alpha,6alpha-triol (4). Their structures were identified through 1D and 2D NMR studies. Relative stereochemistries were established by analysis of chemical shifts, coupling constants, and NOESY correlations. Compounds 3-4 showed antifungal activity against Candida tropicalis, with an inhibition diameter of 13 and 11 mm at 10 microg/disc, respectively.

Seasonal variations in the levels of polar steroids including polyhydroxylated steroids and related glycosides in digestive organs of the starfish Patiria (=Asterina) pectinifera have been studied. The concentration of polar steroids is related to the annual reproductive cycle of the starfish and periods of active feeding. Two peaks in concentrations of polar steroids in pyloric caeca and stomach were found, the first in winter during reorganization and the second in summer during intensive gametogenesis before spawning. Probable biological functions of polyhydroxysteroids and related glycosides are discussed. The data support the hypothesis these compounds are involved in digestion in the starfish.

High sensitivity radioimmunoassay of 3beta, 7alpha-dihydroxy-5-androsten-17-one (7alpha-OH-DHEA) has been developed and evaluated. The method is based on polyclonal rabbit antisera raised against 19-O-(carboxymethyl)oxime bovine serum albumin conjugate and bridge- and position homologous [(125)I]iodotyrosine methyl ester as a tracer. Sensitivity of the assay amounted to 3.12 fmol (0.95 pg)/tube, precision as a mean intra- and interassay coefficient of variation was 7.1 and 10.6%, respectively, and the average recovery of the analyte added to steroid-free serum was 110%. Out of the steroids occurring in human serum which may interfere with the assay, the only important cross-reactants were dehydroepiandrosterone and 3beta, 7beta-dihydroxy-5-androsten-17-one (7beta-OH-DHEA) with cross-reactivities of 1.95 and 1.16%, respectively. The levels of free (unconjugated) 7alpha-OH-DHEA have been determined in 29 sera from healthy volunteers (23 females and 6 males), and from 48 patients (43 females and 5 males) in which dehydroepiandrosterone and its sulfate (DHEA/S) had been measured for various endocrinopathies. The levels in healthy subjects ranged from 0.21 to 6.57 (mean 2.33+/-1.50) nM, while those of the patients from 0 to 5. 99 (mean 1.46+/-1.52) nM. The levels of 7alpha-OH-DHEA in patients significantly correlated with those of DHEA and its sulfate.

3 alpha- or 3 beta-hydroxysteroids, such as bile acids (free and glycine and taurine conjugates), 3 beta-hydoxy-5-cholenic acid, pregnanediol, 5-pregnene-3 beta, 20 beta-diol and 5-pregnene-3-beta,20 alpha-diol, were converted to 3-oxosteroids by enzymatic reaction using immobilized hydroxysteroid dehydrogenase, derivatized with dansylhydrazine to the corresponding dansyl hydrazones and purified by gel permeation chromatography. The dansyl hydrazones were chromatographed on a C18 column with a tetrahydrofuran-containing eluent and detected at the level of a few femtomoles by a peroxyoxalate chemiluminescence post-column reaction using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate as a chemilumigenic reagent. The dansyl hydrazones of chenodeoxycholic acid and deoxycholic acid (free and glycine and taurine conjugates) in particular, which coeluted under the chromatographic conditions above, were separated using an eluent including acetonitrile and 2,6-di-O-methyl-beta-cyclodextrin and detected in the same way.

The liquid chromatographic properties of various 17-hydroxy anabolic steroids are examined under reversed-phase conditions. These anabolic steroids are now listed as controlled drugs in many states due to their abuse potential in athletics, body building, and other areas. These nonesterified steroids are separated on a C18 stationary phase with a 70% methanol in water mobile phase. In a few cases, two compounds display very similar retention properties. However, dual-wavelength detection at 254 and 280 nm allows for their differentiation. Reversed-phase retention parallels steroid lipophilicity based on hydroxyl and methyl group substituents. Also, those steroids containing a dienone substructure are more polar than steroids containing an enone moiety.

A new silylating reagent that forms cyanosyl (cyanoethyldimethylsilyl) derivatives with monohydroxysteroids is introduced and its usefulness for the gas chromatographic analysis of these compounds evaluated. The reactivity of the new reagent, N-methyl-N-cyanoethyldimethylsilyltrifluoroacetamide, has been studied using a series of monohydroxysteroids and compared to that of other reagents forming cyanosyl or alkylsilyl derivatives. The results obtained indicate that at room temperature, in polar solvents, the reagent reacts rapidly and selectively with most hydroxyl groups found in steroids. The cyanosyl derivatives formed have retention properties that are comparable to those of the corresponding alkylsilyl derivatives. However, the former offer advantageous properties for detection since they can be analyzed with a nitrogen-phosphorus detector, and they exhibit mass spectral features that are suitable for identification and quantitation at low levels using selected ion monitoring mass spectrometry. The detection limits for all the monohydroxysteroids studied in this work, as cyanosyl derivatives, are typically on the order of 30-50 pg and the relative response factors are unity within a range of 25 percent.

The chromatographic mobility of steroidal glucosiduronic acids on paper in chloroform-formamide increases as the concentration of ion exchanger in the chloroform phase increases; mobility decreases as the concentration of counterion in formamide increases. Mobility of glucosiduronic esters and of hydroxylated free steroids increases with an increase in concentration of exchanger; small changes in concentration of counterion in the stationary phase do not alter the migration of these nonionizable compounds. Data are presented which suggest that partition of the glucosiduronic acids between the two phases occurs predominantly by an ion-exchange process and that hydrogen bonding plays a secondary role. Partition of the glucosiduronic esters and hydroxylated free steroids appears to occur primarily by a hydrogen-bonding process.

A method for the separation of 3beta-hydroxysterols from other sterols is presented. The method involves precipitating 3beta-hydroxysterols as the digitonides. The digitonide is then decomposed and separated into components using chromatography on highly cross-linked lipophilic polysaccharide gels. The digitonin in the mother liquor is separated from other sterols using the same chromatographic procedure.

The mass spectra of the dimethylphosphinic, dimethylthiophosphinic and dimethylphosphinous ester derivatives of several monohydroxy steroids are reported. The fragmentations of the derivatized steroids largely depend on the nature of the phosphorus-containing ester group. Phenolic ester derivatives exhibit the base peak at the molecular ion, whereas the spectra of the secondary phosphinic esters are dominated by very intense protonated phosphinic acid ions [Me2P(XH)(OH)]+ at m/e 95 (X =O) or at m/e 111 (x = s). The present results also indicate the low ionization potential for the phosphinic ester group. Due to their good gas chromatographic properties, these steroid derivatives appear to be particularly suitable for gas chromatographic mass spectrometric analysis of biochemical materials.

3,5-Bis-trifluoromethylbenzoyl derivatives of eight monohydroxy steroids and eight dihydroxy steroids were synthesised. Under the reaction conditions described, the monohydroxy steroids each gave a single derivative while the dihydroxy steroids, with the exception of corticosterone, showed multiple product formation. The reactivity of hydroxyl groups relative to their stereochemistry is discussed. The electron capture negative ion chemical ionisation mass spectra of these derivatives were recorded. When the derivatised hydroxyl group was alicyclic or aromatic, a molecular ion was normally the base peak in the mass spectrum. Selected ion monitoring of molecular ions indicated that, in certain cases, as little as 1 pg of the parent steroid could be detected. The potential use of this derivative in the quantitative analysis of steroids by gas chromatography-mass spectrometry is discussed.

In 24 persons with healthy liver and 44 patients with without exception morphologically ascertained chronic hepatopathies of different degree of severity the diagnostic valency of the bile acids was tested. In these cases the bile acids estimated in the C-bile proved as sensitive indicators of a chronic lesion of the liver parenchyma. Quantitative deviations of the bile acids, the relation from tri- to dihydroxycholan acids, qualitative alterations in the spectre of the free bile acids as well as changes of the form of conjugation were of importance concerning the functional diagnosis. The concentration of bile acids decreased the more expressed was the chronic lesion of the liver parenchyma. Trihydroxycholan acids and dihydroxycholan acids variably participated in the reduction of the bile acids in different forms of chronic hepatopathies. From the change of the quotient tri- to dihydroxycholan acids causal connections between the degree of severity of the chronic hepatopathy and the dominance of the chenodesoxycholic acid could be derived. Moreover, the conjugation of the bile acids with glycine prevailing in chronic hepatopathies underwent a degradated change. From the examinations results that quantitative and qualitative deviations of the bile acid spectre under defined morphological prerequisites render possible a differentiated judgment of the liver function in chronic hepatopathies.

The separation of twenty-six saturated and two unsaturated C19O3 steroids has been studied by thin-layer chromatography on silica gel F254, with seven mobile phases, and by gas-liquid chromatography on packed columns with four stationary phases; combination of both techniques permitted separation of all the test compounds. The mass spectra of the steroids were obtained by gas chromatography-mass spectrometry and are presented. Fragmentation processes have been studied, and characteristic ions that may be used for multiple ion detection or lead to identification of biologically produced C19O3 steroids are discussed.

While a different timing of circadian rhythms does not necessarily demonstrate the operation of independent mechanisms, it is one step in the isolation of separate interacting rhythmic factors underlying the dynamics of all life. With this step in mind, circadian rhythms are quantified in rat plasma and brain for corticosterone (B), pregnenolone (P), and dehydroepiandrosterone (D) by the rejection of the zero-amplitude assumption with the single cosinor method, from data obtained every 3 hr for 24 hr on groups of three male Sprague-Dawley rats, 11-12 weeks of age. The rats were killed; steroids were extracted from brain and plasma and were radioimmunoassayed. In relation to the acrophase (phi) of B, the phi of P in brain (P = 0.035) and of D in plasma (P = 0.0012) preceded the phi of B. By contrast, in clinically healthy women, the phi of plasma dehydroepiandrosterone sulfate (DHEA-S) lags behind that of cortisol (F), on the average by 6 hr 28 min. Such a lag is also seen in men. A species difference in the time relations of delta 5-3 beta-hydroxysteroids vs. glucocorticoids in plasma is obvious (P less than 0.01). A difference in time relations of human circulating D, DHEA-S, and F between schizophrenic and clinically healthy men renders the time relations of glucocorticosteroids and delta 5-3 beta-hydroxysteroids in brain particularly interesting, as do relations of an egocentric and expansive personality to the rhythm characteristics of DHEA-S. The fact that the acrophases for some of the steroids investigated in brain and plasma, respectively, are differently timed is in keeping with the assumption that they involve partly different mechanisms, even if differences in the metabolic handling of different steroids also remain to be investigated.

A bile acid:3'phosphoadenosine-5'phosphosulfate:sulfotransferase (BAST I) from adult female rat liver cytosol has been purified 157-fold by a two-step isolation procedure. The N-terminal amino acid sequence of the 30,000 subunit has been determined for the first 35 residues. The Vmax of purified BAST I is 18.7 nmol/min per mg protein with N-(3-hydroxy-5 beta-cholanoyl)glycine (glycolithocholic acid) as substrate, comparable to that of the corresponding purified human BAST (Chen, L-J., and I. H. Segel, 1985. Arch. Biochem. Biophys. 241: 371-379). BAST I activity has a broad pH optimum from 5.5-7.5. Although maximum activity occurs with 5 mM MgCl2, Mg2+ is not essential for BAST I activity. The greatest sulfotransferase activity and the highest substrate affinity is observed with bile acids or steroids that have a steroid nucleus containing a 3 beta-hydroxy group and a 5-6 double bond or a trans A-B ring junction. These substrates have normal hyperbolic initial velocity curves with substrate inhibition occurring above 5 microM. Of the saturated 5 beta-bile acids, those with a single 3-hydroxy group are the most active. The addition of a second hydroxy group at the 6- or 7-position eliminates more than 99% of the activity. In contrast, 3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid (deoxycholic acid) is an excellent substrate. The initial velocity curves for glycolithocholic and deoxycholic acid conjugates are sigmoidal rather than hyperbolic, suggestive of an allosteric effect. Maximum activity is observed at 80 microM for glycolithocholic acid. All substrates, bile acids and steroids, are inhibited by the 5 beta-bile acid, 3-keto-5 beta-cholanoic acid. The data suggest that BAST I is the same protein as hydrosteroid sulfotransferase 2 (Marcus, C. J., et al. 1980. Anal. Biochem. 107: 296-304).

We studied the use of an immobilized enzyme, covalently bound to aminopropyl-CPG, in the analysis of individual delta 5-3 beta-hydroxysteroid sulphates. A microcolumn with immobilized 3 beta,17 beta-hydroxysteroid dehydrogenase was prepared and used together with high-performance liquid chromatography (HPLC). The reduced nicotinamide-adenine dinucleotide produced from delta 5-3 beta-hydroxysteroids by this enzyme was fluorimetrically determined. The immobilized enzyme was sufficiently stable for at least one month or for 180 tests when used repeatedly. A clinical trial demonstrated that this HPLC-immobilized enzyme method is superior to the soluble enzyme method, giving reliable and reproducible results at a low cost.

We describe a simple enzymatic method for the sensitive and specific detection and quantitation of families of hydroxy- and oxo-steroids in biological mixtures. Analysis of the profiles of individual steroids may be achieved following their chromatographic separation. The objectives of this analytical system are, therefore, different from conventional methods which are designed to measure single steroids with a high degree of specificity. The method employs highly purified and active bacterial hydroxysteroid dehydrogenases (HSD) which promote stereospecific, nicotinamide nucleotide-dependent oxidations and reductions at specified positions of steroids. In the presence of catalytic quantities of steroids these enzymes promote the transfer of hydrogen (transhydrogenation) between NADH and NAD analogues. A recently purified 17 beta-HSD from an Alcaligenes species (D. W. Payne and P. Talalay, J. biol. Chem., 260, 13468-13655, 1985) shows almost complete specificity for the 17 beta-hydroxy- and 17-oxo-groups of both C18 and C19 steroids. This enzyme catalyzes steroid-dependent transhydrogenation between NADH and the thionicotinamide analogue of NAD (S-NAD). When these components are incubated at pH 8.5 in the presence of minute quantities of steroid substrates, S-NADH (measured at 398 nm where NADH does not absorb) accumulates at a constant rate which is proportional to the concentrations of steroid and enzyme. The linear increase in absorbance with time is a measure of the total concentration of 17 beta-hydroxy- and 17-oxo-steroids, and can be used to detect subpicomol quantities of steroids. The method is illustrated by the detection and identification of free and conjugated androgens in human serum following their separation by high pressure liquid chromatography. The specificity of the transhydrogenase assay is completely dependent on the specificity of the enzyme and is thus applicable to the detection of other hydroxy- and oxo-steroids by making use of HSDs with appropriate specificities (e.g. 3 alpha-HSD for the measurement of 3 alpha-hydroxy- and 3-oxo-steroids). The simple one-step reaction lends itself to automation, needs no auxiliary detection systems, and requires only an inexpensive colorimeter.

The chromatographic behaviour of adrenal 18-hydroxysteroids, and a series of derivatives thereof, has been studied in reversed-phase and normal-phase high-performance liquid chromatography (HPLC). Both 18-hydroxycorticosterone and 18-hydroxy-11-deoxycorticosterone exhibited a marked loss of chromatographic efficiency when separated on incompletely-covered C18 reversed-phase supports (as determined by methyl red adsorption) showed no such effect, nor was it seen when aprotic solvents, such as dioxane, were used. This phenomenon is unique among the wide range of adrenal and testicular steroids that we have studied and affords a useful test, applicable under aqueous conditions, of coverage by the alkylsilane reversed phase, a factor of considerable importance in the successful resolution of complex mixtures of adrenal steroids by reversed-phase HPLC. The retention times of the 20-methoxy, 20-ethoxy, 21-acetoxy, etiolactone, 11 beta, 18-ether and dimeric derivatives of the naturally-occurring 18-hydroxysteroids have been determined in relation to major adrenal steroids. Procedures for extraction and reversed-phase HPLC of 18-hydroxysteroids from tissues without the formation of these less polar forms, which can complicate their separation by other chromatographic techniques, are illustrated with respect to a human malignant adrenocortical tumour which caused hypermineralocorticism.

In order to apply the intensity-matching technique to polyhydroxysteroids in biological fluids, the doublet of [M]+ and [M+3]+ was prepared in the process of derivatization by use of a mixture of trimethylsilylimidazole (TSIM) and TSIM-d3 and was used for mass fragmentography. The molar ratio of TSIM and TSIM-d3 was defined by the reciprocal of the number of hydroxyl groups in steroids. This technique may be useful in identifying low levels of the endogenous polyhydroxysteroids of interest in biological fluids.

Rabbits injected with mixtures of [3H]deoxycorticosterone and [14C]progesterone had significant levels of both 3H and 14C in several tissues and fluids extracted 10-45 min later. The distribution of radioactivity between 21-deoxysteroid, 21-hydroxysteroid, steroid acid and steroid glucuronide fractions was determined by alumina adsorption chromatography. Steroid acids derived from both steroids accumulated in the liver, kidney and urine, but were quantitatively less significant in the bile, duodenum, uterus, spleen and lung and were detected in the blood for the first time. Different 21-hydroxysteroid profiles were detected in the tissue and fluid extracts by reverse and straight phase high pressure liquid chromatography. [3H]Deoxycorticosterone accumulated in the kidney, lung, spleen and uterus, whereas tetra and hexahydro reduced metabolites predominated in the liver, bile and duodenum. By contrast, [14C]progesterone was metabolised to more polar 21-hydroxylated metabolites which were detected in the liver, kidney and urine. These results show the influence of a steroid 21-hydroxyl function, when administered, as opposed to being formed in in vivo, on the metabolic fate and excretory pathways of 21-hydroxysteroids by the rabbit.

Differences in methylene unit values were used for the determination of the hydroxyl group number of a steroid by means of gas chromatography. This index is defined as the difference in the methylene unit value between trimethylsilyl and other dimethylalkylsilyl (DMAS) ether derivatives of hydroxylated steroids, namely dimethylethylsilyl (DMES) and dimethyl-n-propylsilyl (DMPS) ethers. The reactivities of DMES and DMPS imidazoles as silylating agents were nearly equal to that of TMS-I. Mass spectra of these derivatives were characterized by the molecular ion cluster, [M]+., [M-15]+ and [M-29]+ (or [M-43]+). The molecular ion cluster of these derivatives is most useful for estimating the molecular weight. Therefore, these DMAS ethers provide valuable information for structural elucidation of hydroxylated steroids by gas chromatography electron impact mass spectrometry.

Sex steroids and 5-en-3 beta-hydroxysteroids were determined by radioimmunoassay in specific regions of the human brain, in the anterior and posterior pituitary, in one sensory organ, the retina and in the cranial nerves. Progesterone, androstenedione, testosterone and estrone were found in all areas of the brain and in all the cranial nerves but not in all cases. There was no sex difference except in the case of androstenedione where values were higher in women in some brain areas. Estrone values were always higher than those of estradiol in both men and women. No 5 alpha-dihydrotestosterone was detected in any of the samples studied. The values for pregnenolone, dehydroepiandrosterone and their sulfates were much higher than those of the sex steroids in all areas of the brain and in all the cranial nerves. Values for pregnenolone were greater than those of its sulfate while those of dehydroepiandrosterone were in general equal to or higher than those of its sulfate. The values for pregnenolone were greater than those of dehydroepiandrosterone. There were no obvious regional differences in the concentrations of the 5-en-3 beta-hydroxysteroids either in specific areas of the brain or in the cranial nerves. But there was a definite trend for the free dehydroepiandrosterone values to be higher in women. The possible significance of these observations is discussed.

The 25 eV mass spectra of the trimethylsilyl derivatives of a number of 6-hydroxy and 3,6-dihydroxy steroids together with deuterium and 18O-labeled analogs have been examined to determine the influence of the 6-OTMS group on fragmentation patterns. Ions in the cholestane series at m/z 321 and 403 were the most characteristic ions derived from the 6-OTMS function; their relative abundances, although low in the spectra of 6-OTMS steroids themselves, were considerably elevated when a 3-OTMS or 3-oxo group was present. Similar ions were present in the spectra of androstane and pregnane derivatives. No correlation was found between the abundance of these ions and the stereochemistry at C3, C5, or C6. Fragmentation mechanisms and gas chromatographic data are discussed.

In this study the levels of Estriol and their precursors C(16) substituted in cord plasma are examined. The values found during normal pregnancies are compared with those of pregnancies complicated by different pathology. These compounds can be considered as a good index of feto-placental wellbeing.

We used microbial 3 alpha, 7 alpha-, and 12 alpha-hydroxysteroid dehydrogenases to quantify 3 alpha-, 7 alpha-, and 12 alpha-hydroxyl groups in human feces. Samples collected over a 48-h period were lyophilized, extracted, and reconstituted by a modification of the method of DeWael et al. (clin. Chim. Acta 79: 465--470, 1977), then quantified enzymically by use of three group-specific hydroxysteroid dehydrogenase preparations. Total values for the three hydroxyl groups are given for 18 healthy humans. The ratio of 3 alpha-OH groups/7 alpha-OH groups/12 alpha-OH groups was 34.4 (SD 5.9)/25.2 (SD 6.4)/40.4 (SD 7.7) for the 18 samples.

A phenolic trimethylsilyl (TMS) group was selectively exchanged for a dimethylalkylsilyl (DMAS) group on a gas chromatographic column by use of sandwich injection technique with DMAS-imidazole, and a TMS ether derivative of a phenolic steroid was converted into a DMAS ether or a mixed TMS and DMAS ether derivative with over 95% recovery. The selective exchange reaction seemed to be caused by the difference in lability between the ethereal TMS linkages to phenolic and alcoholic hydroxyl groups. This selectivity was found to be useful for discriminating gas chromatographically between the phenolic and alcoholic hydroxyl groups in steroids.

The fragmentation processes occurring in various 9alpha-hydroxy steroids upon electron ionization have been studied. The mechanisms proposed for the formation of the prominent ions in these spectra have been confirmed with the aid of deuterium labelling, measurements on metastable ion decompositions, low eV spectra and high resolution mass measurements. The fragmentation of the corresponding 9-oxo-9,10-seco steroids, which shows analogy to that of the 9alpha-hydroxy compounds in many respects, has also been discussed.

The trichloroacetyl carbamates of 38 corticosteroids and chemically related compounds were prepared, and their NMR spectra in deuterochloroform were obtained. The effects of the introduction of a number of functional groups on the chemical shift of the carbamate proton signals were determined.

Qualitative and quantitative effects of classical reactions on steroids observed by gas-liquid chromatography (GLC) under standardized conditions, including the double internal standard technique are reported. Simple procedures applicable to nanogram amounts of reactants are described. Reactions studied include the conversion of keto groups to hydroxyl groups by NaBH4, and to dioxolone derivatives by 1,2-diethanol; 17 alpha-hydroxylation of C20-ketosteroids; the conversion of hydroxyl groups to trimethylsilyl (TMS) ethers by hexamethyldisilazane; the hydrolysis of dioxolone and TMS derivatives by H+. Effects of Wolff-Kishner reagents, and CrO3 were also studied. GLC chromatograms of reaction mixtures of single- and multistep reactions readily provide information on effects on functional groups at positions 3, 17, 20, and 21 in the pregnane series, and the retention times of many steroids unavailable from commercial and other sources. GLC data analysis provides relationships between steroid structure and retention time from which methods for the computation of retention times and for steroid identification are designed. The accuracy of the computation methods is demonstrated.

Assaying the low concentrations of steroid hormones in extracts of body fluids requires detectors that are both highly sensitive to the steroid and relatively insensitive to interfering compounds usually present in much higher concentrations. To explore the use of moderately specific enzymes in post-column reactors, we immobilized 3 alpha- and beta-hydroxysteroid dehydrogenase on controlled pore glass beads, 37 microns in diameter, and constructed 4.6-mm diameter reactor columns, 3-cm long, packed with one of the two kinds of these beads. Hydroxysteroids eluted from the analytical column were mixed with the coenzyme, nicotinamide adenine dinucleotide (NAD), before passing through the reactor. The effluent from the reactor was passed through the 70-microliters flow cell of a fluorometer in which the fluorescence of the NADH produced in the enzyme-catalyzed oxidation of the hydroxysteroid was monitored. At the conventional high-performance liquid chromatography flow-rates used, oxidation of the steroids was almost complete. The yield depended on both the residence time of steroids in the reactor column and the concentration of organic modifier in the reaction mixture. Maximal yield was obtained with buffer having a low organic solvent concentration and passing through the reactor slowly. In assays of mixtures of epimeric hydroxysteroids, the 3 alpha-hydroxysteroids were detected with the 3 alpha-hydroxysteroid dehydrogenase reactor; the beta-hydroxysteroids were not, confirming the specificity of the enzymatic detection. With the fluorometer used, picomole quantities of steroids could easily be distinguished from noise.