Simultaneous analysis by LC-MS/MS of 22 ketosteroids with hydroxylamine derivatization and underivatized estradiol from human plasma, serum and prostate tissue.

This study describes a validated LC-MS/MS method for assaying 23 steroids within a single run from 150 µl of human plasma, serum or prostatic tissue homogenate. Isotope-labeled steroids were used as internal standards. Samples were extracted with toluene, and ketosteroids were derivatized with hydroxylamine prior to LC-MS/MS analysis. The steroids were separated on a C18 column and methanol was used as an organic solvent with the addition of 0.2 mM ammonium fluoride to improve underivatized estradiol (E2) ionization. Certified reference serums as well as plasma samples, and homogenates of prostate tissue were utilized in the method validation. The specificity of the method was inspected with a total of 27 steroids. The validation proved that the method was suitable for the quantitative analysis of a wide panel of androgens (testosterone, T (3.3 pM-13 nM); androstenedione, A4 (3.3 pM-13 nM); 5α-androstanedione, DHA4 (13 pM-13 nM); dehydroepiandrosterone, DHEA (67 pM-133 nM); dihydrotestosterone, DHT (33 pM-33 nM); 11-ketodihydrotestosterone, 11KDHT (13 pM-13nM); 11-ketotestosterone, 11KT (33 pM-6.7 nM); 11ß-hydroxyandrostenedione, 11bOHA4 (33 pM-13 nM); 11ß-hydroxytestosterone, 11OHT (13 pM-33 nM)), as well as estrogens (estrone, E1 (3.3 pM-13 nM)), progestagens (17α-hydroxypregnenolone, 17OHP5 (32 pM-127 nM); 17α-hydroxyprogesterone, 17OHP4 (67 pM-133 nM); progesterone, P4 (3.3 pM-13 nM); pregnenolone, P5 (6.6 pM-13 nM)), and glucocorticoids (cortisol, F (33 pM-134 nM); cortisone E (66 pM-131 nM); corticosterone, B (33 pM-67 nM); 11-deoxycortisol, S (33 pM-66 nM); 21-hydroxyprogesterone, 21OHP4 (32 pM-13 nM)). Furthermore, E2 (335 pM-134 nM) and 11α-hydroxyandrostenedione, 11aOHA4 (33 pM-33 nM) could be analyzed if the concentration in the sample was high enough. In addition, aldosterone, A (128 pM-64 nM) and 11-ketoandrostenedione, 11KA4 (33 pM-13 nM) could be analyzed semiquantitatively. The limits of quantification for all compounds ranged from 0.9 to 91 pg/ml, and from 0.009 to 0.9 pg/mg tissue. Compared to our previous method, this new method also permits the analysis of the more challenging steroids, like DHT, DHEA and P5, and a panel of 11-ketosteroids.

Changes in reproductive biomarkers in an endangered fish species (bonytail chub, Gila elegans) exposed to low levels of organic wastewater compounds in a controlled experiment.

In arid regions of the southwestern United States, municipal wastewater treatment plants commonly discharge treated effluent directly into streams that would otherwise be dry most of the year. A better understanding is needed of how effluent-dependent waters (EDWs) differ from more natural aquatic ecosystems and the ecological effect of low levels of environmentally persistent organic wastewater compounds (OWCs) with distance from the pollutant source. In a controlled experiment, we found 26 compounds common to municipal effluent in treatment raceways all at concentrations <1.0 microg/L. Male bonytail chub (Gila elegans) in tanks containing municipal effluent had significantly lower levels of 11-ketotestosterone (p=0.021) yet higher levels of 17beta-estradiol (p=0.002) and vitellogenin (p=0.036) compared to control male fish. Female bonytail chub in treatment tanks had significantly lower concentrations of 17beta-estradiol than control females (p=0.001). The normally inverse relationship between primary male and female sex hormones, expected in un-impaired fish, was greatly decreased in treatment (r=0.00) versus control (r=-0.66) female fish. We found a similar, but not as significant, trend between treatment (r=-0.45) and control (r=-0.82) male fish. Measures of fish condition showed no significant differences between male or female fish housed in effluent or clean water. Inter-sex condition did not occur and testicular and ovarian cells appeared normal for the respective developmental stage and we observed no morphological alteration in fish. The population-level impacts of these findings are uncertain. Studies examining the long-term, generational and behavioral effects to aquatic organisms chronically exposed to low levels of OWC mixtures are needed.

Analysis of succinylacetone, as a Girard T derivative, in urine and dried bloodspots by flow injection electrospray ionization tandem mass spectrometry.

Flow injection electrospray ionization tandem mass spectrometric methods for succinylacetone (SA) in 250 microL urine, using d5-SA as internal standard, and in 3 mm dried bloodspots, using 13C4-SA as internal standard, are described. Selectivity and sensitivity of analysis is achieved by the use of a mono-Girard T derivative. Measured SA infant urine normal range (n=20) is 0.013-0.27 micromol/mmol creatinine. Measured SA newborn bloodspot normal range (n=152) is 0-0.30 micromol/L. Bloodspots from children with hepatorenal tyrosinemia type 1, and kept at room temperature for up to 7 years, afforded SA concentrations of 0.9-5.7 micromol/L.

2-hydrazino-1-methylpyridine: a highly sensitive derivatization reagent for oxosteroids in liquid chromatography-electrospray ionization-mass spectrometry.

A derivatization reagent, 2-hydrazino-1-methylpyridine, was developed for the liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) of oxosteroids. The reagent quantitatively reacted with oxosteroids at 60 degrees C within 1h and the resulting derivatives of the mono-oxosteroids provided a 70-1600-fold higher sensitivity compared to intact steroids. However, HMP was unsuitable for di-oxosteroids, such as androstenedione and progesterone. The developed derivatization procedure was applied to the LC-ESI-MS analysis of 5alpha-dihydrotestosterone in human prostate, and allowed the reproducible quantification of nanogram/gram level of the androgen with a 10-mg sample.

Analysis of oxosteroids by nano-electrospray mass spectrometry of their oximes.

A method for the analysis of neutral oxosteroids by electrospray mass spectrometry is described. The oxosteroids are converted into their oximes by treatment with hydroxyammonium chloride in aqueous methanol. Intense peaks corresponding to protonated oxime molecules are observed in nano-electrospray mass spectra. The detection limits for the oximes of progesterone, pregnenolone and dehydroepiandrosterone were 2.5, 5 and 25 pg/microL, respectively, approximately 20 times lower than for the underivatised steroids. The signal intensities were proportional to the concentration of the steroids in the range of 500 to 2.5 pg/microL. Fragmentation by collision-induced dissociation (CID) was studied using oximes of 28 model steroids carrying an oxo group at C-3, C-17 or C-20. Some of the steroid oximes were labelled with deuterium or (15)N. Fragment ions were observed which yielded useful structural information. Upon CID, protonated oximes of 3-oxo-Delta(4)-steroids produced abundant ions by cleavage through the B-ring and by loss of the side chain, while protonated oximes of saturated 3-oxosteroids did not give abundant ions by cleavage through the B-ring. Protonated oximes of 20-oxosteroids unsubstituted at C-21, C-17 or C-16 produced a characteristic ion at m/z 86 containing the side chain, C-16 and C-17. Protonated oximes of steroids containing only a 17-oxo group gave fewer ions of diagnostic value. Coupled with the selective isolation of steroid oximes from a biological matrix this method of derivatisation and CID may be used for the analysis of neutral oxosteroids in biological samples.

Analysis of steroids Part 50. Derivatization of ketosteroids for their separation and determination by capillary electrophoresis.

4-Ene-3-ketosteroids and 17-ketosteroids were quantitatively transformed into the corresponding hydrazones using Girard P and T reagents, respectively. The positively charged derivatives were separated by capillary electrophoresis. The spectrophotometric characteristics of the derivatives permitted their sensitive detection in the 230-280 nm range. The steroids investigated included nortestosterone and its phenylpropionate, norethisterone and its oenanthate, d,l-norgestrel, dehydroepiandrosterone, androstenedione and ethisterone.

Applications of gas chromatography-mass spectrometry in the study of androgen and odorous 16-androstene metabolism by human axillary bacteria.

The known involvement of axillary microflora with under-arm odour (UAO) production led us to determine whether the odorous 16-androstene steroids are formed in the axilla by bacterial metabolism of an odourless precursor such as testosterone. Axillary bacteria from 34 men were selectively cultured for aerobic coryneform bacteria (ACB), Micrococcaceae and propionibacteria. Overnight suspensions of bacteria were incubated separately at 37 degrees C for two weeks with radiolabelled testosterone plus unlabelled testosterone (0.5 mg) and 0.5-mg quantities of 4,16-androstadien-3-one (androstadienone) and 5,16-androstadien-3 beta-ol (androstadienol). After extraction and purification by Sep-Pak cartridges and thin-layer chromatography, the eluted steroids were derivatised as the pentafluorobenzyl oximes (PFBO) and tert.-butyl dimethylsilyl (TBDMS) ethers. Saturated analogues were used as internal standards. Selected-ion monitoring electron-impact mass spectrometry was performed at the m/z corresponding to the M+.ion for the PFBO derivatives and the [M - 57]+ ion for the TBDMS ethers. Only ACB produced classical musk-like UAO (UAO + ve) in an in vitro odour-producing system with 29% being UAO -ve. ACB (UAO +ve) metabolised far more (p = 0.001) testosterone than ACB (UAO -ve), the principal metabolites being 5 alpha(beta)-dihydrotestosterone, 5 alpha(beta)-androstane-3,17-dione and 4-androstene-3,17-dione (4-androstenedione). No non-polar 16-androstenes were formed. Micrococcus luteus (ten strains) metabolised testosterone to 4-androstenedione only; propionibacterium spp. did not metabolise testosterone at all. However, incubation of 16-androstenes with ACB gave evidence for 4-ene-5 alpha(beta)-reduction, 3 alpha(beta)-oxido-reduction and epimerisation. In general the direction of transformations favoured formation of the more odorous 5 alpha-androst-16-en-3-one (5 alpha-androstenone) and 5 alpha-androst-16-en-3 alpha-ol (3 alpha-androstenol) from less odorous steroids. Such transformations, in vivo, would not require de novo synthesis of 5 alpha-androstenone or 3 alpha-androstenol and would be consistent with utilisation by ACB of 16-androstenes already present in small quantities in fresh apocrine secretions, which are odourless, to produce a more powerfully smelling mixture on the axillary skin surface.

Structure-gas chromatographic electron capture sensitivity relationships of some substituted 17alpha-acetoxyprogesterones.

Structure-electron capture sensitivity relationships were established for underivatized 17alpha-acetoxyprogesterones. While progesterone was very insensitive, 17alpha-acetoxyprogesterone had a response of 4.8 X 10(2) C/mole. Methyl groups in the A or B ring of 17alpha-acetoxyprogesterone had no effect. A keto group at C-6 was 25 times more sensitive (1.2 X 10(4) C/mole). A double bond at C-6,7 enhanced the sensitivity sevenfold (3.5 X 10(3) C/mole), but double bonds at C-1,2 or C-9,11 had only slight effect. Substitution at C-16 was important. A methyl group at C-16 had two and three times the sensitivity in the 3-keto delta4 and 3-keto delta4,6 series (1.1 X 10(3) and 1.1 X 10(4) C/mole), respectively. A methylene group at C-16, in contrast showed a six-and twofold greater sensitivity over the C-16 methyl in the two series (7 X 10(3) and 2.2 X 10(4) C/mole), respectively. The most sensitive compound was 6-dehydro-6methyl-16-methylene-17alpha-acetoxyprogesterone (melengestrol acetate). Its sensitivity was 2.2 X 10(4) C/MOL, Comparable to the most sensitive halo esters of steroid alcohols reported in the literature. Its electron capture coefficient was 3-7.6X 10(10) 1/mole. The coef-icient was independent of the detector temperature, indicating low activation energy for electron absorpiton.

Gas-liquid chromatographic studies of reactions and structural relationships of steroids. IV. Substitution in the pregnane side-chain.

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.

Studies on steroids. III. A new type of derivative for electron capture-gas chromatography of ketosteroids.

Pentafluorobenzyloxyamine is presented as a new derivatizing agent for gas chromatography of ketones using electron capture detection. The typical 17-ketosteroid was readily transformed into the O-pentafluorobenzyloxime which on usual trimethylsilylation led to a 3-trimethylsilyl ether derivative exhibiting good gas chromatographic properties. The derivatization procedure was applied to the determination of dehydroepiandrosterone in human plasma by electron capture-gas chromatography employing an internal standard method and in consequence satisfactory results were obtained.

Inhibition of the steroidogenic effects of cholera and heat-labile Escherichia coli enterotoxins by GM1 ganglioside: evidence for a similar receptor site for the two toxins.

The effects of three different ganglioside preparations on cholera enterotoxin (CT) and heat-labile Escherichia coli enterotoxin (ECT)-induced steroidogenesis in Y1 and OS3 adrenal tumor cells in tissue culture were examined. Only with GM1 ganglioside was any inhibition of the toxins' effects noted. Concentrations of the crude ECT preparation that gave similar morphogenic and steroidogenic effects as CT were inhibited by the same amount or less of GM1 as that required to inhibit the effects of CT. The results of competition experiments also demonstrated that previous incubation of GM1 with one toxin could inhibit the ganglioside's ability to inactivate the other toxin. These findings indicate that at least for Y1 and OS3 adrenal tumor cells, GM1 may resemble or be the receptor for both CT and ECT.

0-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride as a sensitive derivatizing agent for the electron capture gas liquid chromatographic analysis of keto steroids.

0-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride was used to prepare oximes of steroids with keto groups in selected positions; 3,17 and 20-monoketo; 3,17 and 3,20-diketo. Some of the 3-keto steroids had hindered 17-hydroxyl groups which were not readily amenable to esterification with perfluoroanhydrides, the most commonly used derivatizing agents for electron capture gas chromatographic analysis of hydroxy steroids. The oximes were readily prepared from 5 ng of each of the compounds tested, and with testosterone it was demonstrated that the derivative could be prepared from as little as 0.1 ng. The derivatives were stable to gas chromatography and extremely sensitive to electron capture detection. The sensitivity ranged from 1.5 X 10(4) coulombs per mole of progesterone. Because of the ease of preparation of the derivatives, their stability in common solvents and analytical manipulative techniques, the reagent would be suitable for the micro analysis of biologically significant keto steroids by electron capture gas chromatography.

Radio-enzymic assay of oxo steroids by their reduction with the tritiated form of reduced nicotinamide--adenine dinucleotide.

A method of analysis is proposed based on the enzyme-catalysed transfer of tritium from [3H]NADH to suitable substrates. Its practicability is demonstrated on the examples of oestrone and progesterone with the respective use of the 3 beta, 17 beta- and 3 alpha, 20 beta-hydroxysteroid oxidoreductase. Specificity is tested by application to the analysis of the plasma of pregnant women and measurement of the 3H/14C ratios on purification of the enzymic reduction products.