Gas Chromatographic-mass Spectrometric Technique Research Articles

Evaluation of chemical profiles and biological properties of Gliricidia sepium (Jacq.) Walp. volatile oils from Nigeria

Volatile oils obtained by hydrodistillation from Gliricidia sepium (Jacq.) Walp. leaf and stem were examined for their chemical composition and biological activity. The oils were analyzed by gas chromatographic-mass spectrometric (GC-MS) techniques. Toxicity, antimicrobial and antioxidant activities were evaluated via brine shrimp lethality assay, agar-diffusion, and DPPH radical-scavenging methods, respectively. A total of 43 and 44 constituents were identified in the leaf and stem oils, correspondingly. The major components recognized in the leaf oil were (E)-hexadecatrienal (16.9%) and pentadecanal (16.0%) while humulene epoxide II (17.5%) and caryophyllene oxide (10.6%) dominated the stem oil. There was no significant activity against the bacteria but moderate inhibition zones (IZ) between 1.8±0.3 and 8.1±0.1 mm were observed against the fungi. The stem oil showed better antifungal activity than the leaf oil but not as active as the standard drug ketoconazole which inhibited the test fungi with IZ range of 10.4±0.4-21.0±1.4 mm at 200 μg. Both oils were toxic to brine shrimp (Artemia salina) giving LC50 of 79.7 μg/mL (leaf) and 38.7 μg/mL (stem). The leaf and stem oils had IC50 of 84.3 μg/mL and 142.2μg/mL, respectively, in the DPPH radical-scavenging assay, indicating moderate antioxidant activity relative to positive controls, butylated hydroxylanisole (IC50=45.1 μg/mL) and α-tocopherol (IC50 =81.6 μg/mL). The results suggest that G. sepium volatile oil may find potential use as a natural antioxidant and antifungal agent.

Assessment of Metabolites Content of Native Alcoholic Beverages Obtained from Jos, Nigeria

Burukutu, (BKT) Pito (PT) and Goskolo (GSK) are drinks consumed routinely within the area of study. Although consumption of GSK is illegal owing to its life threatening toxicity, people still consume it secretly. Whereas BKT and PT are locally prepared using unscientific methods in preparing the drinks, GSK is a mixture containing different compounds and chemicals. BKT and PT are more commonly ingested than GSK. Because methods applied during ‘brewing’ BKT and PT are not scientific, toxic metabolites might be generated. So, the drinkers are prone to their toxic effects. This work ascertained the presence and levels of unwanted metabolites in these drinks using Gas chromatographic-Mass spectrometric (GC-MS) technique. Metabolites could aggravate the toxicity of alcohol and alcohol-related disease conditions. Results, (% relative abundance), showed the significant presence in BKTof6-Octadecanoic acid (38.51), Hexadecanoic acid (20.92), Isopentyl alcohol (12.27), 1,11,13-Octadecatriene (11.60) among others. In PT,9-Octadecanoic acid (39.40), Hexadecanoic acid (15.98), Carbonic acid methylpentyl ester (14.22), 9,17-Octadecadienal (6.37) were the major components. In GSK, 6-Octadecenoic acid (42.43), n-Hexadecanoic (17.51), 1-Butanol (16.52) 9 and 9,12-Octadecadienoic acid (10.72) were the dominant components. BKT, PT, and GSK are composed of compounds/metabolites that are organic in nature, with about 83% of them being organic acids. This leads to a decrease in pH thereby making the drinks acidic. It is concluded that the alcoholic beverages contain metabolites that predispose drinkers to metabolic or respiratory acidosis, due to drop in pH. Furthermore, these metabolites could aggravate the toxicity of alcohol. Other components of the samples include ketones, different types of alcohols, aldehydes, derivatives of benzene and esters.

Gas Chromatographic Study of Bio-active Compounds in Methanolic Extract of Leaf of Crateva adansonii DC

This study investigates the volatile bioactive components in Crateva adansonii plant that has been applied in the treatment of diverse ailments in the Western part of Nigeria and in Africa. The methanolic leaf extract of Crateva adansonii was subjected to analysis using gas chromatographic-mass spectrometric technique. The mass spectra of the compounds were analysed and identity of the compounds confirmed using the data base of the National Institute of Standard and Technology (NIST) library. The compounds identified includesn-Hexadecanoic acid; 9,12-Octadecadienoic acid (Z,Z); 9-Octadecenoic acid; (Z,E)-2,13-Octadecadien-1-ol; 9,17-Octadecadienal; cis-9-Hexadecenal; cis-Vaccenic acid; Z,Z-10,12-Hexadecadienal; 13-Octadecenal; Hexadecanoic acid 2-hydroxy-1-(hydroxymethyl)ethyl ester; E-11-Hexadecenal. Most of the compounds identified have antibacterial, anti-oxidant, anti-inflammatory and hypocholesterolemic properties, which affirms the therapeutic applications of Crateva adansonii as a medicinal plant.

The organic materials in the Five Northern Provinces’ Assembly Hall: disclosing the painting technique of the Qing dynasty painters in civil buildings

The beiwusheng huiguan (‘Meeting hall of the Five Northern Dynasties’) is a building complex from the Qing dynasty (1636–1912 ad) located in Wafangdian, near Ziyang, in the south of the Chinese Province of Shaanxi. Two of the preserved halls are richly decorated with wall paintings dated probably in 1848 ad and representing scenes of the ‘Romance of the Three Kingdoms’ and Confucian moral tales. They are a rare example of well-preserved mural paintings of high artistic value inside civil buildings. The aims of this paper are the chemical characterization and localization of organic materials used as binders and colorants in the wall paintings. A multi-analytical approach, consisting in the combined use of gas chromatographic–mass spectrometric techniques (GC/MS and Py-GC/MS) and high-pressure liquid chromatography with diode array detector (HPLC–DAD), was chosen for these purposes. Proteinaceous materials (animal glue and egg), saccharide material (fruit tree gum) and a siccative oil were identified in different paint layers supplying invaluable information about the painting technique used. Moreover, the analyses of organic dyes allowed identifying indigo and gallic acid in more than one sample adding fundamental information about Chinese artists’ techniques in mural paintings, missing from the previous studies. To shed light on the gilding technique, the distribution of the painting materials was achieved by means of synchrotron radiation Fourier transform infrared spectroscopy (SR micro-FTIR) and X-ray fluorescence (SR micro-XRF). The results obtained from the multi-analytical approach enabled us to determine the organic materials both binders and organic colorants used by Chinese artisans, highlighting the high technical level achieved in nineteenth century. The binding media and the organic colorants identified, as well as their distribution, allowed the discussion on the painting technique used by the artists of the Qing dynasty giving information for the first time about the decoration of Chinese civil buildings.

Simultaneous quantitation of parabens, triclosan, and methyl triclosan in indoor house dust using solid phase extraction and gas chromatography-mass spectrometry

An integrated analytical method for the simultaneous determination of five parabens (methyl-, ethyl-, propyl-, butyl-, and benzyl-), triclosan, and methyl triclosan in indoor house dust was developed based on gas chromatographic-mass spectrometric technique (GC/MS). Analytes were extracted from dust samples by sonication. After sample cleanup by solid-phase extraction (SPE), the extracts were derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and then analyzed by gas chromatography coupled with ion trap mass spectrometry operated in multiple reaction monitoring (MRM) mode. For quantitation, isotope-labelled internal standards were used for each corresponding target analyte. Only 0.05 g of dust sample was needed for the analysis. Method detection limits ranged from 6.5 to 10 ng/g, and absolute recoveries from 74% to 92%. The developed method demonstrated good repeatability and reproducibility, with relative standard deviations (RSDs) less than 16% for all the analytes. The analytes were determined in dust samples collected using two vacuum sampling methods from 63 Canadian homes: a sample of fresh or "active" dust (FD) collected using a Pullman-Holt vacuum sampler, and a composite sample taken from the household vacuum cleaner (HD). Methyl paraben, propyl paraben, and triclosan were detected in all HD and FD samples. HD samples yielded median values for methyl paraben, propyl paraben, and triclosan of 1080, 463, and 378 ng/g, respectively, which were comparable to the FD sample medians of 1120, 618 and 571 ng/g. Ethyl paraben was detected at frequencies of 89% in FD and 73% in HD samples, with median values of 52 and 25 ng/g, respectively. Butyl paraben was detected at frequencies of 44% in FD and 75% in HD samples, with median values of <10 and 59 ng/g, respectively. Benzyl paraben and methyl triclosan were not detected in any of the samples collected by either method. Samples collected according to the fresh dust protocol agreed with the household vacuum samples 90% of the time. Widely scattered concentration levels were observed for target analytes from this preliminary set of 63 Canadian samples, which suggests a wide variability in Canadian household exposures to these chemicals.

Determination of nicotine in hair samples of pre-Columbian mummies

In the literature, there are reports about the presence of cocaine, nicotine and cannabinoids in the hair of ancient mummies from South America or Egypt. Most of the results have been criticised because of the use of improper analytical techniques or contamination of the sample material. Recently an exhibition of 70 mummies from around the globe was organized at the Reiss-Engelhorn-Museen in Mannheim, Germany. It shows exhibits assembled by one of the worldfamous mummy projects, involving various specialists for anthropology, pathology, radiology, molecular biology and toxicology. Within this project, hair samples of eight pre-Columbian mummies were analyzed for drugs using modern routine gas chromatographic-mass spectrometric techniques. The tests revealed negative results, except for nicotine in the hair of three mummies. Nicotine was measured in concentrations of 57.5 ng/mg in the hair of a woman, 14.1 ng/mg in the hair of a child and 11.4 ng/mg in the hair of a further female mummy, but all cases revealed negative results for cotinine. The target analysis was performed with limits of detection of 0.04 ng/mg for nicotine and 0.033 ng/mg for cotinine. The washing solutions yielded negative results for both analytes, nicotine as well as cotinine. In our opinion, even with respect to negative results in the washing solutions, the present results cannot definitely confirm an active consumption with body passage in the life time of the analyzed mummies: An external contamination cannot be excluded, e.g. by transfer from smoking visitors or employees during the early collection history of the objects in the 19th century, as well as in their respective lifetime.

Metabolism and toxicological detection of the designer drug 4-chloro-2,5-dimethoxyamphetamine in rat urine using gas chromatography-mass spectrometry

Studies are described on the metabolism and the toxicological analysis of the amphetamine-derived designer drug 4-chloro-2,5-dimethoxyamphetamine (DOC) in rat urine using gas chromatographic-mass spectrometric techniques. The metabolites identified indicated that DOC was metabolized by O-demethylation at position 2 or 5 of the phenyl ring partly followed by glucuronidation and/or sulfation. The authors' systematic toxicological analysis procedure using full-scan gas chromatography-mass spectrometry after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of an intake of a dose of DOC in rat urine that corresponds to a common drug user's dose. Assuming similar metabolism, the STA procedure described should be suitable as proof of an intake of DOC in human urine.

Isolation and Recovery of Selected Polybrominated Diphenyl Ethers from Human Serum and Sheep Serum: Coupling Reversed-Phase Solid-Phase Disk Extraction and Liquid-Liquid Extraction Techniques with a Capillary Gas Chromatographic Electron Capture Negative Ion Mass Spectrometric Determinative Technique

Polybrominated diphenyl ethers (PBDEs) are isolated and recovered with acceptable percent recoveries from human serum via liquid-liquid extraction and column chromatographic cleanup and fractionation with quantitation using capillary gas chromatography-mass spectrometry with electron capture negative ion and selected ion monitoring. PBDEs are found in unspiked serum. An alternative sample preparation approach is developed using sheep serum that utilizes a formic acid pre-treatment followed by reversed-phase solid-phase disk extraction and normal-phase solid-phase cleanup using acidified silica gel that yields>50% recoveries. When these percent recoveries are combined with a minimized phase ratio for human serum and very low instrument detection limits, method detection limits below 500 parts-per-trillion are realized.

Metabolism and toxicological detection of a new designer drug, N-(1-phenylcyclohexyl)propanamine, in rat urine using gas chromatography–mass spectrometry

Studies are described on the metabolism and the toxicological detection of the phencyclidine-derived designer drug N-(1-phenylcyclohexyl)-propanamine (PCPR) in rat urine using gas chromatographic–mass spectrometric techniques. The identified metabolites indicated that PCPR was metabolized by hydroxylation of the cyclohexyl ring at different positions, hydroxylation of the phenyl ring, N-dealkylation, and combinations of these steps. Parts of the metabolites were excreted in conjugated form. The authors’ systematic toxicological analysis (STA) procedure using full-scan GC–MS after acid hydrolysis, liquid–liquid extraction and microwave-assisted acetylation allowed the detection of an intake of a common drug users’ dose of PCPR in rat urine. Assuming similar metabolism in humans, the STA should be suitable for proof of an intake of PCPR in human urine.

Studies on the metabolism and toxicological detection of the designer drug 4-ethyl-2,5-dimethoxy-β-phenethylamine (2C-E) in rat urine using gas chromatographic–mass spectrometric techniques☆

The phenethylamine-derived designer drug 4-ethyl-2,5-dimethoxy-beta-phenethylamine (2C-E) was found to be mainly metabolized in rats by O-demethylation, N-acetylation, hydroxylation of the ethyl side chain at C2' or at C1' followed by oxidation at C1' to the corresponding ketone, by deamination followed by reduction to the corresponding alcohols or by oxidation to the corresponding acids, and finally combinations of these steps. Most of the metabolites were excreted in conjugated form. The authors' systematic toxicological analysis (STA) procedure using full-scan GC-MS allowed the detection of an intake of a dose of 2C-E in rat urine that corresponds to a common drug users' dose. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-E in human urine.

Identification of Brominated Carbazoles in Sediment Cores from Lake Michigan

During the analysis of brominated flame retardants in sediment cores taken from Lake Michigan, a group of unknown brominated compounds were observed. To identify their chemical structures, a variety of gas chromatographic mass spectrometric techniques were used. With the help of high-resolution and positive chemical ionization mass spectrometry, the molecular formula of the most abundant congener was established to be C12H5Br4N. On the basis of the electron impact and electron capture negative ionization mass spectra, this chemical was suspected of being a tetrabromocarbazole. The structure was confirmed by comparing the mass spectra and gas chromatographic retention times of an authentic standard of 1,3,6,8-tetrabromocarbazole with those of the most abundant unknown. The other unknown compounds may be other bromocarbazole congeners. The time trend determined by measuring these compounds as a function of depth in the Lake Michigan sediment cores indicated that the concentration of 1,3,6,8-tetrabromocarbazole peaked around 1920-1935. This is the first time that bromocarbazoles have been found in the environment.

Mammalian exocrine secretions. XVII: chemical characterization of preorbital secretion of male suni, Neotragus moschatus.

Gas chromatographic and gas chromatographic-mass spectrometric techniques were employed to identify 83 compounds, including alkanes, alkenes, aldehydes, 2-methylalkanes, carboxylic acids, 1-alkyl formates and alken-1-yl formates, benzoic acid, and cholesterol, in the preorbital secretion of the male suni, Neotragus moschatus. Dimethyl disulfide derivatization and lithium aluminum hydride reduction were used to determine the position of double bonds and to confirm the identity of the functional groups in some of the constituents of the secretion.

Determination of Cytochrome P450 Metabolites of Arachidonic Acid in Coronary Venous Plasma during Ischemia and Reperfusion in Dogs

Arachidonic acid (AA) can be metabolized by cytochrome P450 enzymes to many biologically active compounds including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrienoic acids (DHETs), as well as 19- and 20-hydroxyeicosatetraenoic acids (HETEs). These eicosanoids are potent regulators of vascular tone. However, their role in the ischemic myocardium has not been well investigated. In this study, we used a gas chromatographic–mass spectrometric technique to analyze total EETs, DHETs, and 20-HETE released into coronary venous plasma during coronary artery occlusion and reperfusion in anesthetized dogs. Pentafluorobenzyl esters (PFB-esters) of EETs and PFB-esters/trimethylsilyl ethers (TMS-ethers) of DHETs and 20-HETE were detected in the negative ion chemical ionization (NICI) using methane as a reagent gas. Under the conditions used, all four regioisomers of EET eluted from the capillary gas chromatographic column at similar retention times while four regioisomers of DHETs and 20-HETE eluted separately. The detection limits in plasma samples are 5 pg for total EETs, 40 pg for DHET, and 15 pg for 20-HETE. 14,15-DHET is the major regioisomer detected in the plasma samples while other regioisomers of DHETs are probably present at too low a concentration for detection. During the first 5 to 15 min of coronary occlusion, a slight decrease in the concentration of EETs, 14,15-DHET, and 20-HETE from the control values was observed in coronary venous plasma. At 60 min of occlusion, their concentrations significantly increased and remained elevated during 5 to 60 min of reperfusion. The concentrations decreased at 120 min of reperfusion. The NICI GC–MS was successfully used as a sensitive technique to determine cP450 metabolites of AA in plasma during prolonged occlusion–reperfusion periods. Furthermore, the results indicate that these metabolites may play a role in mediating ischemic–reperfusion injury.

Comparison of isotopic dilution methods for determination of theophylline in the plasma and saliva of infants and children

Isotope-dilution mass spectrometry (IDMS) using the direct inlet probe and an isotope dilution gas chromatographic-mass spectrometric (GC-IDMS) technique are presented for determination of theophylline in paired specimens of plasma and saliva from infants and children treated with theophylline for idiopathic apnea of prematurity, or for asthma.15N-theophylline was used as internal standard. A non-invasive means of individualization of the dosage was achieved by analysis of levels of the drug in saliva secreted in response to stimulation with citric acid.

Simple and effective gas chromatographic mass spectrometric procedure for the speciation analysis of organotin compounds in specimens of marine mussels. An evaluation of the organotin pollution of the lagoon of venice.

This paper describes a simple, effective analytical procedure, based on a gas chromatographic mass spectrometric technique, for the speciation analysis of organotin compounds (OTC) in mussel samples. The direct alkylation reaction of the organotin chlorides in the aqueous digestion solution by NaBEt(4) allowed a short analysis time and a good recovery. The evaluation of the yield of each step constituting the analytical procedure indicated that the alkylation step is the most critical one. The proposed method was advantageously utilised to monitor the level of OTC pollution of the Lagoon of Venice. All the sites examined, both near to and far from anthropogenic activities, revealed significant levels of OTC pollution.

Identification of benzo[a]pyrene metabolites in cervical mucus and DNA adducts in cervical tissues in humans by gas chromatography-mass spectrometry

Epidemiological studies indicate that cigarette smoking increases the risk of cervical cancer. To address questions regarding possible mechanisms of tobacco-related cervical carcinogenesis, in a pilot study, using supercritical fluid extraction and a gas chromatographic-mass spectrometric (GC-MS) technique, we detected and characterized benzo[a]pyrene and its metabolites, namely B[a]P-dihydrodiols, phenols and tetraols in cervical mucus samples from eight smokers and non-smokers. Twenty-eight epithelial and stromal cervical tissue samples from seventeen patients undergoing surgery for non-malignant disease were quantitatively analyzed for BPDE–DNA adducts by a GC-MS technique. BPDE–DNA adducts were found in 25 samples. The mean level of BPDE–DNA adducts in epithelial cervical tissues of smokers was nearly two-fold greater than that in self-reported non-smokers; P=0.02. The mean number of BPDE-adducts (±SD) in epithelial cervical tissues of smokers was 3.5±1.06 adducts/108 nucleotides while that in non-smokers was 1.8±0.96 adducts/108 nucleotides. The mean number of BPDE-DNA adducts in stromal cervical tissues of the same subjects was 1.8±0.96 adducts/108 nucleotides in smokers and that in the stromal tissues of non-smokers was 1.4±1.1 adducts/108. These results suggest that polynuclear aromatic hydrocarbons (PAHs) from tobacco smoke and other environmental sources can be transported to the cervix where they are metabolized in the cervical epithelium to ultimate carcinogenic agents, although transport of ultimate carcinogenic metabolites from other organs to the cervix cannot be ruled out. Exposure of cervical epithelia to PAHs and their carcinogenic metabolites suggests a potential role of such carcinogens in the pathogenesis of cervical cancer in humans.

Investigations of S-Transnitrosylation Reactions between Low- and High-Molecular-WeightS-Nitroso Compounds and Their Thiols by High-Performance Liquid Chromatography and Gas Chromatography–Mass Spectrometry

S-Transnitrosylation reactions are supposed to be the basic principle by which nitric oxide-related biological activities are regulatedin vivo.Mechanisms of S-transnitrosylation reactions are poorly understood and equilibria constants for physiologicalS-nitroso compounds and thiols are rare. In the present study we investigated S-transnitrosylation reactions of the thiols homocysteine, cysteine, glutathione,N-acetylcysteine,N-acetylpenicillamine, and human plasma albumin and their correspondingS-nitroso compounds SNhC, SNC, GSNO, SNAC, SNAP, and SNALB utilizing high-performance liquid chromatographic and gas chromatographic–mass spectrometric techniques. These methods allowed to study S-transnitrosylation reactions in mixtures of severalS-nitroso compound/thiol pairs, to determine equilibria constants, and to elucidate the mechanism of S-transnitrosylation reactions. We obtained the following order for the equilibria constants in aqueous buffered solution at pH 7.4: SNhC ≈ SNAC > GSNO ≈ SNALB > SNAP > SNC. Our results suggest that the mechanism of S-transnitrosylation reactions of theseS-nitroso compounds and their thiols involve heterolytic cleavage of the SN bond. Incubation of SNC with human red blood cells resulted in a dose-dependent formation of GSNO in the cytosol through S-transnitrosylation of intracellular GSH by the SNC transported into the cells. This reaction was accompanied with an almost complete disappearance of the SNC fraction transported into the cells. This finding is in full agreement with the equilibrium constantKeqof 1.9 for the reaction SNC + GSH ↔ Cys + GSNO in aqueous buffer.

Genotoxic exposures of potroom workers.

Potroom workers in aluminum reduction plants have increased risks for bladder and lung cancer due to exposure from polycyclic aromatic hydrocarbons (PAH). In this study correlations between measures of the external, internal, and biological effective dose have been studied for PAH. Venous blood samples were obtained from 98 male potroom workers and 55 unexposed male blue-collar workers, for the analysis of aromatic adducts to DNA (deoxyribonucleic acid) in lymphocytes, using the 32P-postlabeling technique. 1-Hydroxypyrene in urine was analyzed with high-pressure liquid chromatography. Personal sampling of both particulate and gas phase PAH was performed during a full workday for the potroom workers and for 5 referents. Individual PAH congeners were determined with liquid chromatographic-mass spectrometric and gas chromatographic-mass spectrometric techniques. The respiratory-zone airborne level of the sum of 22 particulate (median 13.2 micro/m3) and the 7 gas phase PAH-congeners (median 16.3 microg/m3) among the potroom workers was a hundred times higher than among the referents. The urinary concentration of 1-hydroxypyrene before work was 30 times higher for the potroom workers (median 3.43 micromol/mol creatinine) than for the referents. Most airborne PAH congeners correlated with the excretion of 1-hydroxypyrene in urine. The frequency of aromatic DNA adducts did not, however, differ between the potroom workers and the referents, and no correlation was found for 1-hydroxypyrene in urine. Despite an obvious occupational exposure to PAH, no increase in aromatic DNA adducts in lymphocytes was found among the potroom workers.

3-Phenylpropanylcinnamate, a copolymer unit in Siegburgite fossil resin: a proposed marker for the Hammamelidaceae

Siegburgite, an unusual Tertiary aromatic resin found near Bitterfeld (Germany), has been characterised earlier by FTIR spectrometry as polystyrene-like material. Here the molecular weight distribution of the soluble part is determined by size-exclusion chromatography (SEC) to be bomodal with maxima at 400 and 30,000 Da. Using combined gas chromatographic-mass spectrometric techniques (GC–MS) traces of oleanonic and 3-epi-oleanolic acid are found in the low MW fraction of Siegburgite. 3-Phenylpropanylcinnamate is detected in the high MW fraction by pyrolysis (Py)–GC–MS. Although also present in the soluble part of the high MW fraction, this compound is present especially in the insoluble residue, indicating that the ester is incorporated in the polymer chain. Oleanonic acid, 3-epi-oleanolic acid and 3-phenylpropanylcinnamate are constituents of commercially available Gum Storax from modern Liquidambar orientalis Mills. The proposed paleobotanical origin of Siegburgite from the Hammamelidacae family is thus chemically confirmed. In addition, a comparable polystyrene-like resin originating from North America (Squankum New Jersey) was compared to Siegburgite. The polystyrene matrix of this resin is comparable to that of Siegburgite, but no molecular markers confirming its origin could be found.

Use of gas chromatographic-mass spectrometric techniques in studies of androst-16-ene and androgen biosynthesis in human testis; cytosolic specific binding of 5α-androst-16-en-3-one

Homogenates of histologically normal human testis from three men were incubated separately with pregnenolone, 16-dehydropregnenolone, 5alpha-pregnane-3,20-dione, 3beta-hydroxy-5alpha-pregnan-20-one and androsta-5,16-dien-3beta-ol (androstadienol) in the presence of NADPH in a study of androst-16-ene and androgen biosynthesis. After the addition of internal standards and initial extraction and purification, metabolites were identified using gas chromatography-mass spectrometry (GC-MS) and monitoring selectively for three principal ions in each case at the appropriate GC retention time. Quantification was achieved by comparison with calibration lines for authentic steroids, together with the appropriate internal standards, prepared by monitoring three ion fragments for each analyte. In all experiments, androstadienol was found to be the major androst-16-ene metabolite of pregnenolone (seven times the control, i.e. endogenous, quantity; 19.8 +/- 3 ng/100 mg homogenate protein, mean +/- SEM, n = 9). Pregnenolone was also converted to androsta-4,16-dien-3-one (androstadienone) with three times the endogenous quantity (44 +/- 10 ng/100 mg homogenate protein, mean +/- SEM, n = 9) being formed. The formation of testosterone occurred only in trace amounts in the incubations of testis taken from one man (a 69-yr-old) but appreciable yields (six times endogenous levels 90 +/- 7 ng/100 mg homogenate protein, mean +/- SEM, n = 9) were found with testes from two younger men. Only traces of 5alpha-dihydrotestosterone were detected. Using androstadienol as the substrate, androstadienone was shown to be the major metabolite (approximately 10 times greater than control incubations) together with 5alpha-androst-16-en-3alpha- and 3beta-ols at approximately twice the endogenous quantities (5 ng/100 mg homogenate protein). In some incubations with androstadienol, 5alpha-androst-16-en-3-one (5alpha-androstenone) was formed (32 +/- 1 ng/100 mg homogenate protein/h; mean +/- SEM, n = 3); surprisingly, no endogenous 5alpha-androstenone could be detected. No evidence was obtained for the production of testosterone or 5alpha-DHT from androstadienol. Using cytosolic fractions of human testis, specific (displaceable) binding of 5alpha-androstenone was determined, with binding sites of approximately 200 fmol/mg tissue and a Ka of approximately 8 nmol/l.