Some aspects of quantification of neutral steroid metabolites in body fluids of healthy and uremic subjects by capillary gas chromatography

Neutral steroid metabolites enriched from urine and hemofiltrate were identified by gas chromatography/mass spectrometry and quantified by capillary gas chromatography. This study included 20 healthy controls and 37 uremic patients. Before enrichment of steroids from biological material, the standard deviation of the workup procedure and subsequent derivatization into the trimethylsilyl-enol-trimethylsilyl ethers was tested and found to be 2–5% in urine and 12–17% in the more complicated workup procedure of hemofiltrate, but essentially smaller than the biological standard deviation. Compared to the 24 h urinary excretion rates of controls, the excretion rates of androsterone, etiocholanolone, and corticoid metabolites were significantly lower in uremic body fluids, while those of 11-oxygenated androstanolones, degradation products of corticoids, were enhanced in uremic urine. The ratio of corticoid metabolites to 11-oxygenated androstanolones in urine of nondialyzed uremics correlated significantly with their plasma creatinine levels.     

Quantitative analysis of DHEA and androsterone in female urine: investigating the effects of menstrual cycle, oral contraception and training on exercise-induced changes in young women

Dehydroepiandrosterone (DHEA) and its metabolite androsterone (A) are natural steroids secreted in high quantities in human body. To assess the influence of oral contraceptives, menstrual cycle phase, and also physical exercise (acute and chronic such as training) on these metabolites excretions, a collection of 28 female urine specimens was organized. A three-extraction-step method was developed, and the analyses were performed by gas chromatography–mass spectrometry using deuterated 19-noretiocholanolone as the internal standard. Sample hydration state was found to be of great importance for kinetic studies, as it directly influenced the concentrations. No influence of menstrual cycle and training was found for androsterone and DHEA. However, oral contraceptive intake lowered DHEA excretion in urine and A seems to be slightly affected by exercise.     

Determination of the origin of urinary norandrosterone traces by gas chromatography combustion isotope ratio mass spectrometry

On the one hand, 19-norandrosterone (NA) is the most abundant metabolite of the synthetic anabolic steroid 19-nortestosterone and related prohormones. On the other hand, small amounts are biosynthesized by pregnant women and further evidence exists for physiological origin of this compound. The World Anti-Doping Agency (WADA) formerly introduced threshold concentrations of 2 or 5 ng of NA per ml of urine to discriminate 19-nortestosterone abuse from biosynthetic origin. Recent findings showed however, that formation of NA resulting in concentrations in the range of the threshold levels might be due to demethylation of androsterone in urine, and the WADA 2006 Prohibited List has defined NA as endogenous steroid. To elucidate the endogenous or exogenous origin of NA, (13)C/(12)C-analysis is the method of choice since synthetic 19-nortestosterone is derived from C(3)-plants by partial synthesis and shows delta(13)C(VPDB)-values of around -28 per thousand. Endogenous steroids are less depleted in (13)C due to a dietary mixture of C(3)- and C(4)-plants. An extensive cleanup based on two high performance liquid chromatography cleanup steps was applied to quality control and doping control samples, which contained NA in concentrations down to 2 ng per ml of urine. (13)C/(12)C-ratios of NA, androsterone and etiocholanolone were measured by gas chromatography/combustion/isotope ratio mass spectrometry. By comparing delta(13)C(VPDB)-values of androsterone as endogenous reference compound with NA, the origin of NA in doping control samples was determined as either endogenous or exogenous.     

Application of a Matrix of Piezosorption Sensors to Analysis of Ethanol-Containing Gas Mixtures

General relationships were established for operation of piezosorption sensors when exposed to an ethanol-based multicomponent gas mixture. The operation conditions were optimized for the sensors; a matrix comprising six piezosorption elements was designed for analyzing gas mixtures containing low-boiling C₁-C₅ alcohols and ethyl acetate. The sensor matrix is suitable for foodstuff analysis.     

Anomalous Reaction of Ethyl Bromofluoro- and Difluoro-Acetates with Dialkylphosphonites

The Michaelis–Arbuzov reaction of ethyl α -bromo-α -fluoro- and α, α -difluoro-acetates with dialkyl phenylphosphonites leads to the formation of unusual products including a fluorophosphinate. The reaction of ethyl bromofluoracetate with diisopropyl- and dimethyl-phenylphosphonites furnishes a complex mixture of eight and five compounds respectively. Five different compounds are obtained when ethyl bromodifluoroacetate is reacted with diisopropyl phenylphosphonite. Dimethyl phenylphosphonite yields three compounds when heated with bromochloromethane. The probable mechanism of formation of the compounds and their mass spectral characterization using GC-MS, tandem MS-MS and DART? techniques are presented in this paper.     

Unique potentiometric detection systems for HPLC determination of some steroids in human urine

Isocratic HPLC with potentiometric detection is used for the determination of some 17‐ketosteroids (17‐KS), e.g., androsterone, dehydroepiandrosterone and estrone, and their respective sulfated conjugates (17‐KSS). Glassy carbon or composite electrodes containing a mixture of graphite and poly(vinyl chloride), PVC, were used as substrate electrodes. These substrates were covered either by montmorillonite or potassium tetrakis(p‐chlorophenyl) borate containing PVC‐based rubber phase membranes. The neutral 17‐KS compounds were derivatized with Girard's reagent P (GP) to obtain cationic pyridinium acetohydrazones prior to the HPLC/potentiometric detection assay. No side reactions were observed, and the GP itself was not interfering. The method yielded accurate and reproducible results and was applicable to samples containing down to micromolar concentrations. Next, the 17‐KSS compounds, acting as anionic charged molecules, were determined directly in human urine samples with the HPLC/potentiometry combination without preliminary derivatization. For this purpose, a new anion‐sensitive potentiometric electrode was developed using a macrocyclic polyamine containing, PVC‐based, rubber phase membrane. The three 17‐KSS compounds were also determined accurately down to micromolar concentrations. Especially, the main androgen metabolites as dehydroepiandrosterone sulfate and androsterone sulfate could be selectively determined with a developed potentiometric sensor in human urine samples without time‐consuming cleanup and preconcentration step.     

Solid-Phase Extraction Techniques for Assay of Diuretics in Human Urine Samples

Abstract

Solid-phase extraction techniques were evaluated for the treatment of urine samples in the analysis of diuretics before injection into an HP-Hypersyl ODS-C18 column. Six different reversed-phase extraction columns were tested, and the results obtained are compared with those obtained in a classical liquid-liquid extraction with ethyl acetate.

The solid-phase extraction procedures are the best overall choice for all the diuretics tested, due to their versatility, the minor time-consuming, and the good recovery percentages obtained. C18 and C8 packings give the highest recoveries for a majority of the diuretics studied. However, CH or PH columns, due to their greater selectivity, can be used if the elution of the matrix is not complete in the washing solution. This could be more suitable.     

Analysis of exemestane and 17β-hydroxyexemestane in human urine by gas chromatography/mass spectrometry: development and validation of a method using MO-TMS derivatives

Trimethylsilylation of anabolic agents and their metabolites is frequently achieved by using the derivatization mixture N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)/NH(4)I/2-mercaptoethanol. Nevertheless, artifacts were formed when this mixture was employed in the monitoring of exemestane and its main metabolite 17β-hydroxyexemestane prior to GC-MS analysis. These artifacts were identified as the N-methyltrifluoroacetamide (MTFA) and trimethylsiloxyethylmercapto products of the respective trimethylsilyl (TMS) derivatives. Furthermore, artifact formation was evaluated taking the structure (1,4-diene-3-keto-6-exomethylene) of the compounds into account. Although these artifacts are relevant for investigations regarding the derivatization process and may be of interest in many fields, they are detrimental to cope with the requirements of the World Anti-Doping Agency (WADA) in terms of the limits of detection (LODs) required. To overcome this issue, a method using an alternative derivatization was proposed: formation of methyloxime-TMS derivatives through double derivatization using O-methylhydroxylamine/pyridine and MSTFA/TMS imidazole after enzymatic hydrolysis and liquid-liquid extraction. Samples from an excretion study after administration of exemestane to healthy volunteers were analyzed by the proposed method and detection of both exemestane and its main metabolite was possible. This method showed excellent results for both analytes meeting the LODs required for antiestrogenic agents (50 ng/mL) established by WADA. The method was validated for the main metabolite, it was robust and cost-effective for qualitative and quantitative purposes, with LOD and LOQ of 10 ng/mL and 25 ng/mL, respectively.     

Direct quantification of steroid glucuronides in human urine by liquid chromatography-electrospray tandem mass spectrometry

A method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the direct quantification of glucuronides of testosterone (TG), epitestosterone (EPG), androsterone (AG) and etiocholanolone (ETG) has been developed. The method allowed for the direct determination of these analytes avoiding hydrolysis and derivatization, which are usual steps in commonly used methods based on gas chromatography-mass spectrometry (GC-MS). The electrospray ionization and the product ion spectra of the glucuronides have been studied in order to obtain the most specific transitions. The use of the selected transitions is necessary for the determination of the analytes at low ng/ml concentration levels. Two different approaches have been tested for sample preparation: direct injection after filtration and acidic liquid-liquid extraction (LLE) with ethyl acetate. Both approaches have been validated obtaining satisfactory values for accuracy and precision with limits of detection lower than 1 ng/ml for TG and EPG. Ion suppression was more pronounced after LLE probably due to the concentration of interferences from acidic urine. The applicability of the method has been checked by the analysis of 40 urine samples. The results were compared with those obtained with the common GC-MS method. Results have shown a good correlation between both methods with correlation coefficients higher than 0.97. A slope close to 1 was obtained for all analytes except for AG possibly due to losses during the extraction process prior to GC-MS.     

Investigation of matrix effects in inductively coupled plasma-atomic emission spectroscopy using laser ablation and solution nebulization — effect of second ionization potential

Plasma-related non-spectroscopic matrix effects of 31 elements in inductively coupled plasma (ICP)-atomic emission spectrometry were investigated using both laser ablation and solution nebulization as sample introduction techniques. Matrix effects were studied by monitoring the excitation conditions of the plasma using the ionic to atomic spectral line intensity ratios of zinc and magnesium. A new kind of matrix interference was found in the ICP that appears to be related to matrices with elements of low second ionization potential. The matrix effects do not correlate with the first ionization potential of the element. Only those matrix elements with low second ionization potential showed severe matrix effects. Increasing the forward power of the ICP or replacing the carrier gas with a 50%/50% argon–helium mixture did not significantly reduce this matrix effect. However, using 100% helium as the carrier gas greatly reduced the extent of this matrix effect, suggesting that argon is involved in the interference mechanism. The interference mechanism may involve interactions between doubly-charged matrix ions and argon species.     

Unusual observations during steroid analysis

In September 2005, our laboratory detected the presence of 4-androstene-3,17-dione and androsterone in a standard steroid screen of a post-race gelding urine sample received from an overseas authority. All other urine samples from the same batch tested negative. Subsequent gas chromatography/mass spectrometry (GC/MS) confirmatory analyses, however, repeatedly failed to detect any amount of 4-androstene-3,17-dione and androsterone in the suspicious sample. On the other hand, identical results were obtained when the initial GC/MS screening method was repeated on the suspicious sample as well as on the other samples of the same batch, showing the presence of 4-androstene-3,17-dione and androsterone only in the suspicious sample. These unusual and contradictory findings between the screening and confirmatory procedures were investigated, leading to the unequivocal conclusion that the 4-androstene-3,17-dione and androsterone observed during screening were artefacts from the internal standards, [16,16,17-d3]-testosterone and [16,16,17-d3]-5alpha-androstane-3alpha,17beta-diol. The two deuterated internal standards were thought to have undergone first an enzymatic oxidation of the 17beta-hydroxyl group to a 17-keto function by the enzyme 17beta-hydroxysteroid dehydrogenase; complete deuterium-hydrogen exchange at C16 during the methanolysis deconjugation step would then produce the two artefacts. The findings from this study highlight the potential problem of using internal standards in qualitative confirmatory analyses, which may lead to undesirable false positive results.     

Detection of dehydroepiandrosterone misuse by means of gas chromatography- combustion-isotope ratio mass spectrometry

According to World Anti-Doping Agency (WADA) rules (WADA Technical Document-TD2004EAAS) urine samples containing dehydroepiandrosterone (DHEA) concentrations greater than 100 ng ML(-1) shall be submitted to isotope ratio mass spectrometry (IRMS) analysis. The threshold concentration is based on the equivalent to the glucuronide, and the DHEA concentrations have to be adjusted for a specific gravity value of 1.020. In 2006, 11,012 doping control urine samples from national and international federations were analyzed in the Cologne doping control laboratory, 100 (0.9%) of them yielding concentrations of DHEA greater than 100 ng mL(-1). Sixty-eight percent of the specimens showed specific gravity values higher than 1.020, 52% originated from soccer players, 95% were taken in competition, 85% were male urines, 99% of the IRMS results did not indicate an application of testosterone or related prohormones. Only one urine sample was reported as an adverse analytical finding having 319 ng mL(-1) DHEA (screening result), more than 10,000 ng mL(-1) androsterone and depleted carbon isotope ratio values for the testosterone metabolites androsterone and etiocholanolone. Statistical evaluation showed significantly different DHEA concentrations between specimens taken in- and out-of- competition, whereas females showed smaller DHEA values than males for both types of control. Also a strong influence of the DHEA excretion on different sport disciplines was detectable. The highest DHEA values were detected for game sports (soccer, basketball, handball, ice hockey), followed by boxing and wrestling. In 2007, 6622 doping control urine samples were analyzed for 3alpha,5-cyclo-5alpha-androstan-6beta-ol-17-one (3alpha,5-cyclo), a DHEA metabolite which was described as a useful gas chromatography-mass spectrometry (GC-MS) screening marker for DHEA abuse. Nineteen urine specimens showed concentrations higher than the suggested threshold of 140 ng mL(-1), six urine samples yielded additionally DHEA concentrations higher than 100 ng mL(-1), none of them showing positive IRMS findings. These results should be taken into consideration in future discussions about threshold values for endogenous steroids in doping control.     

固相萃取-气相色谱-质谱法分析尿液中邻苯二甲酸单酯和双酯

建立了固相萃取-气相色谱-质谱测定尿液中邻苯二甲酸单酯和双酯的分析方法。尿液经 β-葡萄糖苷酸酶酶解后进行固相萃取净化,用乙腈、乙酸乙酯和乙醚-正己烷(1: 19, v/v)分别洗脱,合并洗脱液,氮气吹干后,用N,O-双三甲基硅基三氟乙酰胺(BSTFA)对邻苯二甲酸单酯进行硅烷化处理,使用气相色谱-质谱法检测。邻苯二甲酸单酯和双酯的线性范围为5~1000 μ g/L,检出限为0.3~1.1 μ g/L,回收率为77.9%~97.7%,相对标准偏差为3.7%~10.9%。应用该方法对50份尿液进行检测,检出邻苯二甲酸二(2-乙基己基)酯(DEHP)等7种邻苯二甲酸单酯和双酯类物质,平均质量浓度为6.0~142.7 μ g/L。该方法准确、可靠、灵敏度高,适用于尿液中邻苯二甲酸单酯和双酯的同时测定。     

Synthesis and interfacial properties of sophorolipid derivatives

Biosurfactants made by fermentation from renewable resources provide “environmental friendly” processes and products. A natural sophorolipid mixture was produced by the yeast Candida bombicola when cultured on glucose and oleic acid. The sophorolipid mixture was chemically modified to form the corresponding sophorolipid alkyl (methyl, ethyl, propyl, and butyl) esters by reaction with the corresponding sodium alkoxides. Interfacial properties of these surfactants, such as surface tension reduction, aggregation, and adsorption, were systematically studied. It was found that the critical micelle concentration of sophorolipid esters decreases to about 1/2 per additional one CH₂ group to the alkyl ester moiety. Interestingly, these surfactants were found to adsorb strongly on alumina but weakly on silica. They have properties that make them attractive candidates for uses in detergents, cosmetics, soil remediation, and enhanced oil recovery.     

Analysis of the Products from the Oxidation of Geting Bituminous Coal by Atmospheric Pressure Photoionization–Mass Spectrometry

Geting bituminous coal was directly oxidized in aqueous sodium hypochlorite. The reaction mixture was sequentially extracted with ethyl ether and ethyl acetate. Both extracts were esterified with diazomethane to obtain methyl esterified products, which were analyzed using high performance liquid chromatography–mass spectrometry with electrospray ionization or atmospheric pressure photoionization. A large number of low- and nonpolar products with relatively high molecular masses were determined using atmospheric pressure photoionization mass spectrometry. Toluene and a toluene/anisole mixture (vol/vol = 95:5) were added to the atmospheric pressure photoionization system as dopants. Toluene induced better ionization than the toluene/anisole mixture in both ion signal intensity and number of detected species. Most of the molecular associated compounds contained heteroatoms.     

Synthesis and Characterization of Poly(ether-block-amide) Membranes

Poly(ether-block-amide) membranes were made via casting a solution on a nonsolvent (water) surface. In this research, effects of different parameters such as ratio of solvent mixture (n-butanol/isopropanol), temperature, composition of coagulation bath (water) and polymer concentration, on quality of the thin film membranes were studied. The mechanism of membrane formation involves solution spreading, solvent–nonsolvent exchange, and partial evaporation of the solvent steps. Solvent- nonsolvent exchange is the main step in membrane formation and determines membrane morphology. However, at higher temperature of polymeric solution greater portion of solvent evaporates. The results showed that type of demixing process (mutual affinity between solvent and nonsolvent) has important role in film formation. Also, addition of solvent to the nonsolvent bath is effective on membrane morphology. The film quality enhances with increasing isopropanol ratio in the solvent mixture. This behavior can be related to increasing of solution surface tension, reduction of interfacial tension between solution and nonsolvent and delayed solvent-nonsolvent demixing. Uniform films were made at a temperature rang of 60–80 °C and a polymer concentration of 4–7 wt%. Morphology of the membranes was investigated with scanning electron micrograph (SEM). Pervaporation of ethyl butyrate/water mixtures was studied using these membranes and high separation performance was achieved. For ethyl butyrate/water mixtures, It was observed that both permeation flux and separation factor increase with increasing ethyl butyrate content in the feed. Increasing temperature in limited range studied resulted in decreasing separation factor and increasing permeation flux.     

Accelerated solid-phase dynamic extraction for the analysis of biogenic volatile organic compounds in air

In this study, accelerated solid phase dynamic extraction (ASPDE) technique was used to identify biogenic volatile organic compounds (BVOCs) emitted from Norway spruce (Picea abies). Compounds that were determined in tree samples are: tricycylene, α-pinene, camphene, β-pinene, myrcene, 3-carene, p-cymene, limonene, cineole, α-phellandrene, α-terpinene, γ-terpinene and terpinolene. ASPDE showed a potential for the analysis of environmental samples as well as for field applications. This technique was further studied by using a gaseous mixture of BVOCs (sabinene, α-pinene, β-pinene, limonene, linalool, and (Z)-hexenyl acetate) and exhibited a good repeatability during all the experiments in the range of 2.5% (α-pinene) and 14.6% (linalool). However, during the analysis of samples it was observed that desorption at high temperature (230°C) can lead to the formation of artifacts, which were not observed at the desorption temperature of 100°C. Further experimental investigations revealed that monoterpenes appeared as unanticipated compounds during desorption of ASPDE samples; these compounds were degradation products of linalool.     

Optimization of operating conditions for the determination of perchlorate in biological samples using preconcentration/preelution ion chromatography

Perchlorate originates as a contaminant in the environment from the use of salts in the manufacture of solid rocket fuels and munitions. Monitoring potential perchlorate contamination in the environment is of interest, however, very few analytical methods have been developed for perchlorate determination in biological samples. Analysis of complex samples by ion chromatography is complicated by matrix components that can interfere with perchlorate determination. However, a recently developed preconcentration/preelution (PC/PE) ion chromatography method has demonstrated the capability to analyze certain complex samples such as high salinity water, milk, and hydroponic fertilizers. The ability of this method to reduce sample background and lower detection limits in ion chromatography for various biological samples was evaluated in this study. The PC/PE method was applicable to the analysis of kidneys, livers, zebrafish, quail eggs, lettuce, and urine. Optimal operating conditions were determined for each matrix. Ranges of optimal wash volumes were shorter when 15 mM NaOH prewash solutions were used compared with 10mM and good recovery was achieved for most matrices with an injection period > or =60s. Prewash solution concentration did not appear to significantly affect matrix background. The PC/PE method was capable of reducing sample background when compared to EPA Method 314.0, which resulted in detection limits, with the exception of zebrafish and urine, that were two-fold lower than those achieved with EPA Method 314.0.     

H NMR aided elucidation of products derived from photodegradation of ethyl 3-azido-4,6-difluorobenzoate in 2,2,2-trifluoroethanol

The major products formed upon photolysis of ethyl 3-azido-4,6-difluorobenzoate in 2,2,2-trifluoroethanol-d₃ has been elucidated by ¹H NMR analysis of the product mixture. Among the products formed and structurally elucidated was a hitherto unreported product formed during photolysis of aryl azides, namely azoxybenzene 19. The structural assignments of the major components of the reaction mixture were aided by comparison with ¹H NMR data from synthetic reference materials and compound isolation. MS, MS/MS, and HPLC analysis as well as UV spectroscopy was also employed in order to confirm and aid the structural analysis.