Derivatization and fragmentation pattern analysis of natural and synthetic steroids, as their trimethylsilyl (oxime) ether derivatives by gas chromatography mass spectrometry: analysis of dissolved steroids in wastewater samples

This paper reports the extension of our multiresidue analysis (MA) procedure with 18 natural and synthetic steroids; permitting the identification and quantification, in total of 81 pollutants from one solution, by a single injection, as their trimethylsilyl (TMS)-oxime ether/ester derivatives, by gas chromatography-mass spectrometry (GC-MS), within 31 min. As a novelty to the field, basic researches, such as fragmentation pattern analysis and derivatization optimization studies were performed for androsterone, transdehydroandrosterone, transandrosterone, mestranol, dihydrotestosterone, ethinylestradiol, testosterone, norethisterone, estriol, 4-androstene-3,17-dione, gestodene, levonorgestrel, etonogestrel, coprostanol, progesterone, cholesterol, medroxy-progesterone-acetate, stigmasterol and β-sitosterol. Results confirmed that (i) the TMS oxime-ether derivatives of the keto steroids provide from 1.40 times (gestodene) up to 4.25 times (norethisterone) higher responses compared to their TMS-ether ones, and (ii) the distribution of syn/anti oximes is characteristic to the ketosteroid species examined. Based on our optimized mass fragmentation, solid phase extraction (SPE) and derivatization studies separations have been performed in the total ion current (TIC) mode, identification and quantification of compounds have been carried out on the basis of their selective fragment ions. Responses, obtained with derivatized standards proved to be linear (hydroxysteroids), or have been calculated from calibration curves (ketosteroids) in the range of 1.88-750ng/L levels. Limit of quantitation (LOQ) values varied between 1.88ng/L and 37.5ng/L concentrations. The most important practical messages of this work are the high androsterone (0.744-4.28μg/L), transandrosterone (0.138-4.00μg/L), coprostanol (2.11-302μg/L), cholesterol (0.308-41μg/L), stigmasterol (1.21-8.40μg/L) and β-sitosterol (1.12-11.0μg/L) contents of influent wastewaters. β-Estradiol (100ng/L) and estriol (54ng/L) were found in one influent sample, only. Reproducibilities, characterized with the relative standard deviation percentages (RSD%) of measurements, varied between 1.73 RSD% (β-estradiol) and 5.4 RSD% (stigmasterol), with an average of 4.82 RSD%.     

Simultaneous determination of C2-C22 non-esterified fatty acids and other metabolically relevant carboxylic acids in biological material by gas chromatography of their benzyl esters

A method for the simultaneous determination of non-esterified short-, medium- and long-chain fatty acids and other types of metabolically relevant carboxylic acids such as hydroxy, keto, aromatic and dicarboxylic acids in biological material by capillary gas chromatography of benzyl ester derivatives is described. Sample preparation avoiding incomplete isolation of carboxylic acids consisted of deproteinization and extraction with ethanol, fixation of carboxylic acids as carboxylates, removal of interfering compounds such as neutral lipids by hexane extraction and amino acids, acyl carnitines and other cations by cation-exchange chromatography, derivatization of keto groups of ketocarboxylic acids into O-methyl oximes and benzyl ester formation by reaction of the potassium carboxylates with benzyl bromide via crown ether catalysis. The sample preparation conditions were investigated, showing the usefulness of this method for quantitative determinations. Chromatograms obtained from human serum, human urine and rat heart ventricle and concentrations of carboxylic acids in these specimens are presented.     

Gas-liquid chromatographic studies of reactions and structural relationships of steroids. Part III. 11alpha-hydroxysteroids of the androstane and pregnane series.

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 which afford excellent yields of the major products are described. Reactions studied include the Wolff-Kishner removal of keto groups, their conversion into hydroxyl groups with sodium-ethanol or sodium borohydride and into dioxolone derivatives with ethylene glycol; the conversion of hydroxyl into keto groups with chromium trioxide and to trimethylsilyl (TMS) ethers by hexamethyldisilazane; the hydrolysis of dioxolone and TMS derivatives by H+. Gas-liquid chromatograms of reaction mixtures of single- and multistep reactions readily provide information on the effects on the 11alpha-hydroxy and other functional groups at positions 3 and 17 (androstane series) and positions 3 and 20 (pregnane series), and the retention times of many steroids unavailable from commercial or 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 calculation methods is demonstrated.     

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.     

Preventive doping control analysis: liquid and gas chromatography time-of-flight mass spectrometry for detection of designer steroids

A new combined doping control screening method for the analysis of anabolic steroids in human urine using liquid chromatography/electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (LCoaTOFMS) and gas chromatography/electron ionization orthogonal acceleration time-of-flight mass spectrometry (GCoaTOFMS) has been developed in order to acquire accurate full scan MS data to be used to detect designer steroids. The developed method allowed the detection of representative prohibited substances, in addition to steroids, at concentrations of 10 ng/mL for anabolic agents and metabolites, 30 ng/mL for corticosteroids, 500 ng/mL for stimulants and beta-blockers, 250 ng/mL for diuretics, and 200 ng/mL for narcotics. Sample preparation was based on liquid-liquid extraction of hydrolyzed human urine, and the final extract was analyzed as trimethylsilylated derivatives in GCoaTOFMS and underivatized in LCoaTOFMS in positive ion mode. The sensitivity, mass accuracy, advantages and limitations of the developed method are presented.     

Determination of Aromatic Amines in Textile After Bromination, by Gas Chromatography Coupled with Electron Capture Detection

A new analytical method is developed for the improved determination of banned aromatic amines in textiles based on the bromination of the aromatic ring system to yield the corresponding bromo derivatives. The derivatization method is very easy to perform and the derivatives can easily extract and separate with gas chromatography. Due to the introduction of the electron withdrawing bromo substituents sensitive detection with very low limits of detection using an electron capture detector is possible. The method was optimized and used for the analysis of textile with quantitative recoveries from spiked samples.     

Mass spectrometry of steroid glucuronide conjugates. II-Electron impact fragmentation of 3-keto-4-en- and 3-keto-5alpha-steroid-17-O-beta glucuronides and 5alpha-steroid-3alpha,17beta-diol 3- and 17-glucuronides

The steroid glucuronide conjugates of 16,16,17-d(3)-testosterone, epitestosterone, nandrolone (19-nortestosterone), 16,16,17-d(3)-nortestosterone, methyltestosterone, metenolone, mesterolone, 5alpha-androstane-3alpha,17beta-diol, 2,2,3,4,4-d(5)-5alpha-androstane-3alpha,17beta-diol, 19-nor-5alpha-androstane-3alpha,17beta-diol, 2,2,4,4-d(4)-19-nor-5alpha-androstane-3alpha,17beta-diol and 1alpha-methyl-5alpha-androstane-3alpha/beta,17beta-diol were synthesized by means of the Koenigs-Knorr reaction. Selective 3- or 17-O-conjugation of bis-hydroxylated steroids was performed either by glucuronidation of the corresponding steroid ketole and subsequent reduction of the keto group or via a four-step synthesis starting from a mono-hydroxylated steroid including (a) protection of the hydroxy group, (b) reduction of the keto group, (c) conjugation reaction and (d) removal of protecting groups. The mass spectra and fragmentation patterns of all glucuronide conjugates were compared with those of the commercially available testosterone glucuronide and their characterization was performed by gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy. For mass spectrometry the substances were derivatized to methyl esters followed by trimethylsilylation of hydroxy groups and to pertrimethylsilylated products using labelled and unlabelled trimethylsilylating agents. The resulting electron ionization mass spectra obtained by GC/MS quadrupole and ion trap instruments, full scan and selected reaction monitoring experiments are discussed, common and individual fragment ions are described and their origins are proposed.     

Comparison of the use of mass spectrometry and methylene unit values in the determination of the stereochemistry of estranediol, the major urinary metabolite of 19-nortestosterone in the horse

The stereochemistry of an isomer of 5-estrane-3,17 alpha-diol, the major metabolite of 19-nortestosterone in horse urine has been established by the use of methylene unit (MU) values. The empirical MU values of the bis-trimethylsilyl (TMS) derivatives of the eight available isomers of 5-androstane-3,17-diol and four isomers of 5-estrane-3,17 beta-diol were determined by capillary gas chromatography using three different columns. From this data the theoretical MU values for the bis-TMS derivatives of the four 5-estrane-3,17 alpha-diol isomers were predicted. Comparison of the experimentally determined MU value of the urinary metabolite with those of the theoretical values established the correct stereochemistry of the steroid. This method has been compared with the use of gas chromatography-mas spectrometry in the determination of the stereochemistry of unknown metabolites.     

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.     

气相色谱法分析尿液样品中阿特拉津及其代谢物

建立尿液样品中阿特拉津(ATZ)及其代谢物脱乙基阿特拉津(DEA)、脱异丙基阿特拉津(DIA)、脱乙基脱异丙基阿特拉津(DEDIA)的气相色谱分析方法。样品通过乙酸乙酯萃取,硫酸钠干燥,弗罗里硅土净化,浓缩后用气相色谱ECD检测器分析。对方法中pH值等条件进行了优化,获得了较好的回收率。方法的检出限为DEDIA:2.5ng/mL,DEA、DIA、ATZ:均为5ng/mL。利用本方法对阿特拉津生产工人的实际尿液样品进行了分析。     

Synthesis of New Substituted 1,2,4‐Triazines from Isonitrosoketones and Terephthalaldehydedihydrazone

This study describes a novel method for the synthesis of 1,2,4‐triazines. Firstly, isonitrosoacetophenone, p‐methylisonitrosoacetophenone, and isonitroso‐1‐acetylnaphthalene were synthesized from the reaction of butylnitrite with acetophenone, p‐methylisonitrosoacetophenone, and 1‐acetylnaphthalene, respectively. Then, symmetric derivatives of 1,2,4‐triazines were prepared from the condensation reaction of keto oximes with terephthalohydrazone. However, the heteroaromatic closure was not observed in the condensation reaction of pyruvic‐aldehyde‐1‐oxime(keto oxime) with terephthalohydrazone. Structures of the obtained products were confirmed by FTIR, ¹H‐NMR, and elemental analyses techniques.     

Determination of benzidines by gas chromatographic separation of derivatives with electron capture detection

Factors considered in the development of a method for the determination of toxic, water-soluble benzidines. involved providing a good technique for their adequate recovery, converting quantitatively the isolated amines to relatively stable derivatives suitable for measurement and storage, coupled with relative ease of preparation of the derivatives, and resolving the prepared compounds during their measurement. Benzidine and dichlorobenzidine are quantitatively extracted from wastewater and measured quantitatively through the preparation of their respective pentafluoropropionamides (PFP) by using PFP-imidazole. Overall recovery efficiency for benzidines from wastewater ranges from 91 to 103%. These derivatives are relatively stable compounds and have detection limits of 0.2 pg or less, when electron capture detection is used with gas chromatography. Derivatives of o-tolidine and dianisidine are also described.     

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.     

Gas chromatography of alpha-keto acids as their O-trimethyl silylquinoxalinol derivatives.

The O-trimethylsilyl (TMS) quinoxalinols are very useful derivatives for the gas chromatography of alpha-keto acids because of their high stability and the absence of stereoisomerism and because of the presence of specific, common and abundant fragments in electron impact mass spectra, which allows the low-level detection of whole groups of keto acids by single-ion detection. In this paper, the chromatographic properties of eleven O-TMS-quinoxalinols on OV-1, OV-17 and Dexsil 300 are reported in terms of methylene units. Also by use of methylene units, the chromatographic isotope effect is analyzed in detail for nine perdeutero-TMS derivatives. The effect is explained by the diminished interaction of the deuterated compounds with the unlabelled liquid phase.     

Derivatization of aromatic amines for analysis in ammunition wastewater I. Derivatization via bromination of the aromatic ring

 A new derivatization method for aromatic amines is presented based on the bromination of the aromatic ring system in an acetic acid medium to yield bromo derivatives. The derivatization method is very simple to perform and overcomes several problems common in the analysis of polar aromatic amines. The derivatives are easy to extract with pentane and can be separated with gas chromatography. Due to the introduction of the electron withdrawing bromo substituents sensitive detection using an electron capture detector is possible. The method was used to investigate the contents of amino- and aminonitrotoluenes in water samples from the site of a former ammunition plant. Received: 17 May 1996/Revised: 1 August 1996/Accepted: 1 August 1996     

Determination of chloramphenicol residue in chicken tissues by immunoaffinity chromatography cleanup and gas chromatography with a microcell electron capture detector

A rapid and sensitive gas chromatography (GC) method was developed to detect chloramphenicol in chicken tissues. The extracted samples were cleaned up using the immunoaffinity column prepared by coupling antichloramphenicol monoclonal antibody with cyanogen bromide-activated Sepharose 4B. The dynamic column capacity of chloramphenicol was 3265 ng/mL gel. The eluate was evaporated to dryness, and residues were derivatized and determined by GC with a microcell electron capture detector. Average recoveries were 86.6 to 96.9% for chicken muscle and 74.3 to 96.1% for chicken liver. The limit of quantitation of the method was 0.05 ng/g for chicken muscle and 0.1 ng/g for chicken liver.     

Derivatization of Drugs and Bioorganics for Improved Detection by Gas Chromatography and Photoionization Detection (GC-PID)

Abstract

Pentafluorophenyldimethylsilylchloride (flophemesyl chloride, FICI) has recently been used for derivatizations of alcohols, leading to significant improvements in detectability by gas chromatography (GC)-photoionization detection (PID) and electron capture detection (ECD). This off-line reaction has now been applied to a number of related alcohol derivatives, naturally occurring bioorganics such as cholesterol, androsterone, and similar sterols and steroids. Standard GC-ECD-PID analytical parameters have been determined, such as detection limits, calibration plots, ECD and PID relative response factors (RRF), ECD/PID relative response factor ratios, etc. for underivatized and derivatized substrates. Packed and capillary column GC conditions have been used with a 10.2 eV PID lamp. Application of the optimized derivatization-GC-ECD-PID method is reported for bioorganics in biological media. The overall results demonstrate the immediate applicability of such an approach for a number of alcohol derivatives, especially those having biological properties.     

Precision of an automated all-glass capillary gas chromatography system with an electron capture detector for the trace analysis of estrogens

Subnanogram detection of steroids has become increasingly important today. One applicable method for gas chromatographic determination of subnanogram quantities of estrogens as halogenated derivatives is electron capture detection. HFB-derivatives of 7 different estrogens were automatically injected onto a prolonged narrow bore wall-coated glass capillary column. Normal split injection could not be used for this trace analysis because of too much loss of sample. Only small amounts of sample were available from which double analysis had to be performed. Cross-contamination of the automatic sampling system as well as precision of retention times and peak areas were determined. The type of injection described showed better quantitative results compared to the splitless injection technique. All details of the system used together with the results are this discussed in this paper.     

肟的无污染氧化

No2;污染治理;二噁烷;肟的无污染氧化     

Determination of estrone and 17 beta-estradiol in human hair by gas chromatography-mass spectrometry

An efficient procedure is described for the determination of estrone and 17 beta-estradiol in hair by gas chromatography-mass spectrometry (GC-MS). The method involves alkyloxycarbonylation with isobutyl chloroformate (isoBCF) of phenolic hydroxy groups after alkaline digestion of hair samples. The resulting isobutyloxycarbonyl derivatives of estrone and 17 beta-estradiol are extracted with hexane and subjected to chlorodifluoroacetyl derivatization in order to protect the remaining alcoholic hydroxy groups. When GC-MS with selected ion monitoring (SIM) was used, the quantitative ions were at m/z 270 and 384 in the electron ionization mass spectra for estrone and 17 beta-estradiol, respectively. The detection limits for SIM of the steroids were 1 and 2 pg, respectively, and the SIM responses were linear with correlation coefficients varying from 0.991 to 0.994 in the concentration range 0.2-4.0 ng g-1 for the estrogens studied. The detection of estrone and 17 beta-estradiol in hair samples was possible in the concentration range of 0.24-1.30 ng g-1. The concentrations of the two estrogens detected were different in male and female hair samples.