Analysis of C19O3 steroids by thin-layer and gas-liquid chromatography and mass spectrometry

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

Separation of androsterone from epiandrosterone, and dehydroepiandrosterone from its 3-hydroxy epimer by thin-layer chromatography.

The application of thin-layer chromatography to the separation of 13 steroids, including androstanes, 4-androstenes and 5-androstenes, using silica gel and 1,2-propanediol-impregnated cellulose is described. After group-wise separation of various C19 steroids on silica gel, the 3-hydroxy epimers of 5alpha-androstanes and 5-androstenes can be separated by thin-layer chromatography on impregnated cellulose plates. The chromatographic procedure is rapid and makes the prior formation of steroid derivatives unnecessary.     

Two methods for determining erucic acid in edible fats and oils: results from a collaborative study on a rapid, open-tubular (capillary) GLC method and comparison with an isolation TLC procedure

The results of an international collaborative study for the determination of 5% erucic acid in four samples of edible fats and oils with differing levels of 22:1 isomers other than erucic acid, based on the use of wall-coated (SILAR-5CP) open-tubular gas-liquid chromatography, are examined. The same samples had been analyzed in a separate collaborative study by an alternative and more complex method based on argentation thin-layer chromatography. Both methods rejected about the same proportion of participating laboratories and a few individual results from otherwise acceptable laboratories. The means and repeatabilities of the two methods were similar, but the gas-liquid chromatographic method showed better reproducibility.     

A simple gas-liquid chromatographic method with electron capture detection for the determination of progesterone in the blood of humans and domestic animals.

A simple routine method is reported for the determination of progesterone in blood by gas-liquid chromatography. An unlabelled internal standard, testosterone acetate, was added to plasma samples. Following preliminary purification by thin-layer chromatography, progesterone in pregnancy plasma was evaluated as the free steroid with a flame ionization detector. The hormone in the plasma of women during the menstrual cycle and of cycling domestic animals was determined as the 3-enol ester heptafluorobutyrate by electron capture detection. The validity of testosterone acetate as an internal standard was proved by simultaneous processing of [7alpha-3H]progesterone and a [4-14C]testosterone acetate and the determination of the isotope ratio in samples. The results of control experiments and normal values are also presented.     

Gas-liquid chromatographic determination of fatty acid composition of cholesteryl esters in human serum using silica Sep-Pak cartridges

A simple and fast analytical procedure for separation and purification of cholesteryl esters of human serum is described. A single lipid extract, together with spiked cholesteryl pentadecanoate, as an internal standard, was passed through a Silica Sep-Pak cartridge. 1.5% diethyl ester in light petroleum was used to elute cholesteryl esters from the column. The separation was verified with thin-layer chromatography on silica gel using light petroleum-diethyl ether-glacial acetic acid (80:20:1) as a solvent. A very clean thin-layer chromatogram of cholesteryl esters without any additional spots of other lipids was obtained. The cholesteryl esters were quantitated by analyzing their fatty acid composition as methyl esters by gas-liquid chromatography. The coefficients of variation were 0.8--4.9% for the major fatty acids (C16:0, C16:1, C18:1, C18:2, C20:4) and 6.7--30.8% for the minor fatty acids (C18:0 and C20:0). The recoveries for cholesteryl palmitate, cholesteryl oleate and cholesteryl linoleate were 90.7, 92.3 and 91.0%, respectively.     

Eine Methode zur Bestimmung von 25 Oestrogenen im Urin mit Hilfe der kombinierten Papier-, Dünnschicht- und Gas-Chromatographie

25 phenolic steroids of which many have already been isolated from human pregnancy urine can be separated from each other and quantitatively determined by a combination of paper, thin-layer and gas chromatography. The method involves the following steps: (1) enzyme hydrolysis of urine, (2) extraction of the hydrolysed urine with ether, (3) washing of the ether extracts with sodium hydrogen carbonate and water at 0° C, (4) multiple chromatography of the residue on formamide-impregnated paper with monochlorobenzene, chloroform, cyclohexane, monochlorobenzene-ethyl acetate (3∶1) and chloroform-ethyl acetate (5∶1), respectively, and in two Bush-Systems, (5) acetylation of the separated oestrogens, (6) thin-layer chromatography of the steroid acetates and (7) quantitative determination of the acetates by gas chromatography, using a flame ionizationdetector. The reliability of the method (accuracy, precision, specificity and sensitivity) has been checked in experiments in which 10 μg of each steroid were added to male urine. The average recovery of the 25 phenolic steroids varied between 50 and 60% and the variation coefficients between 5 and 15%. As shown by microchemical reactions, the methods possesses, even in the presence of urinary pigments, a high specificity. Amounts of 1–2 μg of steroid/24 hr urine can be estimated.     

Development and validation of a method for measuring the glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography

We developed and validated a simple method for measuring the individual glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography with a C18 reversed-phase column using an isocratic solvent system of acidified methanol--potassium phosphate. Without preliminary derivatization or purification, complete separation of the ten major conjugated bile acids present in bile could be achieved in 65 min. Total bile acid concentrations were identical when measured enzymatically and by summing the individual bile acids determined by high-performance liquid chromatography. Bile acid composition determined by gas-liquid chromatography correlated with results by high-performance liquid chromatography. Finally, measurements of individual glycine and taurine conjugates in human bile and in mixtures of bile acid standards by high-performance liquid chromatography and thin-layer chromatography gave similar results. This high-performance liquid chromatographic system permits simultaneous quantification of total and individual bile acids and their glycine and taurine conjugates in bile.     

Chromatographic and spectroscopic methods for the identification of alkaloids from herbarium samples of the genus Uncaria.

A combination of thin-layer chromatography, gas-liquid chromatography, ultraviolet spectroscopy and mass spectrometry techniques for the alkaloid screening of herbarium samples of the genus Uncaria (Rubiaceae) is described. Some sixty alkaloids are distinguished by the screening procedure, and they represent heteroyohimbine, oxindole, roxburghine, simple beta-carboline, pyridino-indolo-quinolizidinone and gambirtannine types.     

On the influence of sorbent activity,mobile phase polarity,and structure of the investigated substances on thermodynamic characteristics of the chromatographic system

Advantages of the thermodynamic characteristics of Chromatographic systems are discussed with respect to previous experimental results. It seems that the principles of the thermodynamic approach can be successfully applied not only to paper and thin-layer chromatography, but also to gas-liquid and high-performance chromatography.     

Gas-liquid chromatography of fecal neutral steriods.

A method is described for the analysis of fecal neutral steriods with a dual-column gas-liquid chromatography (GLC) system. After saponification of the fecal slurry, the neutral steroids were extracted with hexane. The GLC separation of the compounds and quantitation were achieved by simultaneous injection of the derivatized and derivatized aliquots of the extract onto dual colmuns under identical conditions. The neutral steroids of interest were than identified by matching the retention times with those of known standards, and identification was confirmed by use of an interfaced GLC high-resolution mass spectrometry system. The detection limit was 0.003 mg of steroid/g of fecal slurry. The pricision of the method is illustrated by a relative standard diviation of 2-10% and a recovery of neutral steroids from 73-96%. The method was applied to the determination of fecal neutral steroids in a "High protein diet in colon cancer study". A considerably larger level of coprostanone than of coprostanol was observed. Data on neutral steroids in fecal samples from subjects on different diets are the subject of a separate publication.     

Analysis and quantification of ether lipids by chromatographic methods.

Chromatographic methods, especially thin-layer chromatography (TLC) and gas-liquid chromatography (GLC) are widely used in investigations of the occurrence, molecular structure and metabolism of ether lipids. The application of such techniques to structural analysis and quantification, in combination with methods for the degradation and derivatization of ether lipids, is discussed.     

The thermal properties of some metal complexes of diethyldithiocarbamic acid

The cobalt(II), cadmium(II), copper(II), mercury(II), nickel(II) and zinc(II) complexes of diethyldithiocarbamic acid were prepared and investigated by TG, DSC thin-layer chromatography and gas-liquid chromatography. Heats of fusion were determined.Permission to publish this paper has been given by the Government Chemist, Ministry of Technology.     

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.     

Chromatographic analysis of Passifit multicomponent pharmaceutical syrup

Procedures are developed for the qualitative analysis of a new pharmaceutical preparation, Passifit syrup. The conditions for the separation of the active substances were optimized. Valerian and hop extracts were detected by thin-layer chromatography on Kieselgel 60/Kieselguhr F₂₅₄ plates. Menthol (the volatile component of peppermint oil), thymol and its isomer carvacrol (the components of thyme extract), and sorbic acid (preserving agent) were identified by gas-liquid chromatography on a capillary column with an OV-101 stationary phase and a flame-ionization detector. Luteolin, thymol, rutin, and sorbic acid were identified by reversed-phase HPLC on a Zorbax SB C18 column in a gradient elution mode with UV detection at 210 and 330 nm. Rutin was not detected among the components of the syrup.     

Chromatographic identification of pesticides

The chromatographic properties of 51 common pesticides have been measured using seven different chromatographic systems involving gas-liquid chromatography (GLC), high-performance liquid chromatography (HPLC) with diode-array spectrophotometric detection and thin-layer chromatography (TLC) with different spray reagents. Correlation coefficients were calculated for combinations of all systems. The best combination of the chromatographic systems examined for the identification of an unknown compound is GLC on OV-17, HPLC on ODS-Hypersil with acetonitrile-water as eluent and TLC using an isooctane-ethyl acetate solvent system.     

Total Syntheses of Polyhydroxylated Steroids by an Unsaturation-Functionalization Strategy

Highly oxygenated cardiotonic steroids, such as ouabain, possess a wide spectrum of biological functions and remain significant synthetic challenges. Herein, we have applied an unsaturation-functionalization strategy and developed a synthetic method in addressing the C19-hydroxylation issue for efficient synthesis of polyhydroxylated steroids. An effective asymmetric dearomative cyclization allowed the construction of the C19-hydroxy unsaturated steroidal skeleton in only four steps from the Hajos-Parrish ketone ketal 7 . The synthetic sequence featured C3−OH-directed hydrogenation/epoxidation, m-CPBA-triggered epoxidation/S_N2′ nucleophilic substitution, Birch reduction of an enone, and regioselective LiAlH₄ reduction to furnish the polyhydroxy functionalities on the steroid skeleton with high stereochemical control and efficiency. This approach ultimately enabled the total synthesis of 19-hydroxysarmentogenin and ouabagenin in 18 and 19 steps, respectively, overall. The synthesis of these polyhydroxylated steroids offers synthetic versatility and practicality in the search for new therapeutic agents.     

Isolation and purification of lithocholic acid metabolites produced by the intestinal microflora.

Lithocholic acid metabolites produced by the intestinal microflora of rats can be isolated from other endogenous lipids using Sephadex LH-20 column chromatography. Analyses of individual metabolites collected from the column by silica gel coated glass fiber paper chromatography result in the resolution of epimeric 3-hydroxy derivatives. In addition, glass fiber paper chromatography is more sensitive and requires less development time than conventional glass-coated thin-layer plates. Further confirmation of the identity of metabolites is achieved by gas-liquid chromatography, which separates both methyl and ethyl esters of lithocholic and isolithocholic acids.     

某些单体-单体、单体-二聚体体系的活度系数

用气液色谱法测定了苯在二苯醚、联苄、二苄醚、二苯甲烷、二苯乙炔、二苯甲酮、联苯、二苯胺、α-甲基萘、α-氯萘、α-溴萘、氮萘、α-硝基萘、α-萘胺等十四种溶剂中的无限稀活度系数;用单体-二聚体的溶液模型对以上体系进行活度系数的计算,计算值与实验值接近。 用气液色谱法测定了甲苯、乙苯、邻二甲苯、对二甲苯、间二甲苯等五种溶质在环丁砜、二甲基环丁砜、N-甲基吡咯烷酮,5,N-二甲基吡咯烷酮等四种溶剂中的无限稀活度系数;用单体-单体的溶液模型计算溶剂的“溶解度参数”,计算得各溶剂的“溶解度参数”分别为一常数。     

Influence of chamber saturation in thin-layer chromatography on theoretical and experimental parameters of mixture separations

The influence of chamber saturation in thin-layer chromatography on theoretical and experimental parameters of mixture separations were examined. Five different types of chambers for development of chromatograms were taken into consideration. The relation found experimentally differed widely from that calculated using O?cik's equation for different method of carrying out of the chromatographic process. The investigations presented here show the possibility of choosing chromatographic chambers which are optimum for theoretical studies on the process of thin-layer chromatography.