E.C. Horning

Conversion of steroids to trimethylsilyl derivatives for gas phase analytical studies: reactions of silylating reagents.

Abstract Methods for the conversion of human steroids to trimethylsilyl derivatives for gas-phase analytical studies have been investigated. Three useful conditions have been defined. These are (a) BSA at room temperature, (b) BSA:TMCS or BSTFA:TMCS at room temperature or at 60°C, and (c) TSIM:BSA:TMCS at 60–80°. Steroid hydroxyl groups may be classified as unhindered, moderately hindered, or highly hindered, on the basis of the conditions needed for derivative formation. Quantitative conversions to single products were observed in the instances that were studied. Ketone groups may yield enol TMSi ethers under catalyzed silylation conditions. This reaction may be avoided by using methoxime derivatives.

Formation and gas-liquid chromatographic behavior of isometric steroid ketone methoxime derivatives

Abstract The reaction of methoxylamine with steroid ketones to yield methoxime (MO) derivatives may yield two isomers. The isomers are presumably geometric isomers of the syn anti type by analogy with the well-known reaction of oxime formation. In some instances isomers are not found; the course of the reaction apparently depends upon steric interrelationships. Isomer separation occurred both in thin-layer and gas-liquid chromatographic systems. Isomer formation is generally not a desirable effect in quantitative analytical GLC procedures. However, the major C19 and C21 human urinary steroids do not form isomeric MO and MO-TMSi derivatives. The 11-keto group does not react to form a MO derivative, and 17- and 20-keto groups lead to only one MO isomer. Compounds with a 3-keto-Δ4, 3-keto-5α-H, and 16-keto structure gave isomers. The development of GLC analytical methods for compounds containing these structures (cortisone, cortisol, testosterone, progesterone, for example) will require consideration of this effect if MO or MO-TMSi derivatives are employed.

The characterization by gas-liquid chromatography of ethyl β-D-glucosiduronic acid as a metabolite of ethanol in rat and man

Abstract Ethyl β-D-glucosiduronic acid was isolated from the urine of male and female rats, and from the urine of an adult male human alcoholic. The isolation and structural studies were carried out by apparently general gas phase procedures for the study of glucosiduronic acids. The methods involve isolation from urine by extraction and ion exchange chromatography, and identification by gas liquid chromatography and by the combined technique of GLC-mass spectrometry.

Gas chromatographic behavior of long chain alcohols and their derivatives

Abstract The gas chromatographic behavior of a number of long chain and alicyclic alcohols and aliphatic diols and their derivatives has been studied with three stationary phases. It is evident that a wide choice of derivatives possessing excellent gas chromatographic properties is available; these may be used in identification and structure—retention time correlation work. The relative response of the hydrogen flame ionization detector for a number of long chain compounds was determined.

Gas-liquid chromatographic study and estimation of several urinary aromatic acids.

Abstract A procedure for the separation of urinary acids as trimethylsilyl ethermethyl ester and methyl ester derivatives by temperature-programed gas chromatography has been developed. The liquid phase is 10% F-60. The excretion level of individual acids may be measured quantitatively after determination of response factors for the derivatives. A solvent extraction isolation procedure is suitable for aromatic compounds; nonaromatic compounds are extracted reproducibly but not quantitatively. Since the present method leads to derivatives of glucuronides and other nonaromatic acids in addition to aromatic acids, it provides a way of observing a pattern or profile of metabolic relationships that covers a wide range of compounds. Alterations in these patterns may result from environmental change, drug administration, or pathologic circumstances, and in these instances it may be more important to study over-all changes than to estimate several specific compounds. The determination of selected compounds and an analysis of pattern changes (without determination of response factors) can be made from the same chart, so that both qualitative and quantitative observations are obtained for each experiment. The method is being used in studies of drug effects, and for a study of developmental biochemistry for the neonate. The method may also be useful in studies of comparative biochemistry.

SEPARATION AND IDENTIFICATION OF TRYPTAMINE-RELATED INDOLE BASES BY GAS CHROMATOGRAPHIC METHODS.

Abstract The possibility of separating and identifying a number of indole bases related to tryptamine by gas chromatographic techniques has been investigated. Methods were found for the separation of positional isomers of phenolic amines and related compounds in this series. The major indole base of epena, a South American snuff with reported hallucinogenic properties, was found to be 5-methoxy-N,N-dimethyltryptamine.

The Glc Separation of Indole Amines and Indole Alcohols as Heptafluorobutyryl Derivatives

Abstract Indole amines and indole alcohols were converted to heptafluoro-butyryl (HFB) derivatives by an acyl transfer reaction with heptafluoro-butyrylimidazole. The indole NH group as well as all amino and hydroxyl groups were acylated. The HFB derivatives have excellent gas chroma-tographic properties and can be used with either hydrogen flame or electron capture detection systems. Mass spectra of the HFB derivatives of biologic N, N-dialkyl indole amines are very characteristic; these compounds can be identified easily by GLC-MS methods.

Milligram-scale preparative gas chromatography of steroids and alkaloids

Abstract It has been found that milligram-scale preparative chromatography may be carried out for steroid and alkaloid mixtures through the use of thin-film columns with analytical-type packings. The use of temperature programing is helpful. Two examples of experimental separations are given.

Thin layer and column chromatographic group separations of steroids as trimethylsilyl ethers. Isolation for GLC analysis of pregnanediol and estriol in pregnancy urine

Abstract Trimethylsilyl ethers of steroids may be separated by thin-layer and column chromatographic methods. They are suitable derivatives for the group separation of hydroxysteroids from hydroxyketosteroids prior to gas chromatographic analysis. Group separations of two model steroid mixtures are described. An application of the method has been made to the simultaneous analytical separation of estriol and pregnanediol in human pregnancy urine; the determination of these two steroids is often of interest in pregnancy studies.

A comparison of the behavior of the hydrocarbons used as reference standards in the determination of “steroid numbers” and “methylene units” selective retention of steroid hydrocarbons by polar stationary phases

Abstract During an investigation designed to compare the gas-liquid chromatographic behavior of two sets of compounds used as reference standards in structure-retention behavior studies it was observed that with polar stationary phases selective retention exists for steroid hydrocarbons when compared to straight chain hydrocarbons of similar carbon content. With nonpolar stationary phases the steroids are eluted relative to the paraffins on the basis of molecular weight. Such behavior differences can be used to classify the polarity or selectivity of stationary phases.