1. A gas-liquid-chromatographic procedure is described which permits separation and identification on the same chromatogram of a wide range of substances occurring in urine or tissue extracts. The method uses hydrogen flame ionization, which detects organic compounds whether free or conjugated with no requirement for specific reactive groups. 2. For chromatography, carboxyl groups are quantitatively converted into methyl esters or trimethylsilyl esters. Phenolic, alcoholic and potential enolic groups are converted into trimethylsilyl ethers. Separations are carried out on a 6ft. column of either 10% F-60 (a polysiloxane) or 1% F-60, temperature programming at 2 degrees /min. being used over such part of the temperature range 30 degrees -260 degrees as is required. Propionyl derivatives of hydroxy compounds can also be used, but only on a non-quantitative basis. Derivatives and columns have been selected for optimum range of usefulness when large numbers of samples are examined by using automated gas chromatography. 3. The method is applicable to: fatty acids above butyric acid; di- and tri-carboxylic acids; hydroxy acids and keto acids; polyhydroxy and alicyclic compounds such as glycerol, inositol, quinic acid, shikimic acid, ascorbic acid and sugar alcohols; aromatic hydroxy and acidic compounds, both benzenoid and indolic; sesquiterpenes; steroids; glycine conjugates; mercapturic acids; glucuronides. It is not satisfactory for sulphate conjugates, iminazoles or polypeptides. 4. Methylene units provide an accurate and reproducible parameter for characterizing peak position. Methylene unit values are reported for a large variety of substances occurring in, or related to those occurring in, urine and tissue extracts. 5. The nature of derivatives was confirmed by combining gas chromatography with mass spectrometry. Combined gas chromatography-mass spectrometry gives a diagnostic tool of great power in the evaluation of metabolic patterns, and various uses are discussed.

Journal Article↗

PubMedDate not supplied

Comparative metabolism of four allylic barbiturates and hexobarbital by the rat and guinea pig.

A method for the extraction and identification of urinary metabolites of allylic barbiturates by gas chromatography and mass spectrometry is described. The metabolites from rat and guinea pig urine were extracted and separated into two fractions (an acidic and a nonacidic fraction) by chromatography on DEAE-Sephadex before converted into suitable derivatives for gas phase analysis. N,N'-dimethyl derivatives were used except in cases where metabolic N-demethylation was possible, in which case N-ethylation yielded more information. Hydroxyl, keto, and epoxy groups were converted into trimethylsilyl (TMS), alkyloxime, and chloro-TMS derivatives, respectively. This procedure was used to identify metabolites present in urine at concentrations as low as 0.1 microgram/ml. The allyl sidechains of secobarbital, alphenal, allobarbital, and aprobarbital were metabolized to epoxides, diols, and, in the case of secobarbital, to a ketol. Other sidechains were usually hydroxylated. Secobarbital was metabolized to compounds containing hydroxyl groups in both chains. Hexobarbital was metabolized by allylic hydroxylation, and no evidence of the epoxide-diol pathway was observed. The significance of the detection of epoxides of the four allylic barbiturates is discussed.

Animals↗

PubMedDate not supplied

Metabolism of methaqualone by the epoxide-diol pathway in man and the rat.

The metabolism of methaqualone (2-methyl-3-o-tolyl-4(3H)-quinazolinone) has been studied in man and the rat using gas phase analytical methods. Seven new metabolites formed by the epoxide-diol pathway were detected in human urine after methaqualone administration. Five of these compounds were characterized as dihydrodiols and two as hydroxydihydrodiols. The seven dihydrodiol metabolites were present in the nonhydrolyzed fraction isolated from urine. After intraperitoneal administration of methaqualone to the rat (40 mg/kg) the major monohydroxyl metabolites of the drug in hydrolyzed urine were identified as 2-methyl-3-(2'-hydroxymethylphenyl)-4(3H)-quinazolinone (I) and 2-hydroxymethyl-3-o-tolyl-4(3H)-quinazolinone (II). Two dihydroxyl metabolites were also present, but only trace amounts of a dihydrodiol were detected. The major monohydroxyl metabolites of methaqualone detected in human urine after enzymic hydrolysis were I, II, 2-methyl-3-(3'-hydroxy-2'-methylphenyl)-4(3H)-quinazolinone (III), and 2-methyl-3-(4'-hydroxy-2'-methylphenyl)-4(3H)-quinazolinone (IV). Hydroxylation of the tolyl moiety of methaqualone probably occurs by way of an epoxide intermediate. The phenols, III and IV, may be formed from an epoxide or from the dihydrodiol(s) by enzymic or nonenzymic reactions. The results obtained suggest that epoxidation of methaqualone represents a major pathway of metabolism in the human.

Animals↗

PubMedDate not supplied

Formation of methylthio metabolites of indene in the guinea pig and the rat.

The formation of methylthio metabolites of epoxides has been shown to be a significant route of metabolism in some species. Several aspects of this metabolic conversion for indene were examined. Two isomers of hydroxy(methylthio)indane were found in the urine of guinea pigs administered indene (14.3 and 100 mg/kg, ip). The major isomer, 2-hydroxy-1-methylthioindane (I) was present as 6-9% of the administered dose after 24 hr, while lower amounts (0-0.6%) of a minor isomer (II) were observed. A significant amount of isomer I was found as a urinary metabolite of indene oxide (14% of 12.5 mg/kg, ip). To further elucidate the route of formation of I, the glutathione (I-GLU) and mercapturic acid (I-MER) conjugates of indene oxide were synthesized and administered to the guinea pig. The methylthio metabolite I was present as a significant urinary metabolite of both conjugates of indene oxide, comprising 9.6% and 5.7% of the dose of I-GLU (5 mg, ip) and I-MER (4 mg, ip), respectively. These results show that the formation of a hydroxy(methylthio)indane is a significant route of metabolism for indene and indene oxide in the guinea pig, and that this metabolite arises via further metabolism of conjugates in the glutathione pathway. In the rat, isomer I is a minor metabolite. Mechanistic aspects of the formation of these thioether metabolites are discussed.

Acetylcysteine↗