Thin layer chromatography metabolites

A thin-layer chromatography assay was developed for ffie simultaneous determination of the three major hydroxylated metabolites of antipyrine 409,410, and 411 in urine of humans and other animals (82JPP168) (Scheme 95). 

The active drug and metabolites can be detected from the urine by thin-layer chromatography, gas-liquid chromatography, or gas chromatography-mass spectrometry. However, assays are available only at specialized centers. Treatment of acute intoxication with mescaline is virtually identical to the treatment outlined for LSD intoxication. DOM-induced vasospasm responds well to intra-arterial tolazohne or sodium nitroprusside. Major life-threatening complications of hallucinogenic amphetamine derivatives include hyperthermia, hypertension, convulsions, cardiovascular collapse, and self-inflicted trauma. 

Table 1 provides the urinalysis test results for the 4,847 arrestees, While PCP was tested for by an EMIT test only, cocaine, opiates and methadone were tested for by both EMIT and thin layer chromatography (TLC). (The EMIT test for opiates is not specific to morphine, the metabolite of heroin, and can detect the recent use of a variety of opiates. A specimen positive for opiates is most likely to indicate the use of heroin in this population, however.) Our analyses will use only the results from the EMIT tests, because we have learned that the TLC general drug screen is less sensitive for detecting recent use of these illicit street drugs (Wish et al. 1983 Wish et al. 1984). 

Clark et al. [53] subjected primaquine to metabolic studies using microorganisms. A total of 77 microorganisms were evaluated for their ability to metabolize primaquine, of these, 23 were found to convert primaquine to one or more metabolites (thin-layer chromatography analysis). Preparative scale fermentation of primaquine with four different microorganisms resulted in the isolation of two metabolites, identified as 8-(3-carboxy-l-methylpropylamino)-6-methoxyquinoline and 8-(4-acetamido-l-methylbutylamino)-6-methoxyquinoline. The structures of the metabolites were proposed, based primarily on a comparison of the 13C NMR spectra of the acetamido metabolite and the methyl ester of the carboxy metabolite with that of primaquine. The structures of both metabolites were confirmed by direct comparison with authentic samples. 

Saltar and Paasivirta [155] have described a method for the analysis in soils of MCPA (4-chloro-2-methyl phenoxy acetic acid) and two of its main metabolites, 4-chloro-o-cresol and 6-chloromethyl catechol by gas chromatography of their pentafluorobenzyl derivatives (Fig. 9.12). After derivitization of the residue extract, a clean-up procedure was applied. The best recoveries of compounds from soil were obtained when the extraction was performed by shaking with ether-acetone-heptane-hexane (2 1 1 1) from acidified soil and when the clean-up was done by thin layer chromatography (Table 9.17). Detection limits were in the range 20-25ng absolute. 

In recent years, using both thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC), it has been possible to separate and identify a variety of conjugated and non-conjugated metabolites of aromatic hydrocarbons in marine organisms (8,10,12,15,42,43,44). 

Although the metabolism of several phthalate esters has been studied in vitro, essentially all of the in vivo studies have involved DEHP. A summary of these experiments which involved exposure offish to aqueous - C-DEHP is presented in Table IV (11,12). Tissue C was isolated and separated into parent and the various metabolites by preparative thin layer chromatography on silica gel. Metabolites were hydrolyzed where appropriate and identified by gas chromatography-mass spectroscopy. In whole catfish, whole fathead minnow and trout muscle, the major metabolite was the monoester while in trout bile the major metabolite was the monoester glucuronide. The fact that in all cases the major metabolite was monoester or monoester glucuronide despite the differences in species, exposure level and duration, etc. represented by these data, suggests that hydrolysis of DEHP to monoester is important in the biotransformation of DEHP by fish. 

The nature of the radioactivity in the water, soil and fish from the carbon-14 DDT experiment was examined by thin-layer chromatography as shown in Figure 5. The radioactivity in the water was very polar in nature and did not migrate appreciably from the origin. About 78% of the radioactivity in the soil was extracted with methanol. The major metabolite in the extractable fraction was DDD which represented 33% of the total radioactivity. The reductive dechlorination of DDT to DDD is a known pathway under anaerobic conditions and has been shown to be due to microbial metabolism (5). Since carbon-14 DDT was incor-... 

The soluble metabolites excreted from animals dosed by injection were collected on AmberliteR XAD-4 resin, the resin eluted sequentially with diethyl ether, acetone, and methanol, and the solutes separated by thin-layer chromatography on silica gel and quantitated by liquid scintillation counting. 

Salicylic acid and Its metabolite were separated by two methods. The first was thin layer chromatography on cellulose with BAW solvent as for the In vivo metabolism studies. A quicker separation was achieved with a polyamide column. The entire 400 pL from an individual assay was placed on top of a 0.8 x 2.0 cm column packed with Polyamide-6 (Accurate Chemical and Scientific Corp.). The salicylic acid metabolite was eluted with 6 mL water but salicylic acid was retained. 3a70B scintillation fluid (Research Product International Corp.) was used to determine the radioactive content of the entire 6 mL of eluant. Separation of salicylic acid and its metabolite by polyamide column chromatography was verified by thin layer chromatography. 

The ability of an esterase or a -glucosidase to hydrolyze the in vitro generated metabolite was tested. An assay mixture that had been incubated for 22 h (ca. 50% conversion of salicylic acid) was incubated with either 10 units of hog-liver esterase (E.C. 3.1.1.1, Sigma Chemical Co.) at pH 8.0 or 20 units of -glucosidase (E.C. 3.2.1.21, Sigma) at pH 5.0 for 1 h at 37 °C. Salicylic acid and the metabolite were separated by thin layer chromatography with BAW and quantified by liquid scintillation chromatography. 

The radiolabelled compounds were separated by thin layer chromatography on cellulose using 6% acetic acid. of salicylic acid was 0.63 and of the metabolite was 0.76. 

Special attention was given to the characterization of the radioactive metabolites found in the strawberry fruits where no parent compound was detectable. Unidentified, very polar metabolites accounted for 83% of the material while 3% was identified as thiazolidine-2-thione-4-carboxylic acid by two-dimensional thin-layer chromatography. Treatment with diazomethane allowed for GC-MS confirmation of the latter as the methylated derivatives of the thiazolidine. 

Cyclosporin A is slowly but extensively metabolized. The biotransformation pathway and the pattern of the generated metabolites are similar in humans and animals. Approximately 17 single metabolites have been detected so far, all of which are present in considerably lower plasma concentration than cyclosporin A itself [46]. Eleven ether-extractable compounds have been isolated from urine of dog and man and from rat bile and faeces using preparative HPLC and thin-layer chromatography [43]. Structural assignments for these... 

Chloro-2-methyl-phenoxyacetic acid and metabolites Ethenacetone heptane hexane (2 1 1 1) Derivatisation to pentafluorobenzyl derivatised after clean-up by thin layer chromatography then gas chromatography. Limit of detection 20 - 25 ng absolute [402]... 

A qualitative thin-layer chromatography method has been described by Kleef et al. [27] for the detection of rocuronium bromide and its metabolites in biological samples. This method was developed to confirm the identity of rocuronium bromide and its metabolites prior to their determination using HPLC coupled with fluorescence detection. In this method, the dried residue from the extraction process was dissolved in 0.05 ml of 0.01 M HC1. The stationary phase used was silicagel plates, that were developed in a mobile phase consisting of 2% solution of Nal in 2-propanol. The elution process was run for 4 h, and after the elution... 

Lee et al. (1975) also qualitatively studied urinary metabolites of32P-labeled white phosphorus in rats. They used thin-layer chromatography (TLC) at 4 and 24 hours after a single oral dose to show that radioactive urinary metabolites consisted of two classes of compounds. One of the compounds corresponded to inorganic phosphate, the other compound was less polar and suggested an organic phosphate, although the composition of this class of metabolites was not determined. TLC analysis of liver extract also showed two classes of compounds with similar properties. 

Nishikaze, O., Separation and quantitative analysis of adrenocortical hormone and its metabolite (C21) by thin-layer chromatography, Kagaku No Ryoiki Zokan, 64, 37, 1964. 

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