Screening procedure for detection of stimulant laxatives and/or their metabolites in human urine using gas chromatography-mass spectrometry after enzymatic cleavage of conjugates and extractive methylation.

A gas chromatography-mass spectrometry (GC-MS)-based screening procedure was developed for the detection of stimulant laxatives and/or their metabolites in human urine after enzymatic cleavage of conjugates followed by extractive methylation. The part of the phase-transfer catalyst remaining in the organic phase was removed by solid-phase extraction on a diol phase. The compounds were separated by capillary GC and identified by computerized MS in the full scan mode. By use of mass chromatography with the ions m/z 305, 290, 335, 320, 365, 350, 311, 326, 271, and 346, the possible presence of stimulant laxatives and/or their metabolites could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra. This method allowed the detection of the diphenol laxatives bisacodyl, picosulfate, and phenolphthalein and of the anthraquinone laxatives contained in plant extracts and/or their metabolites in human urine samples. The overall recoveries of the stimulant laxatives and/or their metabolites ranged between 33% and 89% with a coefficient of variation of less than 15%, and the limits of detection ranged between 10 and 25 ng/mL (S/N 3) in the full scan mode. After ingestion of the lowest therapeutic dose of sodium picosulfate, its main metabolite, bisacodyl diphenol, was detectable in urine samples for 72 hours. After ingestion of the lowest therapeutic dose of a senna extract, the main metabolite of sennosides, rhein, was detectable in urine samples for 24 hours. This procedure is part of a systematic toxicological analysis procedure for acidic drugs and poisons with the modification of enzymatic cleavage of conjugates.

Phenolphthalein metabolite inhibits catechol-O-methyltransferase-mediated metabolism of catechol estrogens: a possible mechanism for carcinogenicity.

Phenolphthalein (PT), used in over-the-counter laxatives, has recently been identified as a multisite carcinogen in rodents, but the molecular species responsible for the carcinogenicity is not known. A catechol metabolite of PT, hydroxyphenolphthalein (PT-CAT), was recently identified and may be the molecular species responsible for at least part of the toxicity/carcinogenicity of PT. We hypothesize that PT-CAT inhibits the enzyme catechol-O-methyltransferase (COMT) and therefore potentiates genotoxicity by either PT-CAT itself or the endogenous catechol estrogens (CEs) in susceptible tissues. The present studies were conducted to determine the effects of PT treatment and PT-CAT itself on the COMT-mediated metabolism of 4- and 2-hydroxyestradiol both in vitro and in vivo. Female mice were treated with PT (50 mg/kg/d) for 21 days and then euthanized. PT-CAT concentration in urine reached plateau levels by 7 days of exposure. An O-methylated metabolite of PT-CAT was detected in feces. In vitro experiments demonstrated that PT treatment resulted in an increase in free CEs, which are normally cleared by COMT and a concurrent decrease in the capacity of hepatic catechol clearance by COMT. In vitro, PT-CAT was a substrate of COMT, with kinetic properties within the range measured with endogenous substrates. PT-CAT was an extremely potent mixed-type inhibitor of the O-methylation of the catechol estrogens, with 90-300 nM IC50s. The above data, when taken together, suggest that chronic administration of PT may enhance metabolic redox cycling of both PT-CAT and the catechol estrogens and this, in turn, may contribute to PT-induced tumorigenesis.