A mass-spectrometric assay for electrophilic metabolites of natural-product mixtures

The popularity of botanical dietary supplements and other medicinal natural product mixtures in the United States has created a demand for methods to rapidly determine whether such products are safe for human consumption. Botanical natural products that cause toxicity often exert their effects via the action of unstable intermediates that are formed by drug-metabolizing enzymes such as cytochromes P450. As an in vitro test to determine whether the consumption of a natural product mixture might lead to the formation of such reactive species, an ultrafiltration LC-MS-MS assay was developed to screen natural product preparations for the formation of electrophilic mammalian metabolites including quinones and epoxides. Several electrophilic metabolites of botanical products were identified using this screening assay, including o-quinones from Rosmarinus officinalis (rosemary) and Cimicifuga racemosa (black cohosh), and quinoid metabolites of methysticin and 7,8-dihydromethysticin from Piper methysticum (kava). When possible, urine samples from human volunteers who consumed these botanicals were analyzed using LC-MSn for the detection of mercapturic acids and other sulfur-containing conjugates that might provide evidence for the formation of reactive metabolites in humans. In an alternative approach, methysticin was administered via IP injection to CD1 mice, and bile samples from these mice were analyzed using LC-MS for the detection of GSH conjugates that were formed following the bioactivation of methysticin. This analysis was used to help characterize a bioactivation pathway for methysticin that might explain multiple cases of kava-related hepatotoxicity in humans. Finally, the hormone replacement product Prempro™, which contains a mixture of conjugated estrogens and other metabolites derived from horse urine, was screened using this assay. Several GSH conjugates were detected corresponding to low-molecular-weight phenolics including p-cresol. Prempro™ tablets were analyzed subsequently for p-cresol content using LC-MS/MS, and the results indicated the presence of approximately 340 μg of p-cresol sulfate per tablet. In its unconjugated form, p-cresol is known to cause acute toxicity in a variety of target organs. Hence, p-cresol sulfate is a potential source of toxicities that are associated with long-term usage of estrogen/progestin supplements as demonstrated in clinical trials.