Screening botanical extracts for quinoid metabolites

Botanical dietary supplements represent a significant share of the growing market for alternative medicine in the USA, where current regulations do no t require assessment of their safety. To help ensure the safety of such pro ducts, an in vitro assay using pulsed ultrafiltration and LC-MS-MS has been developed to screen botanical extracts for the formation of electrophilic and potentially toxic quinoid species upon bioactivation by hepatic cytochr omes P450. Rat liver microsomes were trapped in a flow-through chamber by a n ultrafiltration membrane, and samples containing botanical extracts, GSH and NADP(H), were flow-injected into the chamber. Botanical compounds that were metabolized to reactive intermediates formed stable GSH adducts mimick ing a common in vivo detoxification pathway. If present in the ultrafiltrat e, GSH conjugates were detected using LC-MS-MS with precursor ion scanning followed by additional characterization using product ion scanning and comp arison to standard compounds. As expected, no GSH adducts of reactive metab olites were found in extracts of Trifolium pratense L. (red clover), which are under investigation as botanical dietary supplements for the management of menopause. However, extracts of Sassafras albidum (Nutt.) Nees (sassafr as), Symphytum officinale L. (comfrey), and Rosmarinus officinalis L. (rose mary), all of which are known to contain compounds that are either carcinog enic or toxic to mammals, produced GSH adducts during this screening assay. Several compounds that formed GSH conjugates including novel metabolites o f rosmarinic acid were identified using database searching and additional L C-MS-MS studies. This assay should be useful as a preliminary toxicity scre en during the development of botanical dietary supplements. A positive test suggests that additional toxicological studies are warranted before human consumption of a botanical product.