Quantification of a botanical negative marker without an identical standard: Ginkgotoxin in Ginkgo biloba

A toxic secondary metabolite, ginkgotoxin (4'-O-methylpyridoxine) has been detected in both ginkgo seeds and leaves. Moreover it has been shown that its content in tissues varies with the time of harvest during the season, thus making the quantification of this compound in commercial preparations a matter of public safety. A new strategy for the analysis of natural products using a combination of solid absorbent-free countercurrent separation to remove most of the chemical complexity of a natural product extract (chemical subtraction), followed by quantitative ¹H NMR of the concentrated analyte was employed to assay ginkgotoxin in Ginkgo biloba preparations. While commercial Ginkgo biloba seeds contained 59 ppm of ginkgotoxin, the compound was below the limit of detection (9 ppm) in a commercial leaf extract. Two ginkgotoxin related positional isomers were synthesized as authentic standards, but neither could be detected in seed or leaf extracts.