Rapid analysis of 4-O-methylpyridoxine in the serum of patients with Ginkgo biloba seed poisoning by ion-pair high-performance liquid chromatography.

We have established a new method of HPLC analysis for the rapid separation from human serum and the quantification of 4-O-methylpyridoxine (MPN), which is contained in Ginkgo biloba seeds, and which, when consumed in large amounts, causes vomiting and convulsions. As a result of using IPCC-MS3 (GL Science, Tokyo, Japan), an ion-pair reagent, in the mobile phase, we succeeded in separating MPN in the deproteinized serum sample which was introduced directly onto the reverse-phase HPLC column. For the calibration curve of MPN standard solution, prepared with fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 400 nm, a good linear relationship was obtained within the HPLC injection range of 10 ng-10 pg (in terms of the injected sample concentration, range: 1.0 microg/ml-1 ng/ml), allowing the detection of minute amounts, with the limit of detection (concentration of injected sample: 500 pg/ml) being 5 pg. In addition, when MPN solution was added to human reference serum to give a concentration of 0.002 microg/ml, the mean recovery rate was 92.5%, with RSD=7.09% (n=5). The time required for one analysis using this method is approximately 30 min, and thus it offers the advantages of greater speed and superior analytical sensitivity over the conventional methods, which require solid-phase extraction. We employed our new method to determine both the serum levels of MPN in 5 patients with Ginkgo biloba seed poisoning and the levels of free-form MPN in such seeds obtained in 8 regions of Japan.