Simultaneous Determination of Flavonols and Terpene Lactones in Beagle Dog Plasma by Ultra-Performance Liquid Chromatography-Tandem - Mass Spectrometry: 1. Method Development

Purpose: To develop an ultra-performance liquid chromatography-tandem mass spectrometry (UPLCMS/ MS) method for the simultaneous determination of 7 major components of Ginkgo leaf (kaempferol, quercetin, isorhamnetin, ginkgolides A, ginkgolides B, ginkgolides C and bilobalide) in dog plasma.
Methods: Beagle dog plasma samples were spiked with internal standard (domperidone), acidified with HCl and extracted twice by liquid-liquid extraction using ethyl acetate. Chromatographic separation was achieved on an Acquity UPLC BEH C18 column (100 x 2.1 mm, 1.7 μm) by gradient elution with a run time of 4.0 min. The specificity, linearity, precision, recovery, matrix effect and stability of the method were determined.
Results: The method showed high selectivity of the flavonols and terpene lactones in plasma samples. The concentration of the 7 target compounds showed good linear relationship with the peak area ratios of each analyte to internal standard. Lower limit of quantification (LLOQ) was 1.232, 0.240, 0.200, 1.330, 0.960, 0.696, 0.470 ng•mL^-1 for kaempferol, quercetin, isorhamnetin, bilobalide, ginkgolides A, ginkgolides B and ginkgolides C, respectively. Recovery of all QC samples ranged from 77.68 to 105.07 %. Matrix effect derived from QC samples was in the range of 85.09 – 113.14 %. The stability of the analytes, calculated as RSD at three concentrations, was < 15 %.
Conclusion: The developed method is simple, rapid and sensitive and can be applied to the determination of kaempferol, quercetin, isorhamnetin, ginkgolides A, ginkgolides B, ginkgolides C and bilobalide in dog plasma.

Keywords
Ultra-performance liquid chromatography-tandem mass spectrometry; Ginkgo biloba; Beagle dog plasma; Kaempferol; Quercetin; Isorhamnetin; Ginkgolides A; Ginkgolides B; Ginkgolides C; Bilobalide