The ex vivo instability of bilobalide containing three γ-lactone rings has been paid less attention by researchers who developed bioanalytical methods for bilobalide. In the present study, a sensitive LC-MS/MS method for the determination of bilobalide in rat plasma was developed with special consideration of ex vivo bilobalide stability. Several important factors affecting the stability of bilobalide in sampling and handling procedures were investigated. To prevent the ex vivo degradation of bilobalide, EDTA instead of heparin was used as an anticoagulant as well as an esterase inhibitor for blood collection and the separation of plasma was performed at 4 °C. 20 μL of plasma sample was acidified with 0.1 M hydrochloric acid, and then extracted with ethyl ether-methylene chloride (2:1, v/v). The extract was chromatographed on a Thermo Hypersil GOLD (100 mm × 2.1 mm, 5 μm) column using acetonitrile-10mM ammonium acetate-formic acid (90:10:0.4, v/v/v) as the mobile phase. The analyte and the internal standard (ginkgolide B) were detected by selected reaction monitoring mode via negative electrospray ionization. The method was fully validated and proved to be linear over a concentration range of 5.0-5000 ng/mL. The intra- and inter-day precisions were less than 5.2% and the accuracy was within 92.5-101%. The extraction recoveries ranged from 80.7% to 86.7%. The proposed method was successfully applied to a preclinical pharmacokinetic study of bilobalide in rats after intragastric administration of a single dose of bilobalide at 7, 14 and 28 mg/kg.
Keywords: Bilobalide; Ex vivo stability; LC–MS/MS; Pharmacokinetics; Rat plasma.
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