Development and validation of a sensitive LC-MS/MS method for the determination of 6-hydroxykynurenic acid in rat plasma and its application to pharmacokinetics study

摘要

A sensitive and specific bioanalytical method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of 6-hydroxykynurenic acid (6-HKA) in a small quantity of rat plasma has been developed and comprehensively validated. Tolbutamide (Tol) was used as the internal standard (IS). An aliquot of 50 mu L rat plasma sample was deproteinized by 150 mu L methanol, and then 100 mu L of its supernatant was mixed with 100 mu L water after centrifugation. Chromatographic separation was performed on a ZORBAX Eclipse Plus C-18 Rapid Resolution HD column (2.1 mm x 100 mm, 1.8 mu m) with a gradient mobile phase consisting of water containing 2 mM ammonium formate and methanol at a flow rate of 0.2 mL/min over a total run time of 5.0 min. The mass spectrometer was operated under multiple reactions monitoring (MRM) mode with the transitions m/z 206.1 -> 160.0 for 6-HKA and m/z 269.0 -> 170.0 for Tol. The linear range was 2.5-250 ng/mL with the square regression coefficient (r(2)) of 0.997. The lower limit of quantification (LLOQ) was 2.5 ng/mL (Relative Error, RE +1.6% and RSD 4.8%, n = 5). The intra- and inter-day precision and accuracy was < 13.3%. The mean recovery of 6-HKA in rat plasma was between the range of 99.3-103.4%. This method was successfully applied to study the pharmacokinetics of 6-HKA in rats after oral administration at a single dose of 20.0 mg/kg and after tail intravenous injection at a single dose of 2.0 mg/kg. Pharmacokinetic parameters bioavailability, C-max, (oral) , T-max, (oral), AUC(0-24h), (oral) AUC(0-infinity), (oral), AUCG(0-24h), (iv) and AUC(0-infinity), (iv) were 3.96%, 152.0 +/- 85.5 ng/mL, 0.4 +/- 0.1 h, 340.0 +/- 136.3 ng/mL*h, 369.5 +/- 135.0 ng/mL*h, 906.6 +/- 324.1 ng/mL*h and 932.9 +/- 336.5 ng/mL*h, respectively.