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

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 μL rat plasma sample was deproteinized by 150 μL methanol, and then 100 μL of its supernatant was mixed with 100 μL water after centrifugation. Chromatographic separation was performed on a ZORBAX Eclipse Plus C₁₈ Rapid Resolution HD column (2.1 mm × 100 mm, 1.8 μm) 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 (r²) 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-∞, oral}, AUC_{0-24h, iv} and AUC_{0-∞, 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.