Determination of 23-hydroxybetulinic acid in mouse plasma by liquid chromatogram-mass spectrometry

OBJECTIVE: To develop a sensitive and specific LC-MS method for the determination of 23-hydroxybetulinic acid in mouse plasma. METHODS: 23-Hydroxybetulinic acid and the internal standard limonin were extracted by liquid-liquid extraction. The organic layer was blowed by N₂ to dryness at 40°C. The residue was reconstituted in methanol. The LC-MS method was performed on an Intersil C₈ column (2.1 mm x 250 mm, 3.5 μm). The mobile phase consisted of 70% acetonitrile and 0.05% triethylamine. The flow rate was at 0.2 mL· min^-1. The temperature of column was 30°C. Negative ion electrospray ionization(ESI) was used to form deprotonated molecules at m/z 471 of 23-hydroxybetulinic acid and 469 of the internal standard limonin. RESULTS: The linear calibration curve was observed in the concentration range of 10-1 000 μg·L^-1 (r = 0.999 8). The limit of quantitation of 23-hydroxybetulinic acid was 10 μg·L ^-1. The relative recoveries were more than 88%. The inter and intra-day precisions(RSD) were below 8.0%. 23-Hydroxybetulinic acid was stable in plasma samples at -20°C for at least 3 weeks. CONCLUSION: The method is proved to be convenient, sensitive, rapid and suitable for pharmacokinetic study.