Quantitative analysis of tenuifolin concentrations in rat plasma and tissue using LC-MS/MS: Application to pharmacokinetic and tissue distribution study

摘要

A sensitive, reliable and accurate reversed-phased liquid chromatography with tandem mass spectrometry (LC-MS/MS) in negative ion mode was developed and validated for the quantification of tenuifolin in rat plasma and tissue. A single step protein precipitation by methanol was used to prepare plasma and tissue homogenate samples. Tenuifolin and polydatin (internal standard, IS) were separated by HPLC using a C18 column and an isocratic mobile phase consisted of acetonitrile and water containing 0.05% formic acid (42:58, v/v) running at a flow rate of 0.2ml/min for 6min. Detection and quantification were performed using a mass spectrometer by the multiple reaction monitoring (MRM) in negative electrospray ionization mode. The transition monitored were m/z [M-H]- 679.4→455.4 for tenuifolin and m/z [M-H]- 389.0→227.2 for IS, respectively. Calibration curves were recovered over a concentration range of 0.5-1000ng/ml for plasma, heart, liver, lung and kidney, 0.5-200ng/ml for spleen, and 0.5-50ng/ml for brain, respectively. The lower limit of quantification was 0.5ng/ml for plasma and tissue homogenates. The inter-day precision (R.S.D.) was less than 12.9% and intra-day precision R.S.D. was less than 13.4%, while the inter-day accuracy (R.E.) was ranged from -7.20 to 6.87% and intra-day accuracy (R.E.) was ranged from -6.20 to 8.04% in plasma and tissue homogenates. This method was successfully applied to the pharmacokinetic and tissue distribution study of pure tenuifolin in rat. The pharmacokinetic study indicated that poor absorption into systemic circulation was observed after rat was administered orally tenuifolin, and the absolute bioavailability was low (0.83±0.28%). The results of tissue distribution showed the higher tenuifolin concentrations were found in liver, kidney and heart, and the small amount of drug was distributed quickly into the brain tissue at 5min after the intravenous injection of tenuifolin. The fact that tenuifolin could cross the blood-brain barrier provided the material basis for pharmacological action of the tenuifolin in the treatment of memory loss.