OBJECTIVE To establish an LC-MS/MS method to simultaneously determinate phellodendrine (PHE) and its glucuronide-Phellodendrine-11-O-β-D-glucuronide(M1), Phellodendrine-2, 11-di-O-β-D-glucuronide(M2) in rat tissue, and investigate the distribution characteristics of PHE, M1 and M2 in various tissue.
METHODS Normal SD rats were administrated with phellodenine at the dosage of 40 mg·kg-1, and the heart, liver, spleen, lung, kidney, brain, muscle, pancreas and small intestine were clipped at 0.117, 0.17, 0.5, 1, 2, 3, 4 hour after administration.Tissue homogenate samples were pretreated via protein precipitation with methanol, aconitine was used as the internal standard (IS). Chromatographic separation was achieved on a ZORBAX Eclipse XDB-C18 column (150 mm×2.1 mm, 3.5 μm) using a gradient elution of 0.1% formic acid in water and methanol at a flow rate of 0.3 mL·min-1.The column temperature was maintained at 35 ℃. Quantification was performed on an API 4000+ triple-quadrupole mass spectrometer equipped with a turbo electrospray ionization (ESI) source in positive ion multiple reaction monitoring (MRM) mode.
RESULTS Excellent linearity of PHE, M1 and M2 was observed in all analytes within the ranges of 1-800, 1-1 000, 5-1 000 ng·mL-1. Intra- and inter-day relative standard deviations (RSDs) were less than 11.07%, and accuracy were (91.58±1.84)%~(110.67±5.20)%. PHE, M1 and M2 were distributed in all the tissues. PHE tissue exposure was in the order of small intestine>liver>kidney>lung>pancreas>muscle>spleen>heart>brain. The manifestations of M1 and M2 were small intestine>kidney>liver>heart>lung>pancreas>muscle>spleen>brain.
CONCLUSION The established method has strong specificity, high accuracy and good stability, and is suitable for the study of tissue distribution of PHE, M1 and M2. The results of tissue distribution showed that PHE, M1, M2 were widely distributed in rats, mainly in small intestine, kidney and liver.
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