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OBJECTIVE: To establish an HPLC method for determing irinotecan (CPT-11) and its metabolite 7-ethyl-10 HCPT in rat liver microsome incubation system, and to optimize the incubation conditions. METHODS: CPT-11and SN-38 were determined by HPLC. Single factor design was used to optimize the incubation conditions. RESULTS: The linear range of CPT-11 and 7-ethyl-10 HCPT in rat liver microsome incubation system were 20-4000 ng · mL-1 and 2-400 ng · mL-1, respectively. The optimal incubation conditions were as follows: 10 μmol · L-1 CPT-11, 0.02 mg liver microsomes and incubation for 15 min. CONCLUSION: The HPLC method is accurate and suitable for the determination of CPT-11and 7-ethyl-10 HCPT in rat liver microsomes. The incubation condition can be applied in drug interaction studies of irinotecan.
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AIM: To establish the technique of precision-cut fibrotic liver slice (PCLS) and grope the optimal cultural conditions for researching the liver xenobiotic metabolism in vitro and the drug interaction. METHODS: Complex factors (higher fat diet, alcohol and CCl 4) were used to make the animal model of liver fibrosis. Fibrotic liver slices were prepared and cultivation system was established. Lactate dehydrogenase (LDH) leakage, glutathione S-transferase (GST) activity and 3[4,5-Dimethythiazole-2-yl]-2,5- diphenyltetrazolium bromide (MTT) reduction were chosen as indexes to assess the viability of the slice in different thickness, medium pH and cultural time. RESULTS: Rats were in earlier hepatic fibrosis after administration for 3 weeks. When the thickness of slices was 300 ?m and medium pH was 7.0, the LDH leakage, GST activity and MTT reduction could maintain on a steady level in 6 h. CONCLUSION: A 300 ?m of thickness, 7.0 of medium pH and 6 h of cultural time are the optimal slicing and culturing conditions for fibrotic liver slice.
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OBJECTIVE: To develop a method of HPLC/MS/MS to determine phenacetin and paracetamol in rat liver microsomal incubation system. METHODS: Samples were separated on XTerra MS C18 column, different ratios of methanol- 0.1% formic acid were used as the gradient eluent, and the flow rate was 0.2mL?min-1. The electrospray ion-quadrupole mass spectrometry and multiple reaction monitor were adopted to detect the concentration of phenacetin and paracetamol. RESULTS: The calibration curves were linear in the ranges of 45～9 000ng?mL-1(r=0.999 8)and 15.2～1 520ng?mL-1(r=0.999 6) for phenacetin and paracetamol respectively; The lowest limits of assay were 9ng?mL-1 and 10ng?mL-1.The average recoveries at three concentrations of phenacetin were (96.2?2.3)%～(98.3?2.4)% and those of paracetamol were (99.6?2.1)%～(100.2?2.6)%; RSD of the intra-day and inter-day were less than 5%. CONCLUSION: The method is rapid, sensitive and suitable for determination of phenacetin and paracetamol in rat liver microsomal incubation system.