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目的:建立人全血中西罗莫司的HPLC测定方法。初步研究在肾移植术后患者体内西罗莫司的药动学。方法:色谱柱为Supelcosil LC-DB C18柱(4.6mm×250mm,5μm),流动相为81.5%的甲醇水溶液,流速1mL.min-1,柱温70℃;检测波长277nm。取人全血1mL,以FK-506为内标,用叔丁基甲醚为提取液,震荡,离心后取上清液常温氮气下吹干,残留物用100μL流动相复溶后进样分析。2例肾移植术后患者,在初次应用口服液(6mg)后,分别在给药前,给药后0.25,0.5,1,1.5,2,3,6,10,18,24h时间点采集静脉血2mL。用已建好的HPLC方法测定样品浓度,得到药-时曲线。结果:本方法在2.5～100.0μg.L-1范围内线性良好,r>0.99。低、中、高浓度质量控制样品的RSD在4.4%～7.3%之间,批间RSD在7.5%～9.5%之间,准确度在93.8%～101.7%之间,定量下限为2.5μg.L-1。结论:本方法灵敏度高,重复性好,可满足西罗莫司的血药浓度监测及人体内药动学研究要求。西罗莫司在患者间的AUC变异较大,谷浓度可反映两者之间的差别。提示肾移植术后应用西罗莫司的患者定期进行谷浓度监测非常必要。
Objective: To establish a method for the determination of sirolimus in human whole blood by HPLC. Preliminary study of the pharmacokinetics of sirolimus in patients after renal transplantation. Methods: The column was Supelcosil LC-DB C18 column (4.6 mm × 250 mm, 5 μm) with a mobile phase of 81.5% aqueous methanol at a flow rate of 1 mL · min-1 and a column temperature of 70 ° C. The detection wavelength was 277 nm. Fetal blood was taken from 1 mL of whole human blood. FK-506 was used as an internal standard. The mixture was triturated with tert-butyl methyl ether (TMB). After centrifugation, the supernatant was air-dried at room temperature under nitrogen and the residue was reconstituted with 100 μL of the mobile phase. Two cases of renal transplantation patients, after the first application of oral solution (6mg), respectively, before administration, after administration of 0.25,0.5,1,1.5,2,3,6,10,18,24 h time point collection of venous Blood 2mL. With the established HPLC method to determine the concentration of the sample, the drug-time curve is obtained. Results: The method was linear in the range of 2.5 ~ 100.0μg.L-1 with r> 0.99. The RSD of low, middle and high quality control samples ranged from 4.4% to 7.3%, the inter-assay RSD was between 7.5% and 9.5%, the accuracy was between 93.8% and 101.7%, and the lower limit of quantitation was 2.5μg.L -1. Conclusion: The method has high sensitivity and good repeatability, which can meet the need of sirolimus monitoring of plasma concentration and pharmacokinetics in human body. The variation of AUC between patients with sirolimus was large, and the trough concentration could reflect the difference between the two. Prompt application of sirolimus after renal transplantation in patients with regular monitoring of trough concentrations is necessary.