中空纤维离心超滤法快速分析血药浓度的研究

A study on rapid analysis of blood concentration by hollow fiber ultrafiltration

目的:建立用同一份血浆样品分析卡马西平(CBZ)游离浓度、总浓度及蛋白结合率的样品前处理方法。方法:加500μL血浆样品至中空纤维离心装置(HFCF-UF)中,4 000 r·min^(-1)离心15 min,取滤液直接注入HPLC测定游离浓度(c_f)后,再向该装置中加入200μL丙酮,混匀5 min,同法进行离心操作,取滤液分析得血浆剩余药物浓度(c_0),经校正,可得血浆药物总浓度(c_t),进而计算蛋白结合率(PPB)。结果:cf和ct在0.535~21.4μg·mL^(-1)的范围内线性关系良好(r^2>0.999),c_t的回收率为98.7%~101.3%,RSD小于1.2%。进行CBZ治疗的20名临床患者的PPB为10.1%~68.9%,其与健康受试者的PPB之间存在统计学差异。结论:该样品前处理方法简单,结果准确,为分析不同生理条件下不同患者的PPB及c_f变化提供有利的手段,进而指导临床开展个性化治疗。

OBJECTIVE To develop a novel method based on hollow fiber centrifugal ultrafiltration for analysis of free concentration,total concentration and protein binding in the same plasma by HPLC.METHODS A total of 500μL plasma sample was subjected to HFCF-UF.After centrifugation at 4 000 r·min^-1 for 15 min,20μL of the ultrafiltrate obtained was injected directly into the HPLC system for the determination of the free concentration.The plasma sample above was then spiked with 200μL of acetone as the mixture and vortexed for 15 min.The ultrafiltrate withdrawn from the same device was assayed for remaining drug concentration in plasma after centrifuge again.Finally the total concentration and protein binding were calculated from the corresponding equations.RESULTS The calibration curves of total and free concentration were linear（r^2〉0.999）over the range of 0.535–21.4μg·mL^-1 and recoveries were in the range of 98.7%–101.3% with RSD no more than 1.2%.The protein bindings of the 20 patients treated with carbamazepine were in the range of 10.1%–68.9%.CONCLUSION This method is simple,accurate and suitable for TDM of drugs with clinical individual variability and highly bound to protein.