HPLC-UV测定人血清中伏立康唑药物浓度的方法学研究及其临床应用

Establishment of an HPLC-UV Method for Determination of Voriconzole in Human Serum

目的建立测定人血清中伏立康唑(voriconazole,VRCZ)浓度的高效液相色谱-紫外检测(HPLC-UV)方法。方法200μL血清样本采用400μL二氯甲烷萃取,采用HPLC测定,以硝西泮为内标,色谱柱为kromasil(4.6 mm×150 mm,5μm),流动相为水-乙腈(体积比60∶40),流速1.0 mL·min^(-1),柱温35℃,进样量20μL,检测波长为254 nm。结果VRCZ在0.2~16μg·mL^(-1)内线性关系良好(Y=0.073X+0.0019,r=0.9992),定量下限(LLOQ)为0.2μg·mL^(-1);日内、日间精密度为1.23%~5.51%,准确度为88.5%~102.36%,提取回收率为92.0%~106.5%,RSD<15%。血清样品在经历3次冻融(-20℃到室温)循环、冷藏放置2周和冷冻放置30 d等条件下均稳定,处理后样品在室温和自动进样器中(4℃)各放置24 h均稳定(RSD<15%),VRCZ储备液于-20℃和工作液于4℃放置10个月,内标储备液于-20℃放置1.5个月稳定(RSD<15%)。测定9个临床样本,范围在0.81~7.96μg·mL^(-1)之间,结论该法操作简便、灵敏、准确,适用于VRCZ血药浓度的测定。

OBJECTIVE To establish a HPLC-UV method for the determination of voriconzole in human serum.METHODS A Kromasil column(4.6 mm×150 mm,5μm)was used,and the mobile phase consisted of H2O-acetonitrile(60∶40).The flow rate was 1.0 mL·min^(-1),the detection wavelength was set at 254 nm,and column temperature was maintainted at 35℃.RESULTS Voriconzole concentration had good linearity(r=0.9991)in the range of 0.2-16μg·mL^(-1),the regression equation was Y=0.073X+0.0019,r=0.9992.The limit of quantitation was 0.2μg·mL^(-1)and the intra-day and inter-day precision is 1.23%-5.51%.The accuracy was 88.5%-102.36%,the extraction recovery was 92.0%-106.5%,and the RSD was<15%.The serum samples were stable after 3 freeze-thaw cycles(-20℃to room temperature),being stored at 4℃for 2 weeks and frozen for 30 days.The processed samples were stable at room temperature and autosampler(4℃)for 24 h(RSD<15%).The voriconazole stock solution and working solution were stable at-20℃and 4℃for 10 months,and the internal standard stock solution was stable at-20℃for 1.5 months(RSD<15%).Nine samples were assayed,with the concentration of 0.81-7.96μg·mL^(-1).CONCLUSION This method is simple,sensitive and accurate,and is suitable for the determination of the blood concentration of voriconzole.