人血浆中伊立替康及其活性代谢物7-乙基-10羟基喜树碱浓度高效液相色谱-荧光测定方法的建立

目的:建立人血浆中伊立替康(CPT-11)及其代谢物7-乙基-10羟基喜树碱(SN-38)的浓度测定方法并进行方法学考证。方法:用Luna 5u CN100A(4.6 mm×150 mm,5μm)色谱柱,乙腈与醋酸铵缓冲溶液(50 mmol.L-1,pH4)为流动相梯度洗脱,CPT-11的检测波长为Ex/Em=368 nm/432 nm,SN-38的检测波长为Ex/Em=368 nm/535 nm。结果:CPT-11保留时间为(9.3±0.3)min,SN-38保留时间为(4.8±0.3)min。空白样品在CPT-11、SN-38及内标喜树碱出峰位置均无干扰。CPT-11在46.9～6 000.0nmol.L-1的范围内线性良好,SN-38在2.0～250.0nmol.L-1的范围内线性良好,r值均为0.998。低浓度点RSD均在20%内,其余浓度点的RSD均在15%内,准确度均在85%～115%之间。血浆样品长期冻存稳定性良好,反复冻融3次及提取后室温放置24 h条件下,样品浓度均无显著变化。结论:使用高效液相色谱-荧光检测方法简便,准确,灵敏,适用于伊立替康及其活性代谢物SN-38的血药浓度检测。

An analytical method built for irinotecan and its active metabolite SN-38 in human plasma using HPLC-FLD

OBJECTIVE To develop and validate a method for simultaneous determination of irinotecan(CPT-11) and its metabolite SN-38 in human plasma by HPLC-FLD. METHODS Chromatographic separation was performed on a Luna 5u CN 100A column(4.6 mm×150 mm,5 μm),using acetonitrile: ammonium acetate(50 mmol·L-1,pH=4) as mobile phase.The detection wavelength of CPT-11 was Ex/Em=368 nm/432 nm,and the detection wavelength of SN-38 was Ex/Em=368 nm/535 nm. RESULTS The retention time of CPT-11 and SN-38 was(9.3±0.3)min and(4.8±0.3)min,respectively.No interfering peaks were observed at the retention time of both targets and interna standards.The assay was linear at the range 46.9-6 000.0 nmol·L-1 for CPT-11(r=0.998) and 2.0-250.0 nmol· L-1 for SN-38(r=0.998).Both the inter-and intra-batch precisions were less than 20% at the low concentration and less than 15% at the middle and high concentrations.The inter-and intra-accuracies were all between 85% and 115%.The spiked plasma samples were stable at-20 ℃ in a long-term stability test.After three freeze-thaw cycles of the spiked samples,the changes in levels of SN-38 and CPT-11 were both within 15%.The changes in levels of SN-38 and CPT-11 in 24 h after extraction were not obvious. CONCLUSION HPLC-FLD method for detection of concentrations of irinotecan(CPT-11) and its active metabolites SN-38 in human plasma is simple,accurate and sensitive.