大鼠血浆中7-乙基-10-羟基喜树碱-10-棕榈酸酯及7-乙基-10-羟基喜树碱HPLC分析方法的建立

Establishment of HPLC method for determination of SN38-PA and SN38 in rat plasma

目的建立同时测定大鼠血浆中7-乙基-10-羟基喜树碱-10-棕榈酸酯（SN38-PA）及其活性代谢产物7-乙基-10-羟基喜树碱（SN38）含量的高效液相色谱法。方法以10-羟基喜树碱（10-CPT）作为内标,甲酸酸化血浆,甲醇沉淀蛋白。采用Agilent C18柱（4.6 mm×250 mm,5μm）色谱柱,以甲醇-0.2%甲酸水溶液为流动相进行梯度洗脱,流速1 ml/min,紫外检测波长372 nm,柱温：30℃。结果大鼠血浆中SN38-PA和SN38线性范围分别为0.25--62.5（r=0.9998）和0.05--12.5μg/ml（r=0.9997）,最低定量限分别为0.18和0.04μg/ml;两组分平均绝对回收率分别为95.89%和97.03%;平均相对回收率分别为99.54%和99.84%。2组分日内、日间精密度的RSD%均〈3%。结论本分析方法快速、简便、准确度和灵敏度高,可用于SN38-PA及其活性代谢产物SN38的体内检测。

Objective To develop an HPLC method for the simultaneous determination of 7-ethyl-10-hydroxycamptothecin-10-palmitic acid ester（SN38-PA）and its active metabolite 7-ethyl-10-hydroxycamptothecin（SN38）in rat plasma. Methods The inter standard was 10-hydroxycamptothecin. The protein in plasma was precipitated with methanol after acidification with formic acid.SN38-PA and SN38 were separated on Agilent C18column（4.6 mm×250 mm,5 μm）with gradient elution by using the mobile phase of methanol-0.2% formic acid solution. The flow rate was 1 ml/min. The detection wavelength was set at 372 nm. The column temperature was maintained at 30℃. Results The linear ranges for SN38-PA and SN38 were 0.25-62.5（r=0.9998） and 0.05-12.5 μg/ml（r =0.9997） respectively. The limits of quantification were 0.18 and 0.04 μg/ml,respectively. The average relative recovery of SN38-PA and SN38 were 95.89% and 97.03%. The average absolutely recovery of SN38-PA and SN38 were 99.54% and99.84%. The RSD for intra-day and inter-day were both less than 3%. Conclusion The method is fast,convenient,accurate and sensitive,so it can be used for determination of SN38-PA and SN38 in vivo.