UHPLC-MS/MS测定大鼠尿液中环磷酰胺及其代谢产物浓度  

Concentration Measurement of Cyclophosphamide and Its Metabolites in Rat Urine Using UHPLC-MS/MS

作　　者：冯格[1,2] 陈力[1,2] 翟健秀 高守红[2] 陈万生[2] 夏天一[2] 谢新芳[1] 熊筱娟[1] 张凤[2] FENG Ge CHEN Li ZHAI Jianxiu GAO Shouhong CHEN Wansheng XIA Tianyi XIE Xinfang XIONG Xiaojuan ZHANG Feng(College of Chemical and Biological Engineering, Yichun University, Yichun 336000, China Department of Pharmacy, Shanghai Changzheng Hospital, Shanghai 200003, China School of Traditional Chinese Meteria, Shenyang Pharmaceutical University, Shenyang 110015, China)

机构地区：[1]宜春学院化学与生物工程学院,江西宜春336000 [2]上海长征医院药材科,上海200003 [3]沈阳药科大学中药学院,沈阳110015

出　　处：《中国现代应用药学》2017年第6期800-805,共6页Chinese Journal of Modern Applied Pharmacy

基　　金：国家自然科学基金资助项目(81573793)

摘　　要：目的基于UHPLC-MS/MS建立测定给予高剂量环磷酰胺(cyclophosphamide,CTX)后大鼠尿液中CTX及其代谢产物脱氯乙基环磷酰胺(dechloroethylcyclophosphamide,DCCTX)、4-酮基环磷酰胺(4-Ketocyclophosphamide,4-Keto CTX)、羧基磷酰胺(carboxyphosphamide,CEPM)浓度的方法。方法大鼠尿液样品经10%甲醇水溶液直接稀释,离心后取上清液,采用UHPLC-MS/MS进行检测分析。色谱柱:Agilent poroshell SB-C18(2.1 mm×75 mm,2.7μm);流动相:甲醇-10 mmol·L-1乙酸铵水溶液,进行梯度洗脱,流速0.25 m L·min^(-1),柱温25℃。采用电喷雾离子源,以多重反应监测模式进行正离子检测。用于定量检测分析的离子对分别为:CTX:m/z 261.10→140.10;DCCTX:m/z 199.20→78.00;4-Keto CTX:m/z 275.10→142.00;CEPM:m/z 293.10→221.10;替硝唑(tinidazole,TNZ):m/z 248.10→121.10。结果 CTX及其代谢产物尿中浓度在40~2 000 ng·m L-1内线性关系良好(CTX、DCCTX、4-Keto CTX、CEPM的r2分别为:0.991 5,0.991 0,0.995 6,0.991 8),最低定量限均为40 ng·m L-1;日内、日间RSD分别为0.67%~12.76%,1.30%~11.92%;由内标归一化的基质因子计算RSD,结果为0.52%~7.60%;该方法的提取回收率为74.00%~102.70%(n=6),方法回收率为85.89%~110.69%(n=5);测定成分和内标的稳定性均良好。结论该方法快捷、准确可靠、重复性好,适用于大鼠高剂量CTX后CTX及其代谢产物尿药浓度的测定及排泄研究。OBJECTIVE To establish an UHPLC-MS/MS method for simultaneous quantification of cyclophosphamide（CTX） and its metabolites in rat urine including dechloroethylcyclophosphamide（DCCTX） 4-Ketocyclophosphamide（4-Keto CTX）, and carboxyphosphamide（CEPM）. METHODS The urine samples were directly diluted by 10% methanol aqueous solution, followed by a centrifugation to get the supernatant for analysis. Chromatographic separation was performed on an Agilent poroshell SB-C18 column（2.1 mm×75 mm, 2.7 μm） using a gradient mobile phase consisting of methanol and 10 mmol·L-1ammonium acetate aqueous solution. The buffer flow rate was 0.25 m L·min^（-1）, and column temperature was maintained at 25 °C. The protonated ions of analytes were detected in the positive multiple reaction monitoring mode with an electrospray ionization source. The precursor ion and the product ion used for quantitative analysis were： CTX： m/z 261.10→140.10; DCCTX： m/z 199.20→78.00; 4-Keto CTX： m/z 275.10→142.00; CEPM： m/z 293.10→221.10; tinidazole（TNZ）： m/z 248.10→121.10. RESULTS Linear ranges for CTX, DCCTX, 4-Keto CTX and CEPM were 40-2 000 ng·m L-1（r2 values were 0.991 5, 0.991 0, 0.995 6, and 0.991 8, respectively）. Limit of quantification for all the analytes was 40 ng·m L-1. The coefficient of RSD for intra-day and inter-day precisions of the quality control samples were 0.67%-12.76% and 1.30%-11.92%. Matrix effect was expressed as internal standard normalized factor. The RSDs all the analytes were between 0.52% and 7.60%. Stability results for all the analytes were in the acceptable levels. CONCLUSION The established method is reliable and reproducible for simultaneous determination of CTX and its metabolites in rat urine after high-dose CTX administration, which is applicable in the cumulative excretion study of these analytes.

关 键 词：环磷酰胺 代谢产物 超高效液相色谱-串联质谱法 尿液 

分 类 号：R969.1[医药卫生—药理学]