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| UHPLC-MS/MS法测定大鼠血浆中环磷酰胺及其代谢物的浓度 |
| 陈力1,2△,熊筱娟2△,高守红1,翟健秀1,3,陈万生1,刘震1,黄宏伟2,张凤1* |
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| (第二军医大学长征医院药材科, 上海 200003;宜春学院化学与生物工程学院, 宜春 336000) |
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| 摘要: |
| 目的 建立利用超高效液相色谱-串联质谱（UHPLC-MS/MS）法同时测定大鼠血浆中环磷酰胺及其代谢产物（羧基磷酰胺、4-酮基环磷酰胺和脱氯乙基环磷酰胺）浓度的方法。方法 以Agilent poroshell SB-C18柱（75 mm×2.1 mm，2.7 μm）为色谱柱，甲醇-10 mmol/L乙酸铵溶液为流动相进行梯度洗脱，流速0.25 mL/min，柱温25℃，进样量5 μL；质谱采用电喷雾离子源（ESI），选择多重反应离子监测（MRM）正离子模式。取8只成年雄性SD大鼠分离获得血浆样品，测定血浆样品中环磷酰胺及其代谢产物的血药浓度和药代动力学参数。结果 方法学验证表明，环磷酰胺、羧基磷酰胺、4-酮基环磷酰胺在浓度为20~4 000 ng/mL范围内线性关系良好（r分别为0.998 0、0.995 3和0.998 6），脱氯乙基环磷酰胺在浓度为5~1 000 ng/mL范围内线性关系良好（r=0.996 8）。日内、日间相对标准差（RSD）对于质控（QC）样品和最低定量限（LLOQ）样品分别小于8.73%和15.38%，基质因子的RSD均在-15%~15%之间，提取回收率均在（66.44±5.53）%~（96.66±1.73）%之间；所有待测化合物的稳定性均表现良好。大鼠血浆中各化合物药代动力学参数如下：环磷酰胺、羧基磷酰胺、4-酮基环磷酰胺和脱氯乙基环磷酰胺的Cmax分别为（207.52±13.20）、（18.47±2.66）、（6.59±1.33）和（8.27±1.44）μg/mL，T1/2分别为（1.28±0.09）、（5.03±0.48）、（6.72±0.47）和（7.47±0.68）h，AUC0→t分别为（372.52±32.79）、（65.70±5.04）、（33.26±11.76）和（45.03±8.93）μg·h·mL-1。结论 该方法操作简单、灵敏、准确、专属性强，适用于高剂量环磷酸铵在大鼠血浆中药代动力学研究。 |
| 关键词: 环磷酰胺 代谢 超高效液相色谱-串联质谱法 药代动力学 |
| DOI：10.16781/j.0258-879x.2016.09.1063 |
| 投稿时间：2016-03-26修订日期：2016-06-15 |
| 基金项目:国家自然科学基金（81573793）. |
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| UHPLC-MS/MS in simultaneous determination of cyclophosphamide and its metabolites in rat plasma |
| CHEN Li1,2△,XIONG Xiao-juan2△,GAO Shou-hong1,ZHAI Jian-xiu1,3,CHEN Wan-sheng1,LIU Zhen1,HUANG Hong-wei2,ZHANG Feng1* |
| (Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China;College of Chemical and Biological Engineering, Yichun University, Yichun 336000, Jiangxi, China) |
| Abstract: |
| Objective To establish a UHPLC-MS/MS method for simultaneous determination of cyclophosphamide (CTX) and its metabolites in rat plasma, including carboxyphosphamide (CPM), 4-ketocyclophosphamide (4-Keto CTX), and dechloroethylcyclophosphamide (DC-CTX). Methods Chromatogram separation was performed on an Agilent poroshell SB-C18 (75 mm×2.1 mm,2.7 μm) column using a gradient mobile phase consisting of methanol and 10 mmol/L ammonium acetate aqueous solution. The flow rate was 0.25 mL/min, column temperature was maintained at 25℃, and the injection volume was 5 μL. The protonated ions of analytes were detected in positive ionization under multiple reaction monitoring mode (MRM) with an electrospray ionization (ESI) source. The plasma samples were obtained from eight adult male SD rats to measure plasma concentrations and pharmacokinetic parameters of cyclophosphamide and its metabolites. Results It was showed that the linear relationships of CTX, CPM and 4-Keto CTX were good in the range of 20-4 000 ng/mL (r values were 0.998 0, 0.995 3 and 0.998 6, respectively), and the linear relationship of DC-CTX was good in the range of 5-1 000 ng/mL (r=0.996 8). Relative standard deviation (RSD) of intra-day and inter-day for the quality control (QC) samples and the lower limit of quantitation (LLOQ) samples were lower than 8.73% and 15.38%, respectively. RSD for matrix factor were in the range of -15%-15%, and the recoveries were in the range of (66.44±5.53)%-(96.66±1.73)%. All analytes showed good stability. The rat plasma pharmacokinetic parameters were as follows:Cmax of CTX, CPM, 4-Keto CTX and DC-CTX were (207.52±13.20) μg·mL-1, (18.47±2.66) μg·mL-1, (6.59±1.33) μg·mL-1 and (8.27±1.44) μg·mL-1, respectively; T1/2 were (1.28±0.09) h, (5.03±0.48) h, (6.72±0.47) h and (7.47±0.68) h, respectively; and AUC0→t were (372.52±32.79) μg·h·mL-1, (65.70±5.04) μg·h·mL-1, (33.26±11.76) μg·h·mL-1 and (45.03±8.93) μg·h·mL-1, respectively. Conclusion The established UHPLC-MS/MS method is simple, sensitive, accurate and selective, which makes it suitable for the comprehensive pharmacokinetic study of high-dose CTX in rat plasma. |
| Key words: cyclophosphamide metabolitsm ultra high performance liquid chromatography with tandem mass spectrometry pharmacokinetics |
