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

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

Key words:  cyclophosphamide  metabolite  UHPLC-MS/MS  urine

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-KetoCTX）、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，2.7 μm）；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 mL·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-KetoCTX：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·mL^-1内线性关系良好（CTX、DCCTX、4-KetoCTX、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·mL^-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。

Abstract:

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-KetoCTX), 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^-1 ammonium acetate aqueous solution. The buffer flow rate was 0.25 mL·min^-1, and column temperature was maintained at 25 ℃. 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-KetoCTX: 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-KetoCTX and CEPM were 40-2 000 ng·mL^-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·mL^-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.