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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 a method for determination of CTX and its metabolite dechloroethylcyclophosphamide (DCCTX) in rat urine after high dose cyclophosphamide (CTX) administration by UHPLC-MS / MS. Amide (4-Ketocyclophosphamide, 4-Keto CTX), carboxyphosphamide (CEPM) concentrations. Methods The urine samples of rats were directly diluted with 10% methanol aqueous solution, centrifuged and the supernatant was taken and detected by UHPLC-MS / MS. The column: Agilent poroshell SB-C18 (2.1 mm × 75 mm, 2.7 μm). The mobile phase consisted of methanol-10 mmol·L-1 ammonium acetate aqueous solution with gradient elution at a flow rate of 0.25 m L · min -1 , Column temperature 25 ℃. Electrospray ionization was used for positive ion detection in multiplex reaction monitoring mode. The ion pairs used for quantitative detection 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 The urinary concentrations of CTX and its metabolites were linear in the range of 40-2 000 ng · m L-1 (r2 of CTX, DCCTX, 4-Keto CTX and CEPM were 0.991 5,0.991 0,0.995 6,0.991 8), and the lowest limit of quantification was 40 ng · m L-1 respectively. The intra-day and inter-day RSD were 0.67% -12.76% and 1.30% -11.92% respectively. RSD was calculated from the normalized matrix factor % ~ 7.60%. The recoveries of the method were 74.00% ~ 102.70% (n = 6). The recoveries were 85.89% ~ 110.69% (n = 5). Conclusion The method is rapid, accurate, reliable and reproducible. It is suitable for the determination of urinary concentration of CTX and its metabolites after high-dose CTX in rats and excretion.