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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-C_(18)柱(75mm×2.1mm,2.7μm)为色谱柱,甲醇-10mmol/L乙酸铵溶液为流动相进行梯度洗脱,流速0.25mL/min,柱温25℃,进样量5μL;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 000ng/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 000ng/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,T_(1/2)分别为(1.28±0.09)、(5.03±0.48)、(6.72±0.47)和(7.47±0.68)h,AUC_(0→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。
Objective To establish a method for the simultaneous determination of cyclophosphamide and its metabolites (carboxyfosfamide, 4-ketocyclophosphamide and dechlorinated ethylphosphatidylcholine in rat plasma by ultra performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) Amide) concentration of the method. Methods A column of Agilent poroshell SB-C_ (18) (75 mm × 2.1 mm, 2.7 μm) was used as the mobile phase. The mobile phase was methanol-10 mmol / L ammonium acetate. The mobile phase was eluted with a gradient of 0.25 mL / , Injection volume 5μL; mass spectrometry using electrospray ionization (ESI), select multiple reactive ion monitoring (MRM) positive mode. Eight adult male Sprague-Dawley rats were divided into two groups to obtain plasma samples. Plasma concentrations and pharmacokinetic parameters of cyclophosphamide and its metabolites in plasma samples were determined. Results Methodological validation showed that cyclophosphamide, carboxyfosfamide and 4-ketocyclophosphamide showed good linearity (r = 0.998 0, 0.9595 3 and 0.998 6, respectively) at concentrations ranging from 20 to 4 000 ng / mL, Chloroethyl cyclophosphamide had good linearity (r = 0.996 8) in the range of 5-1 000 ng / mL. The RSDs were less than 8.73% and 15.38% for the QC samples and the LLOQ samples, respectively. The RSDs of the matrix factors were between -15% and 15% Rates ranged from (66.44 ± 5.53)% to (96.66 ± 1.73)%. The stability of all the tested compounds all performed well. The pharmacokinetic parameters of each compound in rat plasma were as follows: Cmax of cyclophosphamide, carboxyfosfamide, 4-ketocyclophosphamide and dechlorinated ethyl cyclophosphamide were (207.52 ± 13.20), (18.47 ± 2.66 ), (6.59 ± 1.33) and (8.27 ± 1.44) μg / mL respectively, and the T 1/2 was (1.28 ± 0.09), (5.03 ± 0.48), (6.72 ± 0.47) and (7.47 ± 0.68) AUC_ (0 → t) were (372.52 ± 32.79), (65.70 ± 5.04), (33.26 ± 11.76) and (45.03 ± 8.93) μg · h · mL -1, respectively. Conclusion The method is simple, sensitive, accurate and specific. It is suitable for the study of pharmacokinetics of high dose ammonium cyclamate in rat plasma.