HPLC-MS/MS法同时测定儿童微量血浆中达沙替尼和甲氨蝶呤浓度

Simultaneous determination of dasatinib and methotrexate in children with small volume of plasma by HPLC-MS/MS

目的建立液相色谱-串联质谱(HPLC-MS/MS)法同时检测人血浆中达沙替尼和甲氨蝶呤浓度,并用于费城染色体阳性急性淋巴细胞白血病患儿的治疗药物监测。方法分别以稳定同位素伊马替尼-D8和甲氨蝶呤-D3为内标,取10μL血浆样本经甲醇沉淀蛋白,采用Phenomenex Kinetex EVO C18(30 mm×2.1 mm,2.6μm)色谱柱分离,流动相为水(含0.05%甲酸)和乙腈,梯度洗脱;流速为0.5 mL·min^(-1),进样体积为1μL。质谱分析条件:电喷雾离子源正离子模式,多反应监测(MRM)扫描分析,离子通道分别为m/z 488.2→401.2(达沙替尼)和m/z 502.5→394.2(伊马替尼-D8);m/z 455.1→308.2(甲氨蝶呤)和m/z 458.1→311.1(甲氨蝶呤-D3)。结果达沙替尼的线性范围在0.5~200 ng·mL^(-1)(r>0.9900),定量下限(LLOQ)为0.5 ng·mL^(-1);甲氨蝶呤的线性范围在5~2000 ng·mL^(-1)(r>0.9900),LLOQ为5 ng·mL^(-1)。达沙替尼和甲氨蝶呤的批内与批间精密度、准确度、提取回收率、基质效应、稳定性均符合方法学要求。结论本方法操作简便、灵敏、准确,只需要微量血浆,适用于费城染色体阳性急性淋巴细胞白血病患儿的治疗药物监测。

Objective To establish a liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method to simultaneously determine dasatinib and methotrexate in human plasma,and to use the method in clinical therapeutic drug monitoring of dasatinib and methotrexate in children with Philadelphia-positive acute lymphoblastic leukemia.Methods Imatinib-D8 and methotrexate-D3 were separately used as the internal standards and the 10μL plasma samples were used to precipitate protein by methanol.Then,the analytes were gradiently eluted on a Phenomenex Kinetex EVO C18 column(30 mm×2.1 mm,2.6μm)by HPLC-MS/MS.The mobile phase consisted of water(0.05%formic acid)and acetonitrile.The flow rate was 0.5 mL·min^(-1) and the injection volume 1μL.The mass detection was performed in positive ionization mode with electrospray ionization source.Ions monitored in the multiple reaction monitoring mode were m/z 488.2→m/z 401.2 for dasatinib,m/z 502.5→m/z 394.2 for imatinib-D8(internal standard)and m/z 455.1→308.2 for methotrexate,m/z 458.1→311.1 for methotrexate-d3(internal standard),respectively.Results The linearity of dasatinib in human plasma was at 0.5~200 ng·mL^(-1)(r>0.9900),lower limit of quantification(LLOQ)was 0.5 ng·mL^(-1).The linearity of methotrexate in human plasma was at 5~2000 ng·mL^(-1)(r>0.9900),LLOQ was 5 ng·mL^(-1).The intra-day and inter-day precision,accuracy,extraction recovery rate,matrix effect,and stability of dasatinib and methotrexate all met the methodological requirements.Conclusion This HPLC-MS/MS method,which needed small volume of plasma is simple,sensitive and accurate.It is approved appropriate and practical for simultaneously monitoring dasatinib and methotrexate in children with Philadelphia-positive acute lymphoblastic leukemia.