HPLC-MS/MS法测定儿童微量血浆中白消安和甲氨蝶呤的浓度

Determination of busulfan and methotrexate in children with small volume of plasma by HPLC-MS/MS

目的建立高效液相色谱串联质谱(HPLC-MS/MS)法测定血浆中白消安和甲氨蝶呤的浓度,并用于儿童血液病患者的治疗药物监测。方法血浆样本经甲醇沉淀蛋白前处理,采用Phenomenex Kinetex EVO C18色谱柱分离,流动相为水(含2 mmol·L^(-1)乙酸铵+0.07%甲酸)和甲醇,梯度洗脱;流速为0.5 mL·min^(-1)。质谱检测方式为电喷雾离子源正离子模式,多反应监测(MRM)扫描,监测离子对:白消安m/z 264.1→151.0、内标白消安-D8 m/z 272.0→159.0;甲氨蝶呤m/z 455.1→m/z 308.2、甲氨蝶呤-D3(内标)m/z 458.1→m/z 311.1。结果白消安在62.50~4000 ng·mL^(-1)内与峰面积线性关系良好(r=0.9984),甲氨蝶呤在15.63~1000 ng·mL^(-1)与峰面积线性关系良好(r=0.9990)。准确度、精密度、基质效应和稳定性均符合要求。结论本方法操作简便、快速准确、专属性强且稳定性好,适用于血浆中白消安和甲氨蝶呤的快速分析,可用于儿童血液肿瘤患者的临床治疗药物监测。

Objective To develop a high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)to determine busulfan and methotrexate in the plasma and monitor the therapeutic drugs in children with hematological disease.Methods The plasma samples were pretreated by protein precipition by methanol.The analytes were eluted on Phenomenex Kinetex EVO C18 column with the mobile phase consisting of water(2 mmol·L^(-1)ammonium acetate+0.07%formic acid)-methanol at the flow rate of 0.5 mL·min^(-1).Ions monitored in the multiple reaction monitoring(MRM)mode were m/z 264.1→151.0 for busulfan,m/z 272.0→159.0 for busulfan-D8(internal standard),m/z 455.1→308.2 for methotrexate,and m/z 458.1→311.1 for methotrexate-D3(internal standard).Results Calibration plots were established at the concentration range of 62.50~4000 ng·mL^(-1)for busulfan(r=0.9984)and 15.63~1000 ng·mL^(-1)for methotrexate(r=0.9990).Accuracy,precision,matrix effect and stability all met the requirements.Conclusion The method is simple,rapid and suitable for therapeutic drug monitoring of busulfan and methotrexate in the plasma of pediatric patients with neoplastic hematological disorder.