HPLC-MS/MS法快速测定儿童微量血浆中甲氨蝶呤和丙戊酸浓度

Rapid determination of methotrexate and valproate in trace plasma from pediatric patients by HPLC-MS/MS

目的建立高效液相色谱串联质谱(HPLC-MS/MS)法快速测定儿童微量血浆中甲氨蝶呤(MTX)和丙戊酸(VPA)的浓度。方法10μL血浆经甲醇沉淀蛋白后取上清液直接进样分析,内标为MTX-D3和VPA-D6;采用Phenomenex Kinetex C18柱(50 mm×2.1 mm,5μm)分离,以水(含0.01%甲酸)和甲醇为流动相,梯度洗脱,进样量5μL,流速0.5 mL·min^(-1),分析时长2.4 min。电喷雾离子源(ESI)正负离子切换扫描下,采用多反应监测(MRM)模式进行质谱分析,用于定量分析的离子对分别为m/z 455.1→308.2(MTX),143.2→143.2(VPA),458.1→311.1(MTX-D3)和149.2→149.2(VPA-D6)。结果MTX在31.25~2000 ng·mL^(-1)内与峰面积呈良好的线性关系(r=0.9995),定量下限为31.25 ng·mL^(-1);VPA在1.88~120μg·m L^(-1)内与峰面积呈良好的线性关系(r=0.9992),定量下限为1.88μg·m L^(-1);批内、批间准确度在98.05%~104.89%,批内、批间精密度的变异系数(CV)均≤6.73%,平均提取回收率为90.21%~100.55%,基质效应为93.22%~102.73%,溶血效应(溶血程度≤5%)不影响测定结果,血浆样品可分别在冻融3次、室温(25℃)24 h、4℃进样器12 h和冷冻(-20℃)30 d等条件下稳定保存;该法成功用于169例临床患儿血浆样本分析。结论本方法所需样本量小,快速、准确、灵敏、稳定,且不受溶血效应影响(溶血程度≤5%),可应用于儿童MTX和VPA治疗药物监测及联合用药研究。

Objective To establish a rapid HPLC-MS/MS method to determine the concentration of methotrexate(MTX)and valproate(VPA)in trace plasma from pediatric patients.Methods Plasma specimens(10μL)were precipitated by methanol,and then the supernatant was injected for detection.The internal standards included MTX-D3 and VPA-D6.The chromatographic separation was performed with gradient elution on a Phenomenex Kinetex C18 column(50 mm×2.1 mm,5μm)with the mobile phase consisting of water(0.01%formic acid)and methanol,at a flow rate of 0.5 mL·min^(-1).The injection volume was 5μL and the total running time was 2.4 min.For the mass spectrometry,electrospray ionization source was chosen,and both positive and negative ion switching were under multi-reaction monitoring(MRM)mode.Ions monitored were m/z 455.1→308.2 for MTX,143.2→143.2 for VPA,458.1→311.1 for MTX-D3 and 149.2→149.2 for VPA-D6,respectively.Results The calibration curves were linear at 31.25~2000 ng·mL^(-1)(r=0.9995)for MTX and 1.88~120μg·mL^(-1) for VPA(r=0.9992).LLOQ of MTX and VPA was 31.25 ng·mL^(-1) and 1.88μg·mL^(-1) respectively.The intra and inter accuracy of MTX and VPA ranged 98.05%~104.89%.The intra and inter precisions(CV)were less than 6.73%.The average extraction recoveries of the two analytes were 90.21%~100.55%,and the matrix effects were 93.22%~102.73%.The hemolytic effect(hemolytic concentration≤5%)was acceptable.Analytes in the plasma were stable after 3 freeze-thaw cycles and robust for 24 h(at room temperature,25℃),12 h(in the autosampler,4℃)and 30 days(-20℃).This method successfully analyzed the plasma samples from 169 pediatric patients.Conclusion This assay is rapid,sensitive and robust,without interference by hemolytic effect,which can be used in the combination medication study of MTX and VPA and their therapeutic drug monitoring in pediatric patients.