高效液相色谱-串联质谱法测定血液病患者血液甲泼尼龙浓度的应用

Application of HPLC-MS/MS in the Determination of Methylprednisolone Concentration in Patients with Hematological Diseases

目的建立高效液相色谱-串联质谱法(HPLC-MS/MS)测定血液病患者血浆甲泼尼龙浓度,并应用于指导临床合理用药。方法以甲泼尼龙-d3为内标,甲醇沉淀血浆蛋白和萃取甲泼尼龙。月旭Ultimate XB-C18柱(4.6mm×50mm,5μm)为色谱柱,流动相为含0.1%(v/v)甲酸的甲醇溶液(1∶1000)-水溶液[含0.1%(v/v)甲酸+2 mmol/L乙酸铵],流速0.8 ml/min,柱温60℃,进样量5μl。采用电喷雾离子源,正离子模式下进行多反应监测,同时监测甲泼尼龙质荷比(m/z)375.4→339.4(定性离子),甲泼尼龙m/z 375.4→357.3(定量离子),甲泼尼龙-d3 m/z 378.2→360.3。以甲泼尼龙和甲泼尼龙-d3峰面积比为定量依据,计算血浆甲泼尼龙浓度,并评价其性能。结果甲泼尼龙在10~1000ng/ml范围线性关系良好(r2=0.9967),定量下限为10ng/ml;日内、日间精密度实验结果相对标准差(RSD)均小于15%;提取回收率为99.52%~104.79%;甲泼尼龙样品在经历3次反复冻融(-20℃~室温)循环条件下稳定,处理后的样品在室温、4℃环境中放置24h均稳定(RSD<15%)。16例患者甲泼尼龙血药浓度在0~258ng/ml范围内。结论该方法可准确、快速、简便地检测甲泼尼龙浓度,适用于临床甲泼尼龙血药浓度检测。

Objective To establish a high-performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS)method for determining the plasma concentration of methylprednisolone in patients with hematological diseases,and apply it to guide the clinical application.Methods Plasma samples were subjected to methanol-precipitated protein containing internal standard methylprednisolone-d3.The HPLC system was equipped with an Ultimate XB-C18(4.6mm×50mm,5μm particle size),maintained at 60℃,and 5μl of the supernatant was injected.Mobile phases consisting of 0.1%(v/v)formic acid(1∶1000)and 2 mmol/L ammonium acetate in water(B)and 0.1%(v/v)formic acid(1∶1000)in methanol at aflow rate of 0.8 ml/min was used.The electrospray ionization(ESI)source was operating in positive ion mode.Multiple reaction monitoring(MRM)was applied for the detection of the components:Methylprednisolone mass-to-charge ratio(m/z)375.4→339.4(qualitative ions),methylprednisolone m/z 375.4→357.3(quantitative ions),methylprednisolone-d3 m/z 378.2→360.3.The peak area ratio of methylprednisolone to methylprednisolone-d3 was used as the quantitative basis.The concentration of methylprednisolone in plasma was calculated and its performance was investigated.Results The linear range of methylprednisolone was 10~1000ng/ml(r2=0.9967),and the lower limit of quantification was 10 ng/ml.The RSDs of intra-day and inter-day precision results were less than 15%and the relative recovery ranged from 99.52%~104.79%.For methylprednisolone,the samples were stable at-20℃after three repeated freeze-thaw cycles.The prepared samples were stable at room temperature and 4℃for 24h(RSDs<15%).The steady-state blood drug concentrations of methylprednisolone in 16 patients were in the ranges of 1~258 ng/ml.Conclusion The HPLC-MS/MS method can accurately,rapidly and simply detect the concentration of methylprednisolone,and be suitable for clinical application.