HPLC-MS/MS法在白血病患者血浆维生素C水平测定中的临床应用

The clinical application of determination of ascorbic acid concentration in leukemia patient plasma by HPLC-MS/MS

目的建立测定白血病患者血浆维生素C水平的高效液相色谱-质谱联用(HPLC-MS/MS)法,并应用于临床。方法采用HPLC-MS/MS法,L-Ascorbic Acid-1-13 C为内标,XBridge?C18(4.6 mm×50.0mm,5μm)色谱柱,流动相为(0.1%甲酸)甲醇-(2mmol/L乙酸铵-0.1%甲酸)水,梯度洗脱,流速为0.8mL/min,柱温60℃,进样量10μL。采用电喷雾电离源,以多反应监测(MRM)方式进行负离子检测。并对123份白血病患者标本及407份健康志愿者标本进行了检测,测定结果用t检验进行比较分析。结果维生素C的水平在0.5～50.0μg/mL范围内线性关系良好(r>0.990 0),日内及日间精密度(RSD)均小于5%,回收率在90%～110%;与健康人相比,患者体内维生素C水平明显偏低[(8.8±3.0)vs.(2.4±3.9)μg/mL,P<0.01]。结论该方法快速、准确、灵敏度高、操作简单,适用于临床中患者血浆中维生素C水平的检测。建议白血病患者应适当补充维生素C。

Objective To develop a HPLC-MS/MS method for determination of ascorbic acid in leukemia patient,and make it used in clinic trial.Methods HPLC-MS/MS method was adopted.L-Ascorbic Acid-1-13 C as the internal standard,the separation was performed on a XBridge?C18(4.6 mm×50.0 mm,5μm)column with a mobile phase of water(containing 2 mmol/L ammonium acetate and 0.1% formic acid and methanol(containing 0.1%formic acid)at flow rate of 0.8 mL/min and column temperature of 60 ℃.The way of eluting was gradient.The injection volume was 10μL.Mass spectrum detection method was ESI negtive ionmode and multiple reaction monitoring was used.123 leukemia patients and 407 healthy volunteers samples were monitored,and the results were analyzed by t-test.Results The standard curve of ascorbic acid was linear over the range of 0.5-50.0μg/mL(r=0.990 0).RSDs of intra-and inter-day validation were all less than5%.The relative recovery was among the range of 90%-110%.Compared with healthy people,the concentration of vitamin C in leukemia patients was significantly lower[(8.8±3.0)vs.(2.4±3.9)μg/mL,P<0.01].Conclusion This method is rapid,accurate,sensitivity,and simple.It was suitable for determination of ascorbic acid concentration in plasma of leukemia patients.It is suggested that leukemia patients should take vitamin C supplements appropriately.