HPLC-MS/MS法检测白血病患者尿液及血浆中维生素C浓度

Determination of Vitamin C in Urine and Plasma of Leukemia Patients by HPLC-MS/MS

目的:建立高效液相色谱串联质谱法(HPLC-MS/MS)法测定白血病患者尿液中维生素C(VC)浓度,并比较HPLCMS/MS法与干化学法测定结果,分析尿液与血浆中VC浓度的相关性。方法:以L-Ascorbic Acid-1-13C为内标,采用Ultimate XB-C18色谱柱(50 mm×4.6 mm,5μm),柱温:60℃,流速:0.8 ml·min^(-1),流动相:甲醇(0.1%甲酸)-纯水(2 mmol·L^(-1)乙酸胺-0.1%甲酸),梯度洗脱,进样量:5μl。质谱检测方式采用ESI负离子模式,以多反应监测模式(MRM)扫描,定量检测VC m/z 175.0→115.1,L-Ascorbic Acid-1-13C m/z 176.0→115.8。选取127例未服用VC制剂的白血病患者,分别采用HPLC-MS/MS法和干化学法测定其尿液中VC含量,对结果进行比较分析;同时采用HPLC-MS/MS法检测患者血浆中VC浓度,对患者尿液和血浆中VC浓度进行相关性分析。结果:尿液中VC浓度在1~100μg·ml^(-1)范围内线性关系良好(r=0.9971),低、中、高3个浓度质控样品日内及日间RSD均小于10%,方法回收率为85%~110%。与干化学法相比,HPLC-MS/MS法测得的尿液中VC含量明显较低(P<0.01),两种方法检测结果符合率为68.50%。127例患者尿液中VC平均含量为(53.99±56.58)μg·ml^(-1),血浆中VC平均含量为(2.48±2.53)μg·ml^(-1),尿液VC含量明显高于血浆VC含量,结果相关性较差(r=0.451),血浆与尿液中VC浓度平均比值为0.0810(0.0046~0.8667)。结论:与干化学法相比,HPLC-MS/MS法特异性强、灵敏度高,可以准确定量检测白血病患者尿液中VC浓度结果。

Objective:To establish a high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method for the determination of vitamin C(VC)in urine of leukemia patients,compare with dry chemical method for the determination of VC in urine,and analyze the correlation between the concentration of VC in urine and that in plasma.Methods:Using L-ascorbic acid-1-13C as the internal standard,the determination was performed on an Ultimate XB-C18 column(50 mm×4.6 mm,5μm)at the column temperature of 60℃and the flow rate of 0.8 ml·ml^(-1).The mobile phase was methanol(0.1%formic acid)-pure water(2 mmol·L^(-1)ammonium acetate-0.1%formic acid)with gradient elution.The injection volume was 5μl.Mass spectrum detection was in an ESI negative ion mode and multiple reaction monitoring was used to detect VC at m/z 175.0→115.1 and L-ascorbic acid-1-13C at m/z 176.0→115.8.127 leukemia patients without VC treatment were selected.HPLC-MS/MS and dry chemical method was respectively used to determine the concentration of VC in urine of leukemia patients,and the results were compared and analyzed.Meanwhile,pearson correlation coefficient was used to analyze the results of VC concentration in urine and plasma of the same patient.Results:The concentration of VC in urine had a good linear relationship within the range of 1-100μg·ml^(-1)(r=0.9971).The intra-and interday RSDs of the quality control samples at low,medium and high concentrations were all less than 10%,and the method recoveries were 85%~110%.Compared with that detected by dry chemical method,the content in urine of VC detected by HPLC-MS/MS method was significantly lower(P<0.01),and the coincidence rate of the two methods was 68.50%.The concentration of VC in urine of 127 patients was(53.99±56.58)μg·ml^(-1),and(2.48±2.53)μg·ml^(-1) in plasma.The concentration of VC in urine was significantly higher than that in plasma,and the correlation was poor(r=0.451).The average ratio of VC concentration in plasma and urine was0.0810(0.0046~0.8667).Conclusion:Compared with dry chemical method,HPLC-MS/MS method is more specific and sensitive,and can accurately and quantitatively detect VC concentration in urine of leukemia patients.