UHPLC-MS/MS测定人血浆中尿嘧啶与二氢尿嘧啶的浓度

UHPLC-MS/MS determination of uracil and dihydrouracil in human plasma

目的建立同时测定人血浆中尿嘧啶(U)和二氢尿嘧啶(UH_(2))含量的超高效液相色谱串联质谱(UHPLCMS/MS)方法。方法在Agilent 6460A串联质谱仪上采用正离子检测模式,以氯尿嘧啶为内标,3%牛血清蛋白为代血浆基质,样本经乙酸乙酯液液萃取后在Agilent poroshell 120 SB-Aq(2.1 mm×100 mm,2.7μm)色谱柱上采用梯度洗脱进行色谱分离。流动相为5 mmol/L醋酸铵水溶液和乙腈溶液;流速0.3 ml/min;柱温为30℃;进样量为5μl。结果尿嘧啶和二氢尿嘧啶的线性范围为10.0~1500.0 ng/ml,线性关系良好,其相关系数r>0.990,日内与日间精密度偏差均<15%。结论该方法操作简单、选择性好,可用于测定人血浆中尿嘧啶和二氢尿嘧啶的含量。

Objective To establish an UHPLC-MS/MS method for the determination of uracil(U)and dihydrouracil(UH_(2))in human plasma.Methods A positive ion detection mode was adopted on the Agilent 6460A mass spectrometer.Chlorouracil was used as the internal standard.3%bovine serum albumin was used as surrogate plasma matrix.The pretreatment of plasma sample was completed based on liquid-liquid extraction with ethyl acetate.The chromatographic separation was achieved on an Agilent Poroshell 120 SB-Aq(2.1 mm×100 mm,2.7μm)column with gradient elution.The mobile phase was 5 mmol/L ammonium acetate aqueous solution and acetonitrile solution.The flow rate was 0.3 ml/min.The column temperature was 30°C.The injection volume was 5μl.Results The linear range of uracil and dihydrouracil was 10.0-1500.0 ng/ml.Both of uracil and dihydrouracil had good linear relationship with correlation coefficient(r)>0.990.Both of inter-and intra-day precision was<15%.Conclusion The established method is simple,selective,and suitable for the determination of U and UH2 in human plasma.