摘要
目的 建立人血清中叶酸及其代谢产物浓度的超高效液相色谱质谱联用测定方法.方法 采用Agilent EclipsePlus-C18 色谱柱(2.1 mm × 50 mm,1.8 μm),以 0.1％甲酸-水(A)-0.1％甲酸-甲醇溶液(B)为流动相梯度洗脱,流速0.3mL/min,柱温40 ℃,用电喷雾离子化源,正离子方式,多反应监测扫描方式进行监测,内标法定量.结果 3种叶酸循环代谢物标准曲线的线性相关系数为0.995 6～0.999 9,准确度为91.2％～109.2％,精密度均低于10％,最低检测限为1 ng/mL,基质效应可忽略不计,稳定性考察结果均符合要求.对150例健康人群的血样进行测定,叶酸浓度范围为0.00～3.99 ng/mL,平均浓度为(2.29±0.31)ng/mL;5-甲基四氢叶酸浓度范围在1.22～29.07 ng/mL,平均浓度为(6.10±4.75)ng/mL;同型半胱氨酸浓度范围在4.57～68.22 ng/mL,平均浓度为(12.47±8.19)ng/mL.结论 超高效液相色谱质谱联用测定方法快速、灵敏、准确、专属性强、重复性好,适用于人血清中叶酸及其代谢产物浓度的测定.
Abstract
Objective To establish an ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method to determine the concentrations of folic acid and its metabolites in human serum.Methods An Agilent EclipsePlus-C18(2.1 mm x 50 mm,1.8 μm)column was used with 0.1％formic acid-water(A)-0.1％formic acid-methanol solution(B)as mobile phase gradient eluted at a flow rate of 0.3 mL/min,40℃ column temperature,and electrospray ionization source(positive ion mode).Multiple reaction monitoring(MRM)scanning method was used for mo-nitoring.Internal standard method was used for quantification.Results The linear correlation coefficient of the standard curve for three metabolites of the folate cycle was 0.9956 to 0.9999.The accuracy was between 91.2％and 109.2％,and the precision was less than 10％.The minimum detection limit was 1 ng/mL and the matrix effects of the three metabolites in the folate cycle were negligible.The results of stability test were in accordance with the requirements.Blood samples from 150 healthy adults were measured.The folic acid concentration ranged from 0.00 to 3.99 ng/mL,with an average concentration of(2.29±0.31)ng/mL.The 5-methyl tetrahydrofolate concentration ranged from 1.22 to 29.07 ng/mL,with an average concentration of(6.10±4.75)ng/mL.The homocysteine concentration ranged from 4.57 ng/mL to 68.22 ng/mL,with an average concentration of(12.47±8.19)ng/mL.Conclusion The method was rapid,sensitive,accurate,specific,and reproducible.It can be applied to analyzing folic acid and its metabolites in human serum.
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