液相色谱串联质谱法测定人血清色氨酸及其代谢产物的浓度

Determination of tryptophan and its metabolites in human serum using liquid chromatography tandem mass spectrometry

目的建立人血清中色氨酸、犬尿氨酸、喹啉酸浓度测定的液相色谱串联质谱法。方法以甲醇为提取剂,用蛋白沉淀法。色氨酸、犬尿氨酸以犬尿氨酸-13C4,15N为内标,色谱柱:Eclipse XDB-C18(4.6 mm×150 mm,5μm),流动相为甲醇-水(45∶55,均含5 mmol·L^(-1)甲酸铵-0.1%甲酸);喹啉酸以喹啉酸-d3为内标,色谱柱:Eclipse plus C8柱(4.6 mm×100 mm,3.5μm),流动相:甲醇-水(15∶85,0.75 mmol·L^(-1)甲酸铵-0.4%甲酸);流速:0.5 m L·min-1,柱温:35℃;电喷雾离子源,以正离子多反应监测。考察该方法的专属性、标准曲线和定量下限、精密度与回收率、基质效应和稳定性。结果色氨酸、犬尿氨酸、喹啉酸的线性范围分别为1000~3×104,100~3000,10~300 ng·m L^(-1);定量下限分别为1000,100,10 ng·m L^(-1);平均回收率均在80%以上,日内、日间精密度RSD均小于15%。结论本方法操作简便,灵敏度高,分析快速,符合生物样品分析要求,适用于人血清中色氨酸及其代谢产物的浓度测定。

Objective To determine the concentrations of tryptophan and its metabolites in human serum by liquid chromatography tandem mass spectrometry.Methods L-Kynurenine-13C4,15 N was used as internal standard for tryptophan and kynurenine.Eclipse XDB-C18（4.6mm × 150 mm,5 μm） was employed to determine the concentration of tryptophan and kynurenine.The mobile phase consisted of methanol and water（45∶ 55,5 mmol·L^（-1）ammonium formate and 0.1 % formic acid solution）;2,3-Pyridinedicarboxylic acid-d3 was used as internal standard for quinolinic acid.Eclipse plus C8（4.6 mm × 100 mm,3.5μm） was employed to determine the serum concentration of quinolinic acid.The mobile phase consisted of methanol and water（15 ∶ 85,0.75mmol·L^（-1）ammonium formate and 0.4 % formic acid solution）.Flow rate was 0.5 m L·min-1.Column temperature was 35 ℃.Electrospray ionization source was applied and operated in the positive multiple reaction monitoring（MRM） mode.The proteins of plasma sample were precipitated with methanol.The specificity,standard curve and lower limit of quantitation,precision and recovery rate and stability as well as the matrix effect were investigated.Results The linear ranges of tryptophan,kynurenine and quinolinic acid were 1000-3 × 104,100-3000,10-300 ng·m L^（-1）,respectively.The lower limit of quantitation of tryptophan,kynurenine and quinolinic acid were1000,100,10 ng·m L^（-1）,respectively.The average recovery is above 80%.The intra-day and inter-day precision（RSD） were less than 15%.Conclusion The methods is simple,sensitive and accurate,consistent with requirements of biological sample analysis,suitable for clinical determination of tryptophan and its metabolites in human serum.