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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 establish a method for the determination of tryptophan, kynurenine and quinolinic acid in human serum by liquid chromatography-tandem mass spectrometry. Methods methanol as extractant, using protein precipitation method. Tryptophan, kynurenine to kynurenine -13C4,15N as internal standard, the column: Eclipse XDB-C18 (4.6 mm × 150 mm, 5μm), the mobile phase methanol-water (45:55, both Containing 5 mmol·L -1 ammonium formate-0.1% formic acid). Quinolinic acid was synthesized with quinolinic acid-d3 as internal standard. The column was Eclipse plus C8 (4.6 mm × 100 mm, 3.5 μm) Phase: methanol-water (15:85,0.75 mmol·L -1 ammonium formate-0.4% formic acid); flow rate: 0.5 m L · min -1; column temperature: 35 ° C .; electrospray ionization source Ion multiple reaction monitoring. The specificity, standard curve and limit of quantification, precision and recovery, matrix effect and stability of this method were examined. Results The linear ranges of tryptophan, kynurenine and quinolinic acid were 1000 ~ 3 × 104, 100 ~ 3000 and 10 ~ 300 ng · m L -1, respectively. The lower limits of quantitation were 1000, 100 and 10 ng · m L ~ (-1). The average recoveries were above 80%. The intra-day and inter-day RSDs were less than 15%. Conclusion The method is simple, sensitive, rapid, consistent with the requirements of biological sample analysis, and is suitable for the determination of tryptophan and its metabolites in human serum.