ID-LC-MS/MS检测L-色氨酸及其代谢物的方法建立及方法学评价  

Development and validation of ID-LC-MS/MS method for L-tryptophan and its metabolites

作　　者：戴鑫华 范飞[2] 蒙立业 李壹[1] 安云飞[1] 邹远高[1] 张蕾[1] 白杨娟[1] Dai Xinhua;Fan Fei;Meng Liye;Li Yi;An Yunfei;Zou Yuangao;Zhang Lei;Bai Yangjuan(Department of Laboratory Medicine,West China Hospital,Sichuan University,Chengdu 610041,China;West China School of Basic Medical Science&Forensic Medicine,Sichuan University,Chengdu 610041,China)

机构地区：[1]四川大学华西医院实验医学科,成都610041 [2]四川大学华西基础医学与法医学院,成都610041

出　　处：《中华检验医学杂志》2023年第8期822-829,共8页Chinese Journal of Laboratory Medicine

基　　金：四川省重点研发项目(2022YFS0274);四川大学博士后交叉学科创新启动基金(2021SCU12079)。

摘　　要：目的建立同位素稀释液相色谱串联质谱(ID-LC-MS/MS)方法检测血清中L-色氨酸及其代谢物。方法方法学建立及评价。收集2022年11月至2023年1月的华西医院166例健康体检者的血清样本。采用L-色氨酸(Trp)、L-犬尿氨酸(Kyn)和犬尿喹啉酸(KA)的同位素标记物为内标,通过蛋白沉淀处理血清样本,应用LC-MS/MS同时检测Trp、Kyn和KA含量。评价该方法的选择性、特异性、线性、检出限(LOD)、定量限(LOQ)、携带污染、精密度、加标回收率、基质效应和稀释一致性。结果Trp、Kyn和KA的线性相关系数均>0.999,LOD分别为0.10μmol/L、0.01μmol/L和1.00 nmol/L,LOQ分别为0.20μmol/L、0.04μmol/L和2.00 nmol/L,批内精密度和批间精密度均<10%,平均加标回收率与相对基质效应均在100%左右,超线性范围的样本最多可稀释16倍。健康体检者血清中Trp、Kyn、KA含量、Kyn/Trp和KA/Kyn比值分别为(59.55±10.92)μmol/L、(1.85±0.43)μmol/L、(39.89±17.93)nmol/L、(31.64±8.19)×10^(-3)和21.51±6.72。结论建立了基于ID-LC-MS/MS定量检测Trp、Kyn和KA的方法,该方法简便快捷、灵敏度高、结果准确可靠,为临床相关研究提供了可靠支撑。Objective To establish an isotope dilution liquid chromatography-tandem mass spectrometry(ID-LC-MS/MS)method for the determination of L-tryptophan and its metabolites in serum.Methods The methodology was established and evaluated using serum samples collected from 166 healthy subjects undergoing physical examinations at West China Hospital from November 2022 to January 2023 were collected.Isotope-labeled markers of L-tryptophan(Trp),L-kynurenine(Kyn),and kynurenic acid(KA)were used as internal standards.After protein precipitation treatment of serum samples,LC-MS/MS was used to determine Trp,Kyn,and KA simultaneously.The selectivity,specificity,linearity,detection limit(LOD),quantification limit(LOQ),carry-over,precision,recovery rate,matrix effect,and dilution integrity of the method were evaluated.Results The linearity of Trp,Kyn,and KA was demonstrated to be 0.999.The LODs were 0.10μmol/L,0.01μmol/L and 1.00 nmol/L,respectively.The LOQs were 0.20μmol/L,0.04μmol/L and 2.00 nmol/L,respectively.The intra-batch precision and inter-batch precision were below<10%.The average recovery rate and the relative matrix effect were all about 100%.The samples over the upper limit of quantitation can be diluted up to 16 times.The Trp concentration,Kyn concentration,KA concentration,Kyn/Trp ratio,and KA/Kyn ratio in serum of healthy subjects were 59.55±10.92μmol/L,1.85±0.43μmol/L,39.89±17.93 nmol/L,(31.64±8.19)×10^(-3) and 21.51±6.72,respectively.Conclusion An ID-LC-MS/MS method was successfully established for the quantitative determination of Trp,Kyn,and KA in serum.The method proved to be simple,rapid,sensitive,accurate,and reliable,providing robust support for clinical research related to these analytes.

关 键 词：串联质谱法 L-色氨酸 L-犬尿氨酸 犬尿喹啉酸 

分 类 号：O657.63[理学—分析化学] R446.1[理学—化学]