超高效液相色谱串联质谱法检测晨尿中儿茶酚胺及其代谢产物的价值

Determination value of catecholamines and their metabolites in morning urine by ultra high performance liquid chromatography-tandem mass spectrometry

目的建立一种稳定、灵敏、可靠且可以同时检测尿儿茶酚胺及其代谢产物的方法。方法采用超高效液相色谱串联质谱法(UPLC-MS/MS)检测晨尿标本中儿茶酚胺及其代谢产物。采用固相萃取的前处理方法,对液相色谱条件及质谱条件进行优化,并对该方法的检测限、定量下限、线性、精密度、准确度和基质效应及稳定性进行性能验证。检测100例体检健康者晨尿及其中20例体检健康者24 h尿液,根据肌酐检测值对稀释差异进行归一化,考察晨尿与24 h尿液收集这两种不同采样方法之间检测儿茶酚及其代谢产物的相关性,并建立了晨尿检测6种分析物的参考区间。结果6种分析物在相应的浓度范围内均线性关系良好,精密度、准确度、基质效应均<10%,室温放置24 h、4℃放置24 h、4℃放置48 h及—80℃放置7 d稳定性均较好,变化≤6.2%。晨尿与24 h尿液标本检测结果呈正相关(r=0.93~0.97,P<0.0001),6种分析物的参考区间去甲肾上腺素为2.6~140.4μg/g Cr,肾上腺素为0.3~37.2μg/g Cr,多巴胺为43.4~561.8μg/g Cr,3-甲氧酪胺为18.0~165.7μg/g Cr,甲氧基去甲肾上腺素为15.1~241.5μg/g Cr,甲氧基肾上腺素为6.8~192.0μg/g Cr。结论建立了一种方便快速、灵敏、特异的UPLC-MS/MS,一次进样可同时检测晨尿中6种儿茶酚胺及其代谢产物,可为儿茶酚胺的常规检测及临床应用提供一种简单、可靠的新手段。

Objective To establish a stable,sensitive and reliable method for the detection of urinary catecholamines and their metabolites.Methods The catecholamines and their metabolites in morning urine samples were detected by ultra high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).Solid phase extraction was used as the pretreatment method,and optimized the conditions of liquid chromatography and mass spectrometry,and verified the performance of the method,including limit of detection,limit of quantitation,linearity,precision,accuracy,matrix effect and stability.Morning urine of 100 apparently healthy subjects and 24 h urine of 20 cases of them were measured.The difference of dilution was normalized according to the creatinine measured value.The correlation between the results of 6 kinds of analytes detected by morning urine and 24 h urine were analyzed,then established the reference interval of 6 kinds of analytes detected by morning urine.Results Within the corresponding concentration range,the linear relationship of the 6 kinds of analytes was good,the precision,accuracy and matrix effect were all less than 10%.The stability of the analytes was good when placed at room temperature for 24 h,at 4℃for 24 h,at 4℃for 48 h and at-80℃for 7 d,with the change equal or less than 6.2%.The results of morning urine and 24 h urine samples significantly correlated(r=0.93—0.97,P<0.0001).The reference interval of catecholamines were as follows,noradrenaline 2.6-140.4μg/g Cr,adrenaline 0.3—37.2μg/g Cr,dopamine 43.4—561.8μg/g Cr,3-methoxytyramine 18.0—165.7μg/g Cr,normetanephrine 15.1—241.5μg/g Cr,matanephrine 6.8—192.0μg/g Cr.Conclusion A convenient,rapid and sensitive UPLC-MS/MS method for the determination of 6 kinds of catecholamines and their metabolites is established,which is expected to provide a simple,convenient,accurate and sensitive new method for the routine detection and clinical application of catecholamines.