基于UPLC-MS法快速定量检测人血浆中短链脂肪酸

Rapid quantification of short chain fatty acids in human plasma by UPLC-MS

目的:建立一种3-硝基苯肼化学衍生化与UPLC-MS联用技术的快速检测人血浆中短链脂肪酸(SCFAs)含量的方法.方法:用5倍量的甲醇沉淀蛋白后,上清液中加入3-硝基苯肼(3-NPH)与EDC进行衍生化反应,通过考察反应时间、温度、反应物浓度对衍生化产物峰面积的影响,优化反应条件.采用Waters Acquity UPLC BEH C_(18)色谱柱,以乙腈-甲醇(V(乙腈)︰V(甲醇)=2︰1)及水(含0.1%甲酸)为流动相,分离11个SCFAs.采用ESI离子源,多反应监测正离子模式进行检测.结果:最佳反应条件为25 mmol·L^(−1)3-NPH作为衍生化试剂和30 mmol·L^(−1) EDC作为催化剂,40℃反应30 min;11个SCFAs在11 min内能实现基线分离;衍生化产物的峰面积与浓度有良好的线性关系(r>0.999),日内与日间精密度良好(RSD均小于15%),提取回收率在80%~120%范围内.结论:衍生化方法反应条件温和、操作简单;UPLC-MS/MS方法准确、简易、可重复,可用于人血浆中SCFAs的含量测定.

Objective:Thechemicalderivatizationof3-nitrophenylhydrazine(3-NPH)wascoupledwithultra-high-performance liquid chromatography and mass spectrometry(UPLC-MS)for rapid quantification of short chain fatty acids(SCFAs)in human plasma.Methods:Blood plasma was pretreated by 5 times of methanol.Then the extracted SCFAs were derivatized by 3-NPH and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide(EDC).The effects of reaction time,temperature,concentration of 3-NPH and EDC on the peak area of derivatives were studied to optimize the reaction conditions.11 SCFAs were separated on a Waters Acquity UPLC BEH-C_(18) column in Waters Acquity UPLC system with a mobile phase of acetonitrile-methanol(2︰1,V/V)and water containing 0.1%formic acid.The derivatives were detected in positive ESI,multiple reaction monitoring(MRM)mode.Results:The optimal reaction condition was as follows:25 mmol·L^(−1) of 3-NPH as derivatization reagent,30 mmol·L^(−1) of EDC as catalyst,incubated at 40℃for 30 min.11 SCFAs derivatives could be baseline separated in 11 minutes.The peak area showed good linearity with the concentration,of which the correlation coefficient was larger than 0.999.The intra-day and inter-day precision were good with RSD lower than 15%.The extraction efficiencyrangedfrom80%to120%.Conclusion:The derivativation method has advantages of mild reaction conditions and simple operations.The UPLC-ESI-MS/MS method is accurate,easy and reproducible,which can be applied for the quantitation of SCFAs in human plasma.