赵海燕,黄婷婷,陆金玲,陈宇昕,王少兵.基于UPLC-MS法快速定量检测人血浆中短链脂肪酸[J].中南民族大学学报自然科学版,2022,41(3):292-299
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
  
DOI:10.12130/znmdzk.20220306
中文关键词: 短链脂肪酸  人血浆  化学衍生化  3-硝基苯肼  超高效液相色谱-质谱联用技术
英文关键词: short chain fatty acids  human plasma  chemical derivatization  3-NPH  UPLC-ESI-MS/MS
基金项目:国家自然科学基金资助项目(81703464);中央高校基本科研业务费专项资金资助项目(CZD20005)
作者单位
赵海燕 中南民族大学 药学院武汉 430074 
黄婷婷 中南民族大学 药学院武汉 430074 
陆金玲 中南民族大学 药学院武汉 430074 
陈宇昕 中南民族大学 药学院武汉 430074 
王少兵 中南民族大学 药学院武汉 430074 
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中文摘要:
      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 C18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 3-NPH作为衍生化试剂和30 mmol/L 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(5-50000 ng/mL)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.
英文摘要:
      Objective: The chemical derivatization of 3-nitrophenylhydrazine (3-NPH) was coupled with ultra-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-C18 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 of 3-NPH as derivatization reagent, 30 mmol/L 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 ranging from 5 to 50000 ng/mL, 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 efficiency ranged from 80% to 120%.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.
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