双重吸附结合HPLC-MS/MS法测定生物基质中内源性组胺及组氨酸
Quantitative determination of endogenous histamine and histidine in biological matrices by double adsorption based on HPLC-MS/MS
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摘要: 首次采用双重吸附结合高效液相色谱-串联质谱（HPLC-MS/MS）建立了同时定量分析生物基质中组胺及其前体组氨酸的方法。内标为2，5-二羟基苯甲酸（DHB），血浆和脑组织匀浆液经3倍乙腈沉淀蛋白后，取上清液进样分析；并采用氨基色谱柱（ODS-SPXBridge? Amide）对目标成分进行分离分析，以0.1%甲酸和1 mmol/L甲酸铵水和乙腈进行梯度洗脱；质谱检测采用ESI离子源在多反应监测（MRM）模式下定量分析。为了提高检测的专属性和准确度，首次对生物基质采用活性炭和方解石进行双重吸附；并以吸附后的基质进行方法学考察，结果显示，组胺及组氨酸在定量范围内线性良好（相关系数r≥0.999），准确度、精密度、提取回收率、基质效应和稳定性均满足生物样本分析要求，该方法不仅可用于生物样品中组胺和组氨酸的同时定量分析，还为其他内源性物质提供了共性的检测技术。Abstract: An innovative approach to quantitatively analyze the histamine and its precursor histidine simultaneously in biological matrices was established for the first time based on double adsorption combined with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).The internal standard was 2-dihydroxybenzoic acid (DHB).The plasma and brain tissue homogenate was protein precipitated with 3-fold acetonitrile, and the supernatant was then sampled for injection analysis.The chromatographic separation of the target components was achieved on an amino chromatography column (ODS-SPXBridge? Amide).Gradient elution was carried out with the mobile phase consisting of solvent A (0.1% formic acid and 1mmol/L ammonium formate in water) and solvent B (acetonitrile).Mass spectrometry was employed for quantitative analysis with ESI ion source in multiple reaction monitoring (MRM) mode.In order to improve the specificity and accuracy, activated carbon and calcite were used for the double adsorption of biological matrices for the first time.The adsorbed matrix was then used for methodology validation.The results showed that histamine and histidine were linear in the quantitative range (correlation coefficient r ≥ 0.999).Accuracy, precision, extraction recovery, matrix effect and stability all met the requirements of biological sample analysis.All results suggested that the present method could not only be efficiently and reliably used for simultaneous quantitative analysis of histamine and histidine in biological samples, but also provide reference for the detection of other endogenous substances.
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