柱前衍生化液相色谱-质谱联用法测定大鼠血浆中5-羟色胺的浓度

目的:建立应用液相色谱-质谱联用(LC-MS/MS)测定大鼠血浆(包括富含血小板血浆)中5-羟色胺浓度的方法,并将其应用于感染后肠易激综合征模型大鼠体内5-HT的测定。方法:血浆样品在加入甲巯咪唑(内标)后用苯甲酰氯作为衍生试剂,以四硼酸钠为催化试剂,室温下进行柱前衍生。样品在甲醇沉淀蛋白后采用ESI电离源在正离子模式下检测,用Agilent Zorbax eclipse plus C18色谱柱,甲醇-水溶液为流动相洗脱,流速为1.0 ml·min^-1。结果:经衍生后5-HT在大鼠富含血小板血浆及血浆中其线性范围及定量限均符合测定要求,日内、日间精密度及基质效应均<15%;肠易激综合征大鼠血浆中游离5-HT相较正常组大鼠显著增高。结论:本方法快速、灵敏、专属性强且重现性好,可用于大鼠血浆中5-HT浓度的测定。

Determination of serotonin concentration in rat plasma by LC-MS/MS with pre-column derivatization

OBJECTIVE To develop a LC-MS/MS method for determination of serotonin concentration in vivo in plasma (and in rat platelet-rich plasma),and to apply the method to study on serotonin concentration in rats with post-inflammatory irritable bowel syndrome.METHODS Benzoyl chloride was used as derivatization reagent after methimazole (internal standard) was added to the samples.Sodium borate was used as catalytic agent.Pre-column derivatization was carried on at room temperature.Samples were proteins precipitated with methanol.Detection was accomplished with ESI source operating in the positive ionization mode,with an Agilent Zorbax eclipse plus C₁₈ column as the analytical column,and methanol and water as mobile phase,which was delivered at a flow rate of 1.0 ml·min^-1.RESULTS Linearity and LLOQ of serotonin after derivatization in rat plasma both met relevant requirements.Inter and intra precision and matrix effect were <15%.Concentration of serotonin in IBS rats was obviously higher than that in control group.CONCLUSION This assay is a rapid,sensitive,selective and reliable for determination of serotonin in rat plasma.