HPLC-MS/MS法同时测定大鼠回肠中5-羟色胺和5-羟吲哚乙酸含量

Simultaneous determination of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in rat ileum by HPLC-MS/MS method

目的:建立同时测定大鼠回肠中5-羟色胺和5-羟吲哚乙酸含量的方法。方法:采用高效液相色谱-串联质谱(HPLC-MS/MS)法进行检测。色谱柱为X Bridge BEH C_(18)柱(100 mm×2.1 mm,2.5μm);流动相为0.1%甲酸水(A)-0.1%甲酸乙腈(B),梯度洗脱;流速为0.3 m L·min^(-1);柱温为40℃;进样量为5μL;电喷雾电离源(ESI),正离子模式下多反应监测。结果:5-羟色胺在25~1 000 ng·m L^(-1)范围内线性关系良好(r^(2)=0.999 1),定量下限为25 ng·m L^(-1);5-羟吲哚乙酸在50~2 000 ng·m L^(-1)范围内线性关系良好(r^(2)=0.999 5),定量下限为50 ng·m L^(-1)。5-羟色胺和5-羟吲哚乙酸的日内、日间精密度均小于10%,准确度在96.90%~105.22%范围内,提取回收率均大于90%,稳定性良好,基质影响较小。测得大鼠回肠中5-羟色胺的含量为(2 533.62±225.08) ng·g^(-1),5-羟吲哚乙酸的含量为(1 365.19±69.18) ng·g^(-1),5-羟色胺的转化率为0.54±0.03。结论:该实验建立的方法准确灵敏,快捷高效,能够准确测定大鼠回肠中5-羟色胺和5-羟吲哚乙酸的含量,为探究大鼠回肠中5-羟色胺的代谢情况提供可靠的方法支持。

OBJECTIVE To develop a method of simultaneously determining the content of 5-hydroxyserotonin and 5-hydroxyindoacetic acid in rat ileum.METHODS HPLC-MS/MS method was utilized for detecting.Separation was performed on X Bridge BEH C_(18) column (100 mm×2.1 mm,2.5μm) with a mobile phase of 0.1%formic acid water (A)-0.1%formic acid acetonitrile (B)(gradient elution);flow rate was 0.3 m L·min^(-1);column temperature 40℃;sample size 5μL;electrospray ionization source (ESI),multiple reaction monitoring in positive ion mode.RESULTS 5-HT displayed an excellent linear relationship in the range of 25-1 000 ng·m L^(-1) (r^(2)=0.999 1) with a lower limit of quantification 25 ng·m L^(-1).And 5-HIAA demonstrated an excellent linear relationship in the range of 50-2 000 ng·m L^(-1) (r^(2)=0.999 5) with a lower limit of quantification 50 ng·m L^(-1).Intra/inter-day precision RSDs of 5-HT and 5-HIAA were all under 10%with the accuracies of 96.90%-105.22%.The method has a decent stability and little influence of matrix.The content of 5-HT in ileum was (2 533.62±225.08) ng·g^(-1),the content of 5-HIAA (1 365.19±69.18) ng·g^(-1) and the turnover rate of 5-HT (0.54±0.03).CONCLUSION The method established here is accurate,sensitive,rapid and efficient.It can accurately determine the contents of 5-HT and 5-HIAA and provide reliable supports for exploring the metabolism of 5-HT in rat ileum.