Simultaneous determination of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in rat ileum by HPLC-MS/MS method
OBJECTIVE To develop a method of simultaneously determining the content of 5-hydroxyserotonin and 5-hydroxyindoacetic acid in rat ileum.METH0DS HPLC-MS/MS method was utilized for detecting.Separation was performed on X Bridge BEH C18 column(100 mm × 2.1 mm,2.5 pm)with a mobile phase of 0.1％formic acid water(A)-0.1％formic acid acetonitrile(B)(gradient elution);flow rate was 0.3 mL-min-1;column temperature 40℃;sample size 5 pL;electrospray ionization source(ESI),multiple reaction monitoring in positive ion mode.RESULTS 5-HT displayed an excellent linear rela-tionship in the range of 25-1 000 ng·mL-1(r2=0.999 1)with a lower limit of quantification 25 ng·mL-1.And 5-HIAA demon-strated an excellent linear relationship in the range of 50-2 000 ng-mL-1(r2=0.999 5)with a lower limit of quantification 50 ng·mL-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 tumover 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 pro-vide reliable supports for exploring the metabolism of 5-HT in rat ileum.
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