Simultaneous determination of indole metabolites of tryptophan in rat feces by chemical labeling assisted liquid chromatography-tandem mass spectrometry

As the connecting part of diet and host physiology,intestinal microbes can convert the ingested diet into a huge number of physiologically active small molecules.Indole metabolites of tryptophan are precursors or signal molecules for many biologically active substances,which are involved in serotonin and microbial catabolism pathways.To understand the influence of tryptophan metabolism in the intestinal environment on the neurological and immune systems at the molecular level,it is important to establish a high-coverage analytical method to comprehensively analyze the metabolites involved in tryptophan metabolism.However,due to a small molecular weight and poor response during mass spectrometry analysis,as well as weak retention on the reversed-phase chromatography,determination of indole metabolites of tryptophan is challenging.Here,we proposed a method for the simultaneous determination of 20 indole metabolites of tryptophan in a single run on reversed-phase chromatography by chemical labeling coupled to liquid chromatography-tandem mass spectrometry analysis.4-(Dimethylamino)benzaldehyde(DMAB)was used for the labeling of indole metabolites of tryptophan,which could significantly improve the detection sensitivities and retention of these metabolites on reversed-phase chromatography.With the developed method,we realized the sensitive detection and comprehensive analysis of 15 endogenous indole metabolites of tryptophan in rat feces samples with functional dyspepsia intervention by acupuncture.The developed method offers a useful tool for studying tryptophan metabolism-related diseases.

Distribution in tissue and excretion in urine and feces of swertisin after intravenous administration to rats

Swertisin contents in rat urine,feces and tissues were determined by reversed-phase high-performance liquid chromatography（RP-HPLC） method.Chromatographic separations were performed on a C18 column with acetonitrile-water（23：77,v/v） as the mobile phase.The calibration curves were linear over the ranges of 0.175-35.0μg/mL for rat urine,0.5-60.0μg/mL for rat feces,and 0.014 to 53.0μg/mL for all tissues.The inter-and intra-day precisions and accuracy for all measured samples were satisfactory.The fully validated method was applied for tissue distribution and excretion of swertisin in rat urine and bile after intravenous administration.The maximum level of swertisin was found in kidney,which reached 83.87± 6.36μg/g.In rat heart,swertisin was hardly detected under used experimental conditions.Swetisin level in liver,kidney,stomach,smooth muscle and skeletal muscle continued to decrease from 5 to 60min.Swertisin showed increasing tendency in intestine,spleen and testis tissues at scheduled time points.Detectable swertisin was found in brain and lung tissue.Totally 11.9% swertisin dose was cumulatively excreted from urine in 60h after intravenous administration.There was small amount of swertisin in rat feces and the cumulative excretion level reached 4.59% of intravenous dose in 60h.