Quantification of microbiota-related phenols and aromatic acids in mouse feces of a diabetic nephropathy model by simultaneous BDAPE derivatization using ultra-performance liquid chromatography-tandem mass spectrometry.

Abstract

In the intestine, several phenols and aromatic acids are generated by microbiota and are highly related to the formation of uremic toxins. Herein, we developed a new derivatization reagent, 2-bromo-1-[4-(dimethylamino)phenyl] ethyl ketone (BDAPE), that reacted simultaneously with phenols and aromatic acids. Following a reaction within 2h at 60°C in the presence of 200mM potassium carbonate (K2CO3), the obtained BDAPE derivatives were separated on a reversed-phase C18 column and quantified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in positive electrospray ionization mode. This method allowed a lower limit of quantification (LLOQ) of 0.090μΜ for 3-indolepropionic acid (3IPA), indole-3-acetic acid (3IAA), p-cresol (PC), benzoic acid (BA), and phenol (PN); 0.30μΜ for phenylacetic acid (PAA); 0.45μΜ for 4-hydroxyphenylacetic acid (4HPAA); and 0.60μΜ for 3-phenylpropionic acid (PPA). Methodological validation further demonstrated acceptable accuracy (%RE < 16.1) and precision (%RSD < 16.2), suggesting that this is a sensitive and robust method for simultaneous quantification of phenols and aromatic acids. The method was successfully applied to analyze these microbiota-related analytes in mouse feces of a diabetic nephropathy model. Graphical abstract.