A novel strategy for high-specificity, high-sensitivity, and high-throughput study for gut microbiome metabolism of aromatic carboxylic acids

Aromatic carboxylic acids(ACAs) may be as transformed key metabolites via gut microbiome for playing better pharmacological effects. However, it's rare to achieve high-specificity, high-sensitivity, and highthroughput detection simultaneously, especially, for tracing trace ACAs in gut microbiome. In this work,firstly, a novel dual-template and double-shelled molecularly imprinted 96-well microplates(DDMIPs)was designed and amplified signal for p-hydroxybenzoic acid(PBA) and 3,4,5-trimethoxycinnamic acid(TMA). Additionally, the DDMIPs and a stable isotope labeling derivatization(SILD) method combined with the ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry(UHPLC-TQ MS) was firstly stepwise integrated, achieving high-effective, high-sensitive, and highthroughput study of gut microbiome metabolism. The whole strategy showed lower limits of detections(LODs) up to 1000 folds than the traditional method, and revealed a more real metabolism-time profile of PBA and TMA by 3-step signal amplification. The platform also laid the foundation for fast, simple,high-selective, high-effective, and high-throughput metabolism and pharmacological research.