Analysis of 4-Hydroxyphenyllactic Acid and Other Diagnostically Important Metabolites of α-Amino Acids in Human Blood Serum Using a Validated and Sensitive Ultra-High-Pressure Liquid Chromatography-Tandem Mass Spectrometry Method

Abstract

The profile of and dynamic concentration changes in tyrosine, phenylalanine, and tryptophan metabolites in blood are of great interest since they could be considered potential biomarkers of different disorders. Some aromatic metabolites, such as 4-hydroxyphenyllactic, 4-hydroxyphenylacetic, phenyllactic, and 4-hydroxybenzoic acids have previously demonstrated their diagnostic significance in critically ill patients and patients with post-COVID-19 syndrome. In this study, a sensitive method, including serum protein precipitation with methanol and ultra-high-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) detection, was developed and validated for six phenyl- and five indole-containing acids in human serum. The liquid–liquid extraction was also examined, but it demonstrated unsatisfactory results based on analyte recoveries and the matrix effect. However, the recoveries for all analytes reached 100% and matrix effects were not observed using protein precipitation. This made it possible to use deionized water as a blank matrix. The lower limits of quantitation (LLOQs) were from 0.02 to 0.25 μmol/L. The validated method was used for the analysis of serum samples of healthy volunteers (n = 48) to reveal the reference values of the target analytes. The concentrations of the most clinically significant metabolite 4-hydroxyphenyllactic acid, which were revealed using UPLC-MS/MS and a previously developed gas chromatography-mass spectrometry method, were completely comparable. The proposed UPLC-MS/MS protocol can be used in the routine clinical practice of medical centers.

Keywords

• microbial metabolites
• 4-hydroxyphenylacetic acid
• 4-hydroxybenzoic acid
• phenyllactic acid
• phenylpropionic acid
• indole-3-acetic acid