LC-MS/MS quantification of N-lactoyl-phenylalanine and N-lactoyl-tyrosine in human plasma: method validation and clinical application to metformin administration.

N-lactoyl amino acids, including N-lactoyl-phenylalanine (Lac-Phe) and N-lactoyl-tyrosine (Lac-Tyr), have recently attracted interest as endogenous metabolites of potential clinical and metabolic relevance. In particular, Lac-Phe has been implicated in appetite regulation and in the metabolic effects of metformin. However, validated analytical methods for the simultaneous quantification of these endogenous metabolites in human plasma remain limited. In this study, a sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of Lac-Phe and Lac-Tyr in human plasma using a simple protein precipitation procedure with a 50-μL sample volume. To minimize interference from endogenous analytes, an eight-point calibration curve was prepared using an artificial surrogate matrix. The assay showed a calibration range of 0.5-100 ng/mL for both analytes, with a lower limit of quantification of 0.5 ng/mL and a total run time of 5.5 minutes. The method demonstrated acceptable analytical performance for both Lac-Phe and Lac-Tyr, with intra- and inter-day precision values of ≤ 8.8% and mean accuracy ranging from 88.0% to 101.4%. The validated assay was successfully applied to plasma samples from healthy volunteers collected before and after metformin administration. This method provides a reliable analytical tool for the simultaneous quantification of Lac-Phe and Lac-Tyr in human plasma and may facilitate future clinical and pharmacological investigations of lactoyl-amino acid metabolism. Trial Registration: ClinicalTrials.gov Identifier: NCT04335526.