Comprehensive LC-MS/MS quantification of TCA cycle metabolites in rat biofluids and human hepatic organoids: Application to binge ethanol-induced metabolic dysregulation

Endogenous tricarboxylic acid (TCA) cycle metabolites are critical biomarkers of metabolic perturbation; however, their accurate quantification is complicated by the absence of analyte-free matrices, variable endogenous backgrounds, and matrix effects. We developed and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for 10 TCA metabolites across rat urine (Method 1), rat plasma (Method 2), and human hepatic organoid medium (Method 3). Water was used to prepare calibrators for each matrix, and the validity of this approach was rigorously evaluated with assessments of parallelism and matrix effects. Validation demonstrated selectivity, sensitivity, accuracy, and precision with acceptable linearity across the calibration ranges in all matrices. The developed methods were applied to both in vivo and in vitro models of ethanol exposure in rats (4 g/kg ethanol, 40% v /v, oral gavage at 12 h intervals for 3 days) and human hepatic organoids (200 mM ethanol for 24 h). The analyses revealed that ethanol induced distinct matrix-specific perturbations of the TCA cycle. In rats, the disruption pattern was consistent with a bottleneck in specific TCA-related enzyme activities, whereas in human hepatic organoids, the alterations highlighted impaired energy metabolism and compromised hepatic function. These findings highlight the importance of accurate quantification of TCA cycle metabolites in both target organs and peripheral fluids. Matrix-specific LC-MS/MS methods enable reliable quantification of endogenous TCA cycle metabolites and support mechanistic interpretation of ethanol-related metabolic dysregulation. • Targeted LC-MS/MS quantification of 10 TCA metabolites across three matrices. • Matrix-specific gradients achieved baseline separation of citric and isocitric acids. • Parallelism evaluation demonstrated the suitability of water as the surrogate matrix. • Applied to ethanol exposure models, revealing matrix-specific TCA disruptions.