Pre-Analytical Considerations in the simultaneous quantification of Ketone Bodies, Lactate, pyruvate and TCA cycle intermediates.

Background Accurate quantification of small metabolites such as ketone bodies (KB: β-hydroxybutyrate (BHB), acetoacetate (AcAc)), pyruvate (Pyr), lactate (Lac) and tricarboxylic acid (TCA) cycle intermediates is essential for diagnostics, therapy monitoring and metabolic research. These metabolites serve as energy substrates and signaling molecules, with their interpretation often relying on physiologically meaningful ratios (Lac/Pyr, BHB/AcAc). However, their chemical instability and susceptibility to rapid post-collection metabolism pose significant pre-analytical challenges.Method We developed an LC-MS/MS method for the simultaneous quantification of KB, Pyr, Lac and TCA cycle intermediates, and systematically evaluated pre-analytical factors affecting their stability and accuracy. We compared lithium-heparin (LH), ethylenediaminetetraacetic acid (EDTA), sodium fluoride/EDTA (NaF/EDTA) and sodium citrate (NaCit) collection tubes and deproteinized whole blood (depWB) using perchloric acid. Stability was assessed in plasma at RT over 24 h, as well as in LH and depWB at various temperatures (RT, 4 °C, -20 °C) over 7 days.Results Pyr, Lac, AcAc and fumarate were most labile, while BHB and citrate were stable across matrices. LH-plasma with prompt centrifugation showed minimal metabolic alterations, while NaF/EDTA effectively stabilized Lac but compromised Pyr and TCA cycle intermediates. DepWB improved Lac/Pyr ratio reliability but introduced higher variability and matrix effects. NaCit induced unexpected metabolic shifts, suggesting in-vitro TCA cycle activity.Conclusions Our findings highlight the critical impact of anticoagulants and processing conditions on metabolite stability. LH-plasma provides the best compromise for quantifying KB, Pyr and TCA cycle intermediates when processed rapidly, while depWB remains preferable for accurate Lac/Pyr ratio determination despite its higher variability.