Rapid, sensitive and facile measurement of intra- and extracellular lactate and pyruvate in cells by LC-MS/MS

Lactate and pyruvate are essential metabolites of cellular function. They are involved in mitochondrial function and cellular energetics. Their quantification in extra- and intracellular matrices is crucial to understand mitochondrial alterations that could be associated with certain pathologies, such as cancer. In this study, we present an optimized protocol for the simultaneous quantification of lactate and pyruvate in both intra- and extra-cellular compartments by LC-MS/MS. Lactate and pyruvate were separated on a HILIC stationary phase with isocratic elution in less than 4 min and using a mobile phase of acetonitrile/H₂O (60/40). Recoveries were in the range of 84.15 %-102.85 % and relative standard deviation of intra-day an inter-day precision was lower than 6.53 %. The method was validated according to the accuracy profile method. The lower limit of quantification (LLOQ) was 1.78 μM for lactate and 0.18 μM for pyruvate. This method was applied to extract and quantify the intracellular and extracellular lactate and pyruvate from two respiratory cell lines, BEAS-2B and A549, cultivated in a 24-well plate. The developed technique is fast, sensitive, specific, reproducible and could be adaptable to various cellular models.