Conclusions
The developed method is highly sensitive and robust. It has been validated for use with human serum and plasma, overcoming stability concerns and providing a reliable and efficient quantitative estimation of ketone bodies.
Methods
A novel 6.5-minute liquid chromatography-mass spectrometry-based assay was developed, enabling the precise measurement of alpha-, beta- and gamma-hydroxybutyrate, beta-hydroxyisobutyrate, and acetoacetate. This method was fully validated for human serum and plasma samples by investigating extraction efficiency, matrix effects, accuracy, recovery, intra- and inter-precision, linearity, lower limit of quantitation (LLOQ), carryover, specificity, stability, and more. From 107 normal samples, reference ranges were established for all analytes and the beta-hydroxybutyrate/acetoacetate ratio.
Results
All five analytes were adequately separated chromatographically. An extraction efficiency between 80 and 120 % was observed for all KBs. Accuracy was evaluated through spike and recovery using 10 random patient samples, with an average recovery of 85-115 % for all KBs and a coefficient of variation of ≤ 3 %. Coefficients of variation for intra- and inter-day imprecision were < 5 %, and the total imprecision was < 10 %. No significant interferences were observed. Specimens remained stable for up to 6 h on ice or 2 h at room temperature. Conclusions: The developed method is highly sensitive and robust. It has been validated for use with human serum and plasma, overcoming stability concerns and providing a reliable and efficient quantitative estimation of ketone bodies.