Evaluation of an enzymatic one-step assay for Beta-Hydroxybutyrate blood testing on clinical chemistry platforms using HILIC-MS/MS as a non-enzymatic reference method

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BACKGROUND: β-Hydroxybutyrate (β-HB), the predominant circulating ketone body, serves as alternative energy source during periods of glucose deprivation. Accurate quantification of β-HB is essential for the clinical assessment and monitoring of ketosis and diabetic ketoacidosis (DKA). METHODS: We evaluated the analytical performance of the DiaSys β-Hydroxybutyrate 21 FS assay (BHB 21 FS), a one-step enzymatic assay based on β-hydroxybutyrate dehydrogenase-catalyzed oxidation of β-HB to generate NADH, which is photometrically measured at 340 nm. Performance characteristics, including linearity, repeatability, and total precision, were assessed in serum samples following CLSI guidelines. The BHB 21 FS assay was benchmarked against two commercial two-step assays and a non-enzymatic reference method (HILIC-ESI-MS/MS). RESULTS: The BHB 21 FS assay showed a linear measuring range from 0.05 up to 6.0-8.0 mmol/L and CLSI-compliant total precision (CV ≤3.3 %), notably at the clinically relevant 3 mmol/L cut-off (mean CV: 1.93 %). Method comparison demonstrated excellent correlation with EKF BHB LiquiColor® across three different analyzers (r = 0.999; bias ≤1 %), and high concordance with DiaSys BHB FS (r = 0.998; bias 5.0 %) and the HILIC-MS/MS reference method (r = 0.998; bias 4.0 %). Interference testing indicated robust performance even in highly hemolytic, lipemic, or icteric samples (≤10 % deviation). On platforms with active cooling compartments, calibration and onboard stability were maintained for 10-16 weeks. CONCLUSION: The DiaSys BHB 21 FS assay combines robust analytical performance and high concordance with both enzymatic and mass spectrometric methods. These features support its use as a reliable one-step assay for routine clinical assessment of ketosis and ketoacidosis.