Analytical Validation of β‐Hydroxybutyrate Quantification in Feline Serum with the BT3500 Analyzer

Background: Quantitative measurement of serum β-hydroxybutyrate (β-OHB) is essential for detecting and monitoring ketosis and ketoacidosis in diabetic cats, particularly in the era of sodium-glucose cotransporter two (SGLT2) inhibitors, where euglycemic ketoacidosis may occur. The BT3500 analyzer has not been analytically validated for β-OHB quantification in feline serum. Objectives: Analytically validate the Randox Ranbut enzymatic β-OHB assay on feline serum using the BT3500 chemistry analyzer, including evaluation of sample stability under refrigerated and frozen storage conditions. Methods: Residual feline serum samples and pooled sera were used to assess intra-assay and inter-assay precision, spike-recovery accuracy across five concentration increments, linearity under serial dilution, and analytical sensitivity (LoB, LoD, LoQ). Short-term stability was evaluated at 4°C for 7 days and long-term stability at -20°C at predefined time points up to 360 days. Results: Intra-assay coefficient of variation (CV%) ranged from 1.68% to 14.48% and inter-assay CV% from 3.08% to 10.20%, with higher imprecision at low concentrations. Recoveries ranged from 110% to 122% (mean 114% ± 4%). Linearity was excellent (slope 1.012; R2 ≥ 0.9998). LoB, LoD, and LoQ were 0.044, 0.091, and 0.150 mmol/L, respectively. β-OHB was stable at 4°C up to 6 days and at -20°C up to 180 days, while decreases exceeding the acceptance limit were observed at 360 days. Conclusions: The Randox β-OHB assay on the BT3500 demonstrated acceptable analytical performance in feline serum and is suitable for routine clinical use; frozen storage beyond 180 days may impact result accuracy.