Validation of an LC–MS/MS Method for Urinary Cortisol in Dogs and Reference Interval, with an Exploratory Comparison to Immunoassay

Cortisol measurement in dogs commonly supports the diagnosis of hypercortisolism or hypoadrenocorticism. Unlike serum cortisol, urine cortisol is less affected by daily fluctuations, and collection is non-invasive, making the urine cortisol-to-creatinine ratio (UCCR) a useful diagnostic tool. Immunoassays are commonly used but lack specificity for free, biologically active cortisol. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) is recommended to overcome this limitation. This study aimed to validate an LC-MS/MS method for urinary cortisol measurement, establish a reference interval for UCCR in dogs, and compare the results with the commercial immunoassay system Immulite® 2000 Xpi (Siemens, Cary, NC, USA). A comprehensive method validation was performed. Across different concentrations, the intra- and inter-assay coefficients of variation (CV) ranged from 2.39% to 9.45%, accuracy ranged from 87.7% to 105.5%, and bias varied from −12.3% to 5.53%, which were all within acceptable limits. The method demonstrated excellent linearity (r2 ≥ 0.98), and stability under various conditions was satisfactory (RSD: −12.4% to 13.6%). Interference from endogenous and exogenous corticosteroids was negligible. The established reference interval for UCCR was 0.21–2.84 × 10−6. The exploratory comparison with the immunoassay revealed systematic and proportional differences between methods. The LC-MS/MS method demonstrated excellent analytical performance in terms of accuracy and specificity, and showed suitability for clinical application.