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.

Feline Infectious Peritonitis (FIP) has been transformed from a fatal disease to a treatable condition following the introduction of GS-441524, a nucleoside analogue targeting feline coronavirus replication. However, the widespread use of unregulated compounded formulations and the absence of validated analytical tools for therapeutic drug monitoring (TDM) represent critical gaps in clinical FIP management. This study describes the development and full ICH M10-compliant validation of a high-throughput LC-MS/MS method for the quantification of GS-441524 in feline serum, incorporating an automated protein precipitation protocol and a PBS-BSA surrogate matrix in accordance with 3Rs principles. The method met all acceptance criteria across validated parameters, including linearity (0.1–50 µg/mL), accuracy (bias within ±12.5%), precision (CV ≤10.9%), selectivity, extraction recovery (87.5–107.9%), and stability under clinically relevant storage conditions. Matrix equivalence between PBS-BSA and authentic feline serum was confirmed, enabling routine calibration without animal-derived materials. The validated method was applied to clinical TDM in cats undergoing GS-441524 treatment for FIP, providing preliminary evidence of inter-individual pharmacokinetic variability. The compounded formulations administered to the TDM cohort were independently verified by LC-MS/MS, confirming drug content within ±15% of labelled claims and excluding pharmaceutical quality as a confounding factor in the interpretation of serum drug concentrations.

Feline Infectious Peritonitis (FIP) has been transformed from a fatal disease to a treatable condition following the introduction of GS-441524, a nucleoside analogue targeting feline coronavirus replication. However, the widespread use of unregulated compounded formulations and the absence of validated analytical tools for therapeutic drug monitoring (TDM) represent critical gaps in clinical FIP management. This study describes the development and full ICH M10-compliant validation of a high-throughput LC-MS/MS method for the quantification of GS-441524 in feline serum, incorporating an automated protein precipitation protocol and a PBS-BSA surrogate matrix in accordance with 3Rs principles. The method met all acceptance criteria across validated parameters, including linearity (0.1–50 µg/mL), accuracy (bias within ±12.5%), precision (CV ≤ 10.9%), selectivity, extraction recovery (87.5–107.9%), and stability under clinically relevant storage conditions. Matrix equivalence between PBS-BSA and authentic feline serum was confirmed, enabling routine calibration without animal-derived materials. The validated method was applied to clinical TDM in cats undergoing GS-441524 treatment for FIP, providing preliminary evidence of inter-individual pharmacokinetic variability. The compounded formulations administered to the TDM cohort were independently verified by LC-MS/MS, confirming drug content within ±15% of labelled claims and excluding pharmaceutical quality as a confounding factor in the interpretation of serum drug concentrations.

Pheochromocytomas (PHEO) are rare adrenal medullary tumours characterised by catecholamine secretion. Accurate diagnosis relies on the sensitive measurement of metanephrine (MN) and normetanephrine (NMN); however, the application of liquid chromatography–mass spectrometry (LC–MS/MS) in veterinary medicine remains limited. This study aimed to develop and analytically validate an LC–MS/MS method for the simultaneous quantification of urinary MN and NMN in dogs, establish reference intervals (RIs) for MN- and NMN-to-creatinine ratios (uMN:CR and uNMN:CR), and preliminarily assess the diagnostic utility of uNMN:CR for PHEO detection. The RIs were 2.2–78.9 nmol/mmol for uMN:CR and 4.4–77.4 nmol/mmol for uNMN:CR. Dogs with PHEO showed significantly higher uNMN:CR values compared with dogs with other adrenal masses, non-adrenal disease, or healthy controls. Using the upper RI limit as a threshold, sensitivity and specificity were 91.7% and 35.7%, respectively. Receiver operating characteristic analysis identified an optimal uNMN:CR cut-off of 203.7 nmol/mmol, yielding 78.6% sensitivity, 83.3% specificity, and an overall diagnostic accuracy of 85% (95% CI: 0.68–1.00). These findings support LC–MS/MS as a reliable approach for urinary metanephrine analysis and uNMN:CR as a valuable biochemical tool for PHEO identification in dogs.