Bioassay and liquid chromatography–mass spectrometry are not equivalent in itraconazole therapeutic drug monitoring in dogs

Objective: To compare itraconazole therapeutic drug monitoring (TDM) results by culture-based bioassay versus liquid chromatography-mass spectrometry (LC-MS) in dogs with blastomycosis and determine how the choice of assay might influence dosage adjustments. Methods: This was a retrospective study. Ninety-seven samples from 14 client-owned dogs with blastomycosis were collected from October 2023 through May 2025 and analyzed by bioassay and 3 LC-MS-derived variables: itraconazole alone (ITZ), the hydroxyitraconazole metabolite, and the sum of itraconazole and hydroxyitraconazole. Linear mixed models were used to assess the relationship between dosage and itraconazole concentration by bioassay and LC-MS and between bioassay and the LC-MS variables. A clinical concordance grid was used to assess agreement between bioassay and ITZ by LC-MS regarding dosage-adjustment decisions. Results: There was no significant association between itraconazole dosage and serum concentration. There was a significant relationship between concentration by bioassay versus LC-MS (P < .01). The association was weak for ITZ (r2 = 0.40) but was moderate for the hydroxyitraconazole metabolite (r2 = 0.48) and the sum of itraconazole and hydroxyitraconazole (r2 = 0.48). Of 97 samples, 62 (64%) demonstrated agreement in dosage adjustment, and 35 (36%) did not. Conclusions: Dosage of itraconazole does not reliably predict serum itraconazole concentration, requiring TDM for effective dosage-adjustment decisions. Bioassay and LC-MS are not interchangeable assays for itraconazole TDM in dogs. Clinical Relevance: TDM should be considered during long-term itraconazole therapy to optimize dose. A single assay method (bioassay or LC-MS) should be used consistently throughout the entire course of antifungal therapy.