Ultrahigh performance liquid chromatography mass spectrometry for the determination of azithromycin in canine serum and skin tissue

A simple and reproducible UHPLC-MS assay was developed and validated for quantifying azithromycin in canine serum and skin tissue homogenates. The method employed a Waters Acquity UHPLC with a Quattro Micro triple-quadrupole mass spectrometer operating in positive electrospray mode. Because azithromycin exhibited weak fragmentation between 5 and 30 eV, quantification was performed using the precursor ion ([M + H]⁺, m/z 749.25) in selected-ion recording (SIR) mode, while roxithromycin ([M + H]⁺, m/z 838.6) served as the internal standard. Chromatographic separation on an HSS C18 column (2.1 × 100 mm, 1.7 μm) followed protein precipitation with acetonitrile for sample cleanup. The assay was linear over 0.02-1.2 μg/mL (r² > 0.99), with accuracy and precision within ±15% and recovery >85% in both matrices. The lower limit of quantification was 0.02 μg/mL, meeting ICH M10 validation criteria. Application to clinical samples from dogs with Pythium insidiosum and Paralagenidium karlingii infections showed mean azithromycin concentrations of 0.702 μg/mL in serum and 51.8 μg/g in diseased skin following tissue homogenization and dilution prior to analysis, demonstrating extensive tissue accumulation consistent with azithromycin's pharmacokinetic properties. This validated assay provides a sensitive and cost-effective approach for veterinary and translational pharmacokinetic investigations.