Quantitative LC-MS/MS profiling of N-Acetylcysteine in chicken plasma: Method validation and pharmacokinetic characterization.

N-Acetylcysteine (NAC), an essential precursor in glutathione synthesis, exhibits broad therapeutic potential but remains pharmacokinetically (PK) poorly characterized in poultry. To address this gap, we developed and validated a novel LC-MS/MS method that incorporates an isotope-labeled internal standard (d³-NAC) and a surrogate matrix (5 mg/mL bovine serum albumin). This approach effectively overcame interference from endogenous thiols through one-step methanol protein precipitation followed by DTT reduction, with chromatographic separation on a C18 column using 0.1 % formic acid-acetonitrile gradient elution (0.3 mL/min). Method validation demonstrated excellent linearity (0.01-4 μg/mL, R² > 0.99), sensitivity (LOD: 0.005 μg/mL; LLOQ: 0.01 μg/mL), accuracy (84.9-114.77 %), and precision (RSD < 10.61 %). Pharmacokinetic studies in broilers revealed distinct profiles under intravenous (IV, 10 mg/kg) and oral administration (10, 20, 40 mg/kg): (1) IV injection achieved rapid high exposure (AUC₀₋ₜ: 14.573 ± 2.2 h·μg/mL) with prolonged elimination (t₁/₂: 3.59 ± 2.59 h). (2) Oral dosing showed dose-proportional absorption (tₘₐₓ: 0.65-0.81 h) but low bioavailability (F: 17.04-22.56 %), though higher doses accelerated absorption (t₁/₂ increased from 1.31 ± 0.19 h at 10 mg/kg to 3.40 ± 2.29 h at 40 mg/kg). These findings establish two clinical application paradigms: (1) IV administration is optimal for acute respiratory crises due to its immediate high plasma concentration. (2) Oral delivery suits chronic supplementation, with dose escalation enhancing absorption efficiency. The validated LC-MS/MS platform provides a robust solution for veterinary drug analysis in biologically complex matrices, while the PK insights directly inform rational NAC regimens in poultry respiratory disease management.