LC–MS/MS Quantification of the Anti-Tuberculosis Macrolide Arenicolide A in Rat Plasma: Method Validation and Pharmacokinetic Application
Abstract
Background/Objectives: Arenicolide A (ArA), a 26-membered macrolide from Micromonospora sp. GR10, is active against Mycobacterium tuberculosis, including multidrug-resistant strains. We developed and validated a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for ArA in rat plasma and applied it to pharmacokinetic studies. Methods: Plasma (50 µL) was processed by protein precipitation, and analyzed using 4 min isocratic C18 chromatography with positive-ion multiple reaction monitoring (m/z 827.5 → 765.5 for ArA; m/z 237.1 → 194.1 for carbamazepine, internal standard). Validation was performed with reference to the International Council for Harmonisation M10 guideline. Pharmacokinetics were evaluated in male Sprague–Dawley rats after intravenous (1 mg/kg) or oral (5 or 10 mg/kg) administration. Passive permeability, aqueous solubility and rat microsomal depletion were also evaluated. Results: The assay was linear over 2.0–2000 ng/mL (r2 > 0.996), with a lower limit of quantification of 2.0 ng/mL. Within- and between-run relative errors were −5.81 to 2.30% and −3.20 to −0.10%, with precision ≤9.01% and ≤10.38%. Extraction recovery was 87.7–96.5%, and matrix effects were consistent across six plasma lots. ArA was stable under pre- and post-preparative conditions. Following intravenous administration, ArA showed a terminal half-life of 0.78 ± 0.08 h and plasma clearance of 26.1 ± 2.5 mL/min/kg. Absolute oral bioavailability was 16.8% and 14.2% at oral doses of 5 and 10 mg/kg, respectively. ArA exhibited high passive permeability, limited aqueous solubility of approximately 0.1 mg/mL, and slow microsomal depletion. Conclusions: This validated method enabled characterization of ArA pharmacokinetics and oral bioavailability. This method supports a reliable analytical platform for further formulation and preclinical evaluation of ArA as an anti-tuberculosis lead.