Development and Validation of a Rapid LC-MS/MS Method for Quantifying Eravacycline in Epithelial Lining Fluid: Application to a Prospective Pulmonary Distribution Study in HAP/VAP Patients.

Background: Eravacycline exhibits potent activity against multidrug-resistant pathogens and holds promise for the management of hospital-acquired and ventilator-associated pneumonia (HAP/VAP). However, sensitive and robust bioanalytical methods to quantify eravacycline in human pulmonary epithelial lining fluid (ELF) for pharmacokinetic (PK) and pulmonary penetration studies in these infections remain limited. Methodology: A simple, rapid, and sensitive LC-MS/MS method was developed for the quantification of eravacycline in bronchoalveolar lavage fluid (BALF). Using urea as a volume normalizer, ELF concentrations were calculated from the eravacycline concentrations in BALF. This method was applied in a clinical study evaluating the pulmonary penetration after intravenous infusion in patients with HAP and VAP. Results: The developed LC-MS/MS method exhibited good linearity in the range of 1-200 ng/mL for quantifying eravacycline in BALF. In BALF, intra-day precision ranged from 1.4% to 6.0%, and inter-day precision from 1.6% to 9.9%, with accuracy between 98.0% and 102.4%. Matrix effects were within 97.4% to 107.6% for BALF samples from six different individuals, with extraction recoveries ranging from 103.5% to 107.2%. Stability studies demonstrated that eravacycline remained stable under various conditions, including storage at room temperature, freeze-thaw cycles, long-term (-70 °C) storage, and post-treatment handling. The method was successfully applied to clinical samples from four HAP or VAP patients, with measured eravacycline pulmonary penetration ratios of 4.29, 17.40, 5.22 and 4.70, indicating efficient pulmonary distribution. The measured eravacycline concentrations ranged from 0.0243 to 0.0436 μg/mL in BALF. The corresponding urea-corrected ELF concentrations ranged from 0.570 to 1.617 μg/mL. Conclusions: This study described a detailed and validated method for quantifying eravacycline concentrations in ELF from patients, providing a reliable analytical approach for investigating the pulmonary distribution of eravacycline.