Development and validation of a liquid chromatography–tandem mass spectrometry method for the quantitative determination of gamithromycin in animal plasma, lung tissue and pulmonary epithelial lining fluid

Abstract

A sensitive and specific method for the quantitative determination of gamithromycin in animal plasma, lung tissue and pulmonary epithelial lining fluid (PELF) using liquid chromatography combined with heated electrospray ionization tandem mass spectrometry (LC–MS/MS) was developed. The sample preparation was rapid, straightforward and consisted of a deproteinization and phospholipid removal step using an Oasis® Ostro™ 96-well plate (chicken, turkey and calf plasma) or HybridSPE®-Phospholipid SPE cartridges (pig plasma and turkey lung tissue), while a liquid–liquid extraction with diethyl ether in alkaline medium was used for PELF of turkey poults. Chromatography was performed on a C18 Hypersil GOLD column using 0.01M ammonium acetate in water with a pH of 9, and acetonitrile as mobile phases. The MS/MS instrument was operated in the positive electrospray ionization mode and the following selected reaction monitoring transitions were monitored for gamithromycin (protonated molecule>product ion): m/z 777.45>619.35 and m/z 777.45>157.80 for quantification and identification, respectively. The method was validated in-house: matrix-matched calibration graphs were prepared and good linearity (r≥0.99) was achieved over the concentration ranges tested (2.5–10,000ngmL−1 for chicken, pig and calf plasma; 5.0–2500ngmL−1 for turkey plasma; 50–10,000ngg−1 for turkey lung tissue and 20–1000ngmL−1 for turkey PELF). Limits of quantification (LOQ) were 2.5ngmL−1 for chicken, pig and calf plasma and 5.0ngmL−1 for turkey plasma, while the limits of detection (LOD) ranged between 0.007 and 0.07ngmL−1. For lung tissue and PELF, respective LOQ and LOD values of 50ngg−1 and 0.76ngg−1 (lung tissue) and 20ngmL−1 and 0.1ngmL−1 (PELF) were obtained. The results for the within-day and between-day precision, expressed as relative standard deviation (RSD), fell within the maximal RSD values. The accuracy fell within −30% to +10% (concentrations 1–10ngmL−1) or −20% to +10% (concentrations>10ngmL−1 or ngg−1) of the theoretical concentration.The method was successfully applied for the quantitative determination of gamithromycin in plasma samples of chickens, turkeys, pigs and calves; and in lung tissues and PELF of turkeys, all derived from pharmacokinetic studies in these animal species.