De Baere Siegrid, Devreese Mathias, Watteyn Anneleen, Wyns Heidi, Plessers Elke, De Backer Patrick, Croubels Siska
Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium.
J Chromatogr A. 2015 Jun 12;1398:73-82. doi: 10.1016/j.chroma.2015.04.022. Epub 2015 Apr 17.
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.
建立了一种灵敏且特异的方法，用于使用液相色谱结合加热电喷雾电离串联质谱法（LC-MS/MS）定量测定动物血浆、肺组织和肺上皮衬液（PELF）中的加米霉素。样品制备快速、简便，对于鸡、火鸡和小牛血浆，使用Oasis(®) Ostro™ 96孔板进行脱蛋白和去除磷脂步骤；对于猪血浆和火鸡肺组织，使用HybridSPE(®)-磷脂SPE柱；而对于小火鸡的PELF，在碱性介质中用乙醚进行液液萃取。色谱分析在C18 Hypersil GOLD柱上进行，流动相为pH 9的0.01M醋酸铵水溶液和乙腈。MS/MS仪器在正电喷雾电离模式下运行，对加米霉素监测以下选择反应监测转换（质子化分子>产物离子）：m/z 777.45>619.35用于定量，m/z 777.45>157.80用于鉴定。该方法在内部进行了验证：制备了基质匹配校准曲线，在所测试的浓度范围内（鸡、猪和小牛血浆为2.5 - 10,000 ng mL⁻¹；火鸡血浆为5.0 - 2500 ng mL⁻¹；火鸡肺组织为50 - 10,000 ng g⁻¹；火鸡PELF为20 - 1000 ng mL⁻¹）实现了良好的线性（r≥0.99）。鸡、猪和小牛血浆的定量限（LOQ）为2.5 ng mL⁻¹，火鸡血浆的定量限为5.0 ng mL⁻¹，而检测限（LOD）在0.007至0.07 ng mL⁻¹之间。对于肺组织和PELF，分别获得了50 ng g⁻¹和0.76 ng g⁻¹（肺组织）以及20 ng mL⁻¹和0.1 ng mL⁻¹（PELF）的定量限和检测限。日内和日间精密度结果以相对标准偏差（RSD）表示，均在最大RSD值范围内。准确度在理论浓度的-30%至+10%（浓度为1 - 10 ng mL⁻¹）或-20%至+10%（浓度>10 ng mL⁻¹或ng g⁻¹）范围内。该方法成功应用于鸡、火鸡、猪和小牛血浆样品以及火鸡肺组织和PELF中加米霉素的定量测定，所有样品均来自这些动物物种的药代动力学研究。
