UPLC-MS/MS测定牛血浆中加米霉素方法的建立

Establishment of Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Determination of Gamithromycin in Cattle Plasma

关键词:  加米霉素  超高效液相色谱串联质谱  血浆  牛

Key words:  Gamithromycin  UPLC-MS/MS  plasma  cattle

摘要:

建立牛血浆中加米霉素含量的UPLC-MS/MS检测方法。血浆样品经甲醇沉淀蛋白，外标法定量, 使用Waters ACQUITY UPLC HSS T3色谱柱，以甲醇：10 mM乙酸铵缓冲液为流动相，流速0.3 mL?min-1；柱温：50℃；进样量：10 μL。三重四级杆质谱仪电喷雾离子源（ESI），正离子扫描、多反应监测（MRM）。考察方法的线性范围、准确度与精密度、稳定性和稀释可靠性。结果显示，加米霉素出峰时间在1 min左右，本方法在1 ng?mL-1~100 ng?mL-1浓度范围内呈良好的线性关系，相关系数（r）≥0.999，检测限（LOD）和定量限（LOQ）分别为0.5 ng?mL-1和1 ng?mL-1。3个浓度（1 ng?mL-1、50 ng?mL-1和100 ng?mL-1）的空白血浆添加样品批内和批间平均回收率分别在85.2%～96.4%和87.7%～94.0%之间，相对标准偏差（RSD）分别在1.4%～13.9%和2.9%～8.6%之间。准确度和精密度、稳定性及稀释可靠性均符合生物样品分析的相关要求。本方法可用于牛的血浆中加米霉素的浓度测定及药代动力学研究。

Abstract:

To establish a UPLC-MS/MS method for the determination of gamithromycin in cattle plasma, the protein in the plasma samples was precipitated by methanol, and the gamithromycin concentration was quantified by external standard method. Using methanol:10 mM ammonium acetate buffer as mobile phase, and the quantitative analysis is carried out by triple quadrupole mass spectrometer with positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) mode. The linear range, accuracy and precision, stability and dilution reliability of the method were investigated. The results show that the linear relationship in the concentration range of 1 ng?mL-1~100 ng?mL-1 was illustrious, correlation coefficient (r) was greater than 0.999, and the LOD and LOQ was 0.5 ng?mL-1 and 1 ng?mL-1, respectively. At the added concentration of 1 ng?mL-1, 50 ng?mL-1, and 100 ng?mL-1, the within-run and between-run mean recovery rate of gamithromycin were ranged from 85.2% to 96.4% and 87.7% to 94.0%, respectively, and the relative standard deviation (RSD) were ranged from 1.4% to 13.9% and 2.9% to 8.6%, respectively. The accuracy, precision, stability, and dilution reliability all meet the relevant requirements of biological sample analysis. The method is simple, sensitive, rapid, and accurate, which is appropriate for determine the concentration of gamithromycin in cattle plasma and use in pharmacokinetics study.