超高效液相色谱快速检测万古霉素浓度方法的建立和性能评价

目的建立一种基于超高效液相色谱(UPLC)技术快速测定血清万古霉素浓度的方法并评价其性能。方法以去甲万古霉素为内标,以0.3 mol/L硫酸锌水溶液为蛋白沉淀剂,对血清样本进行前处理。色谱柱为Phenomenex Kinetex C18柱(100 mm×2.1 mm,2.6μm),流速为0.5 mL/min,检测波长为220 nm,柱温40℃。流动相A为25 mmol/L磷酸二氢钾缓冲液(pH值为2.5),流动相B为乙腈,进行梯度洗脱,并对方法的线性、准确度(加标回收率)、精密度、携带污染率、定量限和检测限等分析性能进行评价。分别采用UPLC和高效液相色谱(HPLC)测定50例患者血清万古霉素浓度,并对测定结果进行方法学比对和评价。结果UPLC测定万古霉素的线性范围为2.0~99.6μg/mL,定量限和检测限分别为1.0和0.1μg/mL。低、中、高浓度样本的平均批内变异系数(CV)分别为3.28%、2.21%、2.59%,批间CV分别为5.73%、2.75%、0.82%。低浓度(2.0μg/mL)、中浓度(19.9μg/mL)、高浓度(79.6μg/mL)加标样本的平均加标回收率分别为104.06%、99.80%、100.19%。中浓度样本的携带污染率为0.57%,高浓度样本的携带污染率为0.19%。UPLC和HPLC测定患者血清万古霉素浓度具有良好的相关性(r=0.9919)。结论建立了基于UPLC技术的万古霉素血药浓度监测方法,该方法的重现性好、快速、简便、灵敏度高、准确度高,适用于临床常规万古霉素药物浓度监测。

Establishment on UPLC and its performance evaluation for the rapid determination of vancomycin concentration

Objective To establish ultra performance liquid chromatography(UPLC)for the rapid determination of vancomycin concentration,and to evaluate the performance.Methods Norvancomycin was used as internal standard,serum sample was treated with 0.3 mol/L aqueous solution of zinc sulfate,and a simple protein precipitation method was used for pre-treating serum samples.The chromatography was carried out on Phenomenex Kinetex C18 column(2.6μm,100 mm×2.1 mm),the flow rate was 0.5 mL/min,UV detector wavelength was 220 nm,and column temperature was 40℃.Using mobile phase acetonitrile-25 mmol/L potassium dihydrogen phosphate buffer(pH value was 2.5),gradient elution was performed.The linearity,accuracy(recovery rate),precision,carry-over rate,limit of quantitation and limit of detection were evaluated.A total of 50 clinical samples were determined by the present UPLC method and the validated high performance liquid chromatography(HPLC)method,and the results were compared using linear regression analysis.Results The linear range for serum concentration of vancomycin was 2.0-99.6μg/mL,with 0.1μg/mL detection limit and 1.0μg/mL quantitation limit.The within-run coefficients of variation(CV)of low-,middle-and high-levels were 3.28%,2.21%and 2.59%,and the between-run CV were 5.73%,2.75%and 0.82%,respectively.The recovery rates of low-level(2.0μg/mL),middle-level(19.9μg/mL)and high-level(79.6μg/mL)were 104.06%,99.80%and 100.19%,respectively.The carry-over rate of middle-and high-levels were 0.57%and 0.19%,respectively.There was a correlation between the 2 methods(r=0.9919).Conclusions The established UPLC for the determination of vancomycin concentration is reproducible,simple,sensitive and accurate,which is suitable for monitoring clinic serum vancomycin concentration and experimental subject research.