Li Xin, Wang Feng, Xu Bin, Yu Xiaowei, Yang Yang, Zhang Li, Li Huande
Clinical Pharmaceutical Research Institute, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China; The Third Hospital of Changsha, Changsha, Hunan 410015, China.
Clinical Pharmaceutical Research Institute, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Oct 15;969:181-9. doi: 10.1016/j.jchromb.2014.08.002. Epub 2014 Aug 10.
A robust two-dimensional liquid chromatography (2D-LC) method for determining the free and total concentrations of vancomycin in plasma was developed and validated. The 2D-LC system, which exhibited a strong capacity for inhibiting interference, comprised a unique RP1-IEX-RP2 column system and an "Assistant Flow" configuration. Ultrafiltration technology was employed to separate free vancomycin from the protein-bound fraction in human plasma. The influence of ultrafiltration conditions on the free vancomycin concentration was evaluated. The calibration curve was linear over the 0.195-49.92μg/ml range for the free and total vancomycin concentrations. The within- and between-run precision ranges were 1.5-3.9% and 2.0-4.7% for the total concentration, 1.4-3.3% and 2.4-4.0% for the free concentration, respectively. Ultrafiltration was susceptible to variations in the experimental conditions, including the centrifugation time, the centrifugal force, and the nominal molecular weight limit of the ultrafiltration membrane. A total of 101 serum samples from 84 elderly patients were analyzed by this method. The free vancomycin concentration was 5.88±3.75μg/ml (range: 0.240-16.79μg/ml), the total concentration was 12.36±5.36μg/ml (range: 2.16-27.14μg/ml), and the unbound fraction was 45.6±18.8% (range: 11.1-96.9%). There was a poor correlation between the free and total vancomycin concentrations (R(2)=0.596, p<0.05). This method appears to be sensitive, precise, selective, and suitable for use in protein-binding studies of vancomycin.
开发并验证了一种用于测定血浆中万古霉素游离浓度和总浓度的稳健二维液相色谱（2D-LC）方法。该二维液相色谱系统具有强大的干扰抑制能力，由独特的RP1-IEX-RP2柱系统和“辅助流”配置组成。采用超滤技术从人血浆中的蛋白结合部分分离出游离万古霉素。评估了超滤条件对游离万古霉素浓度的影响。游离和总万古霉素浓度的校准曲线在0.195 - 49.92μg/ml范围内呈线性。总浓度的批内和批间精密度范围分别为1.5 - 3.9%和2.0 - 4.7%，游离浓度的批内和批间精密度范围分别为1.4 - 3.3%和2.4 - 4.0%。超滤易受实验条件变化的影响，包括离心时间、离心力和超滤膜的标称分子量极限。用该方法分析了84例老年患者的101份血清样本。游离万古霉素浓度为5.88±3.75μg/ml（范围：0.240 - 16.79μg/ml），总浓度为12.36±5.36μg/ml（范围：2.16 - 27.14μg/ml），未结合分数为45.6±18.8%（范围：11.1 - 96.9%）。游离和总万古霉素浓度之间的相关性较差（R(2)=0.596，p<0.05）。该方法似乎灵敏、精确、具有选择性，适用于万古霉素的蛋白结合研究。
