定量亲和色谱-质谱研究美洛昔康与β-环糊精的相互作用

Determination of the interaction kinetics between meloxicam and β-cyclodextrin using the quantitative high-performance affinity chromatography coupled with mass spectrometry

药物与环糊精的结合速率常数、解离速率常数是药物从给药系统中解离、吸收的基本动力学常数,但现有研究多集中于结合平衡常数K。本文基于定量亲和色谱技术(quantitative high-performance affinity chromatography),发展了测定药物-环糊精超分子表观解离速率常数(koff,app)的定量亲和色谱-质谱方法。采用质谱检测器,经色谱峰拟合获得不同流速下药物和不保留物质尿嘧啶在β-环糊精柱上的保留时间与半峰宽,计算药物与不保留物质尿嘧啶的塔板高度(HR与HM,C);根据药物和不保留物质尿嘧啶的扩散系数差异与静态流动相传质阻抗,计算获得理论不保留物质的塔板高度HM,T。以(HR-HM,T)对uk/(1+k)2进行线性回归,根据斜率计算得到多流速条件下美洛昔康和对照药物(对乙酰氨基酚)的koff,app分别为0.13±0.00和4.83±0.10 s-1。根据美洛昔康的结合平衡常数K(12.53 L·mol-1),计算得到表观结合速率常数kon,app为1.63 L·mol-1·s-1。本法简便快速、重复性好,为评价药物与β-环糊精相互作用动力学提供了新的方法。

The association rate constant and dissociation rate constant are important parameters of the drug-cyclodextrin supermolecule systems, which determine the dissociation of drugs from the complex and the further in vivo absorption of drugs. However, the current studies of drug-cyclodextrin interactions mostly focus on the thermodynamic parameter of equilibrium constants（K）. In this paper, a method based on quantitative high performance affinity chromatography coupled with mass spectrometry was developed to determine the apparent dissociation rate constant（koff,app） of drug-cyclodextrin supermolecule systems. This method was employed to measure the koff,app of meloxicam and acetaminophen. Firstly, chromatographic peaks of drugs and non-retained solute（uracil） on β-cyclodextrin column at different flow rates were acquired, and the retention time and variance values were obtained via the fitting the peaks. Then, the plate heights of drugs（HR） and uracil（HM,C） were calculated. The plate height of theoretical non-retained solute（HM,T） was calculated based on the differences of diffusion coefficient and the stagnant mobile phase mass transfer between drugs and uracil. Finally, the koff,app was calculated from the slope of the regression equation between（HR-HM,T） and uk/（1＋k）2,（0.13 ± 0.00） s-1 and（4.83 ± 0.10） s-1 for meloxicam and acetaminophen（control drug）, respectively. In addition, the apparent association rate constant（kon,app） was also calculated through the product of K（12.53 L·mol-1） and koff,app. In summary, it has been proved that the method established in our study was simple, efficiently fast and reproducible for investigation on the kinetics of drug-cyclodextrin interactions.