Institute for Risk Assessment Sciences, Utrecht University, PO Box 80177, NL-3508 TD Utrecht, The Netherlands.
J Chromatogr A. 2011 Nov 25;1218(47):8529-35. doi: 10.1016/j.chroma.2011.09.064. Epub 2011 Sep 29.
The addition of proteins to in vitro systems influences the free concentration of the test compound in the medium. The objective of this study was to set up a negligible depletion-solid phase microextraction method, coupled to high-performance liquid chromatography (nd-SPME-HPLC) to measure the free concentration of chlorpromazine (CPZ) in medium containing albumin. The nd-SPME method was optimized for coating thickness (polyacrylate coating) and exposure time, and potential effects from the addition of bovine serum albumin (BSA) were studied. It was shown that the addition of albumin did not cause fouling or influenced the uptake kinetics of CPZ into the fiber. At a realistic in vivo albumin concentration of 40 g/L of albumin, 94% of CPZ was protein bound. This is in line with findings in vivo, reporting a protein binding for CPZ of 92-99%. The nd-SPME-HPLC method described in this study can be used to measure the free concentration of chlorpromazine in medium containing proteins. These findings can be used to correct in vitro data for protein binding of chlorpromazine and this information is essential for the extrapolation to in vivo data.
向体外体系中添加蛋白质会影响测试化合物在介质中的游离浓度。本研究的目的是建立一种可忽略的耗尽固相微萃取方法，结合高效液相色谱（nd-SPME-HPLC），以测量含有白蛋白的介质中氯丙嗪（CPZ）的游离浓度。nd-SPME 方法针对涂层厚度（聚丙烯酸酯涂层）和暴露时间进行了优化，并研究了添加牛血清白蛋白（BSA）的潜在影响。结果表明，白蛋白的添加不会引起污垢，也不会影响 CPZ 进入纤维的吸收动力学。在实际的 40g/L 白蛋白体内白蛋白浓度下，94%的 CPZ 与蛋白质结合。这与体内的发现一致，报告 CPZ 的蛋白结合率为 92-99%。本研究中描述的 nd-SPME-HPLC 方法可用于测量含蛋白质的介质中氯丙嗪的游离浓度。这些发现可用于校正 CPZ 的体外蛋白结合数据，这些信息对于外推到体内数据至关重要。
