Bioanalytical Sciences, Bristol-Myers Squibb Co., Route 206 and Province Line Road, Princeton, NJ 08543, USA.
J Pharm Biomed Anal. 2012 Nov;70:401-7. doi: 10.1016/j.jpba.2012.06.011. Epub 2012 Jun 23.
To support a first-in-human (FIH) clinical study in healthy volunteers, a human plasma assay, a 20-fold more sensitive method than the validated non-clinical LC-MS/MS assays, was requested. For the clinical assay, a LLOQ of 0.050 ng/mL for Compound A and 0.100 ng/mL for Compound B was desired to accurately determine the analyte concentrations in human plasma samples across all treatment groups. A design of experiment (DOE) investigation was performed in an effort to optimize the extraction procedure of the bioanalytical assay used to support the first in human study and future clinical studies. Three factors, extraction buffer pH (two pHs), volume ratio of organic solvent to plasma (two ratios), and extraction shake time (three times), were selected for the DOE. Both analytes were analyzed at a low concentration, 0.150 ng/mL, and a stable isotope label internal standard was used for each analyte. To estimate the recovery of each analyte from the extraction, the response ratio of each analyte over the respective internal standard was used, and to estimate matrix effects, the absolute response (peak area) of each analyte was used. The results of the DOE indicated that the three factors tested had a more significant effect on the extraction of the metabolite, Compound B, compared to that of the parent, Compound A. The extraction buffer pH had the greatest influence on Compound B and the volume of extraction solvent had an influence on both analytes. Unexpectedly, a longer extraction time caused an apparent decrease in the overall recovery for both analytes. This was presumably due to an increased extraction of interfering matrix components. Optimal conditions were achieved for the combined analysis of both compounds using the DOE approach.
为支持健康志愿者的首次人体（FIH）临床研究，请求开发一种人血浆分析方法，该方法比经过验证的非临床 LC-MS/MS 分析方法灵敏 20 倍。对于临床分析，希望 Compound A 的定量下限（LLOQ）为 0.050ng/mL，Compound B 的 LLOQ 为 0.100ng/mL，以便准确测定所有治疗组的人血浆样品中的分析物浓度。进行了实验设计（DOE）研究，以优化用于支持首次人体研究和未来临床研究的生物分析测定的提取程序。选择了三个因素，即提取缓冲液 pH（两种 pH 值）、有机溶剂与血浆的体积比（两种比例）和提取摇动时间（三次），用于 DOE。两种分析物均以低浓度（0.150ng/mL）进行分析，并且使用每种分析物的稳定同位素标记内标。为了估计每种分析物从提取中的回收率，使用每个分析物相对于各自内标的响应比进行估计，为了估计基质效应，使用每个分析物的绝对响应（峰面积）进行估计。DOE 的结果表明，在所测试的三个因素中，与母体化合物 A 相比，测试的三个因素对代谢物 Compound B 的提取具有更显著的影响。提取缓冲液 pH 对 Compound B 的影响最大，提取溶剂的体积对两种分析物都有影响。出乎意料的是，较长的提取时间似乎导致两种分析物的整体回收率明显下降。这可能是由于更多地提取了干扰基质成分。通过 DOE 方法，实现了两种化合物的联合分析的最佳条件。
