Xue Y-J, Liu Jane, Pursley Janice, Unger Steve
Pharmaceutical Candidate Optimization, Pharmaceutical Research Institute, Bristol-Myers Squibb, New Brunswick, NJ 08903, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Feb 2;831(1-2):213-22. doi: 10.1016/j.jchromb.2005.12.023. Epub 2006 Jan 18.
A 96-well single-pot protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of muraglitazar, a PPAR alpha/gamma dual agonist, in human plasma. The internal standard, a chemical analogue, was dissolved in acetonitrile containing 0.1% formic acid. The solvent system was also served as a protein precipitation reagent. Human plasma samples (0.1 mL) and the internal standard solution (0.3 mL) were added to a 96-well plate. The plate was vortexed for 1 min and centrifuged for 5 min. Then the supernatant layers were directly injected into the LC/MS/MS system. The chromatographic separation was achieved isocratically on a Phenomenox C18(2) Luna column (2 mm x 50 mm, 5 microm). The mobile phase contained 20/80 (v/v) of water and acetonitrile containing 0.1% formic acid. Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API 3000. The standard curve, which ranged from 1 to 1000 ng/mL, was fitted to a 1/x weighted quadratic regression model. This single-pot approach effectively eliminated three time consuming sample preparation steps: sample transfer, dry-down, and reconstitution before the injection, while it preserved all the benefits of the traditional protein precipitation. By properly adjusting the autosampler needle offset level, only the supernatant was injected, without disturbing the precipitated proteins in the bottom. As a result, the quality of chromatography and column life were not compromised. After more than 600 injections, there was only slightly increase of column back-pressure. The validation results demonstrated that this method was rugged and provide satisfactory precision and accuracy. The method has been successfully applied to analyze human plasma samples in support of a first-in-man study. This method has also been validated in monkey and mouse plasma for the determination of muraglitazar.
已开发并验证了一种用于测定人血浆中PPARα/γ双重激动剂muraglitazar的96孔单管蛋白沉淀-液相色谱/串联质谱（LC/MS/MS）方法。内标为化学类似物，溶解于含0.1%甲酸的乙腈中。该溶剂系统也用作蛋白沉淀剂。将人血浆样品（0.1 mL）和内标溶液（0.3 mL）加入96孔板中。将板涡旋1分钟，然后离心5分钟。接着将上清液直接注入LC/MS/MS系统。在Phenomenox C18(2) Luna柱（2 mm×50 mm，5μm）上进行等度色谱分离。流动相由20/80（v/v）的水和含0.1%甲酸的乙腈组成。采用Sciex API 3000进行正离子电喷雾串联质谱检测。标准曲线范围为1至1000 ng/mL，采用1/x加权二次回归模型拟合。这种单管方法有效消除了进样前耗时的三个样品制备步骤：样品转移、吹干和复溶，同时保留了传统蛋白沉淀的所有优点。通过适当调整自动进样器针偏移水平，仅注入上清液，而不会扰动底部沉淀的蛋白质。结果，色谱质量和柱寿命未受影响。经过600多次进样后，柱背压仅略有增加。验证结果表明该方法耐用，具有令人满意的精密度和准确度。该方法已成功应用于分析人体血浆样品，以支持一项首次人体研究。该方法也已在猴和小鼠血浆中进行验证，用于测定muraglitazar。
