Xue Y-J, Liu Jane, Unger Steve
Preclinical Candidate Optimization, Pharmaceutical Research Institute, Bristol-Myers Squibb, New Brunswick, New Jersey 08903, USA.
J Pharm Biomed Anal. 2006 Jun 7;41(3):979-88. doi: 10.1016/j.jpba.2006.02.006. Epub 2006 Mar 14.
A single-pot liquid-liquid extraction (LLE) with hydrophilic interaction liquid chromatography/tandem mass spectrometry (HILIC/MS/MS) method has been developed and validated for the determination of muraglitazar, a hydrophobic diabetes drug, in human plasma. To 0.050 ml of each plasma sample in a 96-well plate, the internal standard solution in acetonitrile and toluene were added to extract the compound of interest. The plate was vortexed, followed by centrifugation. The organic layer was then directly injected into an LC/MS/MS system. Chromatographic separation was achieved isocratically on a Thermohypersil_Keystone, Hypersil silica column (3 mmx50 mm, 3 microm). The mobile phase contained 85% of methyl t-butyl ether and 15% of 90/10 (v/v) acetonitrile/water with 0.3% trifluoroacetic acid. Post-column mobile phase of 50/50 (v/v) acetonitrile/water containing 0.1% formic acid was added. Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API 4000. The standard curve, ranged from 1 to 1000 ng/ml, was fitted to a 1/x weighted quadratic regression model. This single-pot LLE approach effectively eliminated time-consuming organic layer transfer, dry-down, and sample reconstitution steps, which are essential for a conventional liquid-liquid extraction procedure. The modified mobile phase was more compatible with the direct injection of the commonly used extraction solvents in LLE. Furthermore, the modified mobile phase improved the retention of muraglitazar, a hydrophobic compound, on the normal phase silica column. The validation results demonstrated that this method was rugged and suitable for analyzing muraglitazar in human plasma. In comparison with a revised-phase LC/MS/MS method, this single-pot LLE, HILIC/MS/MS method improved the detection sensitivity by more than four-fold based upon the LLOQ signal to noise ratio. This approach may be applied to other hydrophobic compounds with proper modification of the mobile phase compositions.
已开发并验证了一种采用亲水作用液相色谱/串联质谱法（HILIC/MS/MS）的单管液液萃取（LLE）方法，用于测定人血浆中疏水类糖尿病药物muraglitazar。向96孔板中每份0.050 ml血浆样品中加入乙腈和甲苯中的内标溶液，以萃取目标化合物。将板涡旋，然后离心。然后将有机层直接注入LC/MS/MS系统。在Thermohypersil_Keystone Hypersil硅胶柱（3 mm×50 mm，3 µm）上进行等度色谱分离。流动相包含85%的甲基叔丁基醚和15%的90/10（v/v）乙腈/水以及0.3%的三氟乙酸。加入含0.1%甲酸的50/50（v/v）乙腈/水的柱后流动相。采用Sciex API 4000通过正离子电喷雾串联质谱进行检测。标准曲线范围为1至1000 ng/ml，拟合为1/x加权二次回归模型。这种单管LLE方法有效消除了传统液液萃取程序中耗时的有机层转移、吹干和样品复溶步骤。改良后的流动相与LLE中常用萃取溶剂的直接进样更兼容。此外，改良后的流动相提高了疏水化合物muraglitazar在正相硅胶柱上的保留。验证结果表明该方法耐用，适用于分析人血浆中的muraglitazar。与反相LC/MS/MS方法相比，基于定量下限信噪比，这种单管LLE、HILIC/MS/MS方法的检测灵敏度提高了四倍以上。该方法经流动相组成适当修改后可应用于其他疏水化合物。
