Ceccato A, Boulanger B, Chiap P, Hubert P, Crommen J
Department of Analytical Pharmaceutical Chemistry, University of Liège, Belgium.
J Chromatogr A. 1998 Sep 11;819(1-2):143-53. doi: 10.1016/s0021-9673(98)00547-0.
A fully automated liquid chromatographic (LC) method for the simultaneous determination of methylphenobarbital enantiomers and phenobarbital in human plasma has been developed. The method is based on the use of a precolumn packed with an internal-surface reversed-phase packing material (LiChrospher ADS) for sample clean-up coupled to LC analysis on a cellulose tris(4-methylbenzoate) based chiral stationary phase (Chiralcel OJ-R). A 100-microliter plasma sample was injected directly on the precolumn packed with LiChrospher RP-18 ADS using a mixture of pH 5.0 phosphate buffer-methanol (97:3, v/v) as washing liquid. The analytes were then eluted in the back-flush mode with the LC mobile phase. The enantiomeric separation of methylphenobarbital was achieved on Chiralcel OJ-R). The retention times were modelled using a D-optimal design with ten experimental points in order to optimise the LC mobile phase for the separation of phenobarbital from the enantiomers of mephobarbital. The factors selected were the acetonitrile content, the pH and the sodium perchlorate concentration in the mobile phase. A Derringer's desirability function was used to find an optimal and robust solution within the experimental domain. The mobile phase selected consisted of a mixture of pH 7.0 phosphate buffer-acetonitrile (60:40, v/v). The elution profiles of phenobarbital, methylphenobarbital and blank plasma samples on the precolumn and the time needed for analyte transfer from the precolumn to the analytical column were then determined. Finally, the method developed was validated.
已开发出一种全自动液相色谱（LC）方法，用于同时测定人血浆中的甲基苯巴比妥对映体和苯巴比妥。该方法基于使用填充有内表面反相填充材料（LiChrospher ADS）的预柱进行样品净化，并与基于纤维素三（4-甲基苯甲酸酯）的手性固定相（Chiralcel OJ-R）上的LC分析相结合。使用pH 5.0磷酸盐缓冲液-甲醇（97:3，v/v）混合物作为洗涤液，将100微升血浆样品直接注入填充有LiChrospher RP-18 ADS的预柱中。然后用LC流动相以反冲模式洗脱分析物。甲基苯巴比妥的对映体分离在Chiralcel OJ-R上实现。使用具有十个实验点的D-最优设计对保留时间进行建模，以优化LC流动相，以便从美芬妥英对映体中分离苯巴比妥。选择的因素是流动相中的乙腈含量、pH值和高氯酸钠浓度。使用Derringer合意函数在实验范围内找到最优且稳健的解决方案。选择的流动相由pH 7.0磷酸盐缓冲液-乙腈（60:40，v/v）的混合物组成。然后测定了苯巴比妥、甲基苯巴比妥和空白血浆样品在预柱上的洗脱曲线以及分析物从预柱转移到分析柱所需的时间。最后，对所开发的方法进行了验证。
