Nováková Lucie, Matysová Ludmila, Solichová Dagmar, Koupparis Michael A, Solich Petr
Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic.
J Chromatogr B Analyt Technol Biomed Life Sci. 2004 Dec 25;813(1-2):191-7. doi: 10.1016/j.jchromb.2004.09.045.
The performance of monolithic HPLC columns Chromolith (made by Merck, Germany) and conventional C18 columns Discovery (Supelco, Sigma-Aldrich, Prague, Czech Republic) was tested and the comparison for two topical preparations Ketoprofen gel and Estrogel gel was made. The composition of mobile phases - for Ketoprofen analysis a mixture of acetonitrile, water and phosphate buffer adjusted to pH 3.5 (40:58:2) and for Estrogel analysis a mixture of acetonitrile, methanol, water (23:24:53) - was usually not optimal for analyses at all types of columns. Thus an adjustment of components ratio was necessary for sufficient resolution of the compounds analysed. Various flow rates (1.0-5.0 ml/min) and mobile phases (usually increasing ratio of water content) were applied. Determination of active substances, preservatives and impurities and comparison of retention times and system suitability test parameters was accomplished. For Estrogel gel, following chromatographic conditions were found: using Chromolith Flash RP-18e monolith column, mobile phase was acetonitrile, methanol, water (13:24:63, v/v/v) and flow-rate 3.0 ml/min. Using monolith column ChromolithSpeedROD RP-18e, the mobile phase was acetonitrile, methanol, water (18:24:58, v/v/v) and flow-rate 4.0 ml/min. For the monolith column Chromolith Performance RP-18e, the mobile phase was acetonitrile, methanol, water (23:24:53, v/v/v), flow-rate 3.0ml/min. Analysis of Ketoprofen gel gave the best results using following analytical conditions: for monolith column Chromolith Flash RP-18e, mobile phase as a mixture of acetonitrile, water, phosphate buffer pH 3.5 (30:68:2, v/v/v) was used, at flow-rate 2.0 ml/min. For ChromolithSpeedROD RP-18e monolith column, acetonitrile, water, phosphate buffer pH 3.5 (35:63:2, v/v/v) was used as a mobile phase at flow-rate 3.0 ml/min. Chromolith Performance RP-18e gave the best results using mobile phase acetonitrile, water, phosphate buffer pH 3.5 (30:68:2, v/v/v) at the flow-rate 5.0 ml/min. It was proved that monolith columns, due to their porosity and low back-pressure, can save analysis time by about a factor of three with sufficient separation efficiency. Thus, for example 11 min long analysis can be performed in 4 min with comparable results.
对整体式高效液相色谱柱Chromolith（德国默克公司生产）和传统C18柱Discovery（捷克共和国布拉格西格玛奥德里奇公司旗下的Supelco品牌）的性能进行了测试，并对两种外用制剂酮洛芬凝胶和雌二醇凝胶进行了比较。流动相的组成——用于酮洛芬分析的是乙腈、水和pH值调至3.5的磷酸盐缓冲液的混合物（40:58:2），用于雌二醇分析的是乙腈、甲醇、水的混合物（23:24:53）——通常对于所有类型的色谱柱分析来说都不是最佳的。因此，为了对所分析的化合物进行充分分离，有必要调整各组分的比例。采用了各种流速（1.0 - 5.0 ml/min）和流动相（通常是增加水含量的比例）。完成了活性物质、防腐剂和杂质的测定以及保留时间和系统适用性测试参数的比较。对于雌二醇凝胶，发现了以下色谱条件：使用Chromolith Flash RP - 18e整体柱时，流动相为乙腈、甲醇、水（13:24:63，v/v/v），流速为3.0 ml/min。使用ChromolithSpeedROD RP - 18e整体柱时，流动相为乙腈、甲醇、水（18:24:58，v/v/v），流速为4.0 ml/min。对于Chromolith Performance RP - 18e整体柱，流动相为乙腈、甲醇、水（23:24:53，v/v/v），流速为3.0 ml/min。使用以下分析条件对酮洛芬凝胶进行分析可得到最佳结果：对于Chromolith Flash RP - 18e整体柱，使用乙腈、水、pH值3.5的磷酸盐缓冲液的混合物（30:68:2，v/v/v）作为流动相，流速为2.0 ml/min。对于ChromolithSpeedROD RP - 18e整体柱，使用乙腈、水、pH值3.5的磷酸盐缓冲液（35:63:2，v/v/v）作为流动相，流速为3.0 ml/min。Chromolith Performance RP - 18e在流速为5.0 ml/min时，使用乙腈、水、pH值3.5的磷酸盐缓冲液（30:68:2，v/v/v）作为流动相可得到最佳结果。事实证明，整体柱由于其孔隙率和低背压，在具有足够分离效率的情况下，可以将分析时间节省约三分之二。例如，原本11分钟的分析可以在4分钟内完成，且结果相当。
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