Lee I H, Pollack S, Hsu S H, Miksic J R
Department of Clinical Drug Disposition, Rhône-Poulenc Rorer Central Research, Horsham, Pennsylvania 19044.
J Chromatogr Sci. 1991 Apr;29(4):136-40. doi: 10.1093/chromsci/29.4.136.
The retention properties of calcitonins on a reversed-phase column are examined using salmon calcitonin as the model compound. The effect of the concentration of organic modifier, buffer strength, pH of the mobile phase, and ion-pair reagent are studied. In the absence of an ionic modifier in the eluent the calcitonin peak shapes are not symmetrical. The addition of 0.1% trifluoroacetic acid (TFA), however, results in good peak characteristics without the need to add nonvolatile salts. The retention of the calcitonins was found to be very sensitive to the concentration of the organic modifier (acetonitrile) present in the mobile phase. A change of pH between 2 and 5 has only a slight effect of the k' of salmon calcitonin, but the k' increases significantly at higher pH values. The addition of a phosphate buffer to the mobile phase and an increase in the buffer concentration (0-0.2 M) causes a decrease in the retention of salmon calcitonin. Evidence shows that reproducible, quantitatively measurable data can be obtained using reversed-phase chromatography if the ion-pairing reagent and organic modifier concentrations are carefully controlled. The system also shows a good selectivity for the calcitonin series. Therefore, both highly selective methods (qualitative) as well as quantitative methods for analytical, pharmaceutical, and manufacturing use can be developed by adjusting the high-performance liquid chromatography (HPLC) conditions as discussed.
以鲑鱼降钙素为模型化合物，研究了降钙素在反相柱上的保留特性。考察了有机改性剂浓度、缓冲强度、流动相pH值和离子对试剂的影响。在洗脱液中不存在离子改性剂时，降钙素的峰形不对称。然而，加入0.1%的三氟乙酸（TFA）可得到良好的峰形，而无需添加非挥发性盐。发现降钙素的保留对流动相中有机改性剂（乙腈）的浓度非常敏感。pH值在2至5之间的变化对鲑鱼降钙素的k'只有轻微影响，但在较高pH值时k'显著增加。向流动相中加入磷酸盐缓冲液并增加缓冲液浓度（0-0.2 M）会导致鲑鱼降钙素的保留率降低。有证据表明，如果仔细控制离子对试剂和有机改性剂的浓度，使用反相色谱法可以获得可重复的、定量可测的数据。该系统对降钙素系列也具有良好的选择性。因此，通过调整所讨论的高效液相色谱（HPLC）条件，可以开发出用于分析、制药和生产用途的高选择性方法（定性）以及定量方法。
