Gritti Fabrice, Sehajpal Jyotsna, Fairchild Jacob
Waters Corporation, 34 Mapple Street, Milford, MA 01757, USA.
Waters Corporation, 34 Mapple Street, Milford, MA 01757, USA.
J Chromatogr A. 2017 Mar 17;1489:95-106. doi: 10.1016/j.chroma.2017.02.003. Epub 2017 Feb 6.
The peak distortion observed in hydrophilic interaction chromatography (HILIC) may be caused by the sample diluent to mobile phase mismatch. The United States Pharmacopeia (USP) method for organic impurities in cetirizine HCl tablets calls for such a mismatch, having a higher concentration of strong solvent in the sample diluent than in the mobile phase. A significant peak deformation is reported for cetirizine (a second-generation antihistamine) when it is purified on a Ethylene Bridged Hybrid (BEH) HILIC column (4.6mm×100mm, 2.5μm particles) using an acetonitrile-water eluent mixture and a sample diluent containing 7% and 9% water (in volume), respectively. The mechanism and physical origin of such peak distortion are related to (1) the diluent-to-eluent excess of water that propagates along the column at a velocity similar to that of the analyte, (2) the significant drop of the Henry's constant of the analyte upon increasing water concentration in the eluent, (3) the sample volume injected, and (4) to the pre-column sample dilution factor that depends on the characteristics of the LC instrument used. This proposed mechanism is validated from the calculation of the concentration profiles of cetirizine and water by using the equilibrium-dispersive (ED) model of chromatography. The observed distortion of cetirizine peaks is successfully predicted from the measurement of (1) the excess adsorption isotherm of water from acetonitrile onto the BEH HILIC adsorbent, (2) the retention factor of cetirizine as a function of the volume fraction (7, 8, and 9%) of water in the mobile phase, and (3) of the pre-column sample dispersion related to the instrument used (HPLC or UHPLC). The results of the calculations enables the user to anticipate the impacts of the diluent-to-eluent mismatch in water content, the injection volume, the analyte retention under infinite dilution, and of the pre-column sample dispersion on the amplitude of peak distortion in HILIC. Appropriate and permitted alterations of the USP method are then suggested based on a sound physico-chemical approach.
亲水作用色谱法（HILIC）中观察到的峰变形可能是由样品稀释剂与流动相不匹配引起的。美国药典（USP）中盐酸西替利嗪片有机杂质的检测方法就存在这种不匹配情况，即样品稀释剂中强溶剂的浓度高于流动相。据报道，当使用乙腈 - 水流动相混合物和分别含有7%和9%水（体积比）的样品稀释剂在乙烯桥联杂化（BEH）HILIC柱（4.6mm×100mm，2.5μm粒径）上纯化西替利嗪（第二代抗组胺药）时，会出现明显的峰变形。这种峰变形的机理和物理根源与以下因素有关：（1）稀释剂与流动相之间水过量，其以与分析物相似的速度沿色谱柱传播；（2）随着流动相中水浓度增加，分析物的亨利常数显著下降；（3）进样体积；（4）柱前样品稀释因子，该因子取决于所用液相色谱仪器的特性。通过使用色谱的平衡扩散（ED）模型计算西替利嗪和水的浓度分布，验证了所提出的机理。通过测量以下参数成功预测了观察到的西替利嗪峰变形：（1）水从乙腈到BEH HILIC吸附剂的过量吸附等温线；（2）西替利嗪的保留因子与流动相中水的体积分数（7%、8%和9%）的函数关系；（3）与所用仪器（HPLC或UHPLC）相关的柱前样品分散情况。计算结果使用户能够预测水含量方面的稀释剂与流动相不匹配、进样体积、无限稀释下分析物保留以及柱前样品分散对HILIC中峰变形幅度的影响。然后基于合理的物理化学方法提出了USP方法的适当且允许的变更建议。
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