Prieto-Blanco María Carmen, Planas-Franco Angela, Muniategui-Lorenzo Soledad, González-Castro María José
Universidade da Coruña, Grupo QANAP, Instituto Universitario de Medio Ambiente (IUMA), Departamento de Química, Facultade de Ciencias, Zapateira, 15071, A Coruña, Spain.
Universidade da Coruña, Grupo QANAP, Instituto Universitario de Medio Ambiente (IUMA), Departamento de Química, Facultade de Ciencias, Zapateira, 15071, A Coruña, Spain.
Talanta. 2023 Apr 1;255:124228. doi: 10.1016/j.talanta.2022.124228. Epub 2022 Dec 29.
In this work, a retention behavior based on mixed-mode reversed-phase (RP)/hydrophilic interaction liquid chromatography (HILIC) was observed for benzalkonium chloride (BAK) using a core-shell column functionalized with biphenyl groups. Although in the literature, the U-shaped retention was reported for polar compounds in mixed functionalized phases, in the present work, the behavior was dependent upon the chemical structure of the analyte with mixed functionality (ammonium group, a benzyl group and an alkyl chain) and on the high selectivity of the chromatographic column. The bimodal retention was observed for the four BAK homologues using a content of acetonitrile from 65 to 95% in the mobile phase. The data were adjusted to polynomial equations which allow for modeling and predicting the U-shaped retention. The salt concentration (50 and 100 mM), anion (formate and acetate) and cation (ammonium and triethylammonium) of the salt, pH (4 and 5) in the mobile phase were studied in order to understand their influence on the two retention modes. Significant electrostatic interactions were involved in the two retention modes, especially with a content of acetonitrile higher that 90%. Linear relationships between the retention factors of the four homologues were found in a wide range of %acetonitrile when the salt and triethylamine concentration, pH and nature of salt were changed. The differences found on the retention of the homologues, when increasing the alkyl chain length, were more significant in the RP mode due to predominant hydrophobic interactions. A pH decrease and a salt concentration increase caused a retention decrease for both modes. A decrease on of the retention was observed when acetate anion was replaced by formate anion. The different order of the polynomial equations according to the used mobile phase confirmed its relevant role in the interactions with the analytes and stationary phase. A mobile phase was selected (85% acetonitrile, pH 4 and 100 mM ammonium formate) for the BAK determination in cutaneous, otic and ophthalmic formulations with different active pharmaceutical ingredients and excipients. Low sample volume (500 μL) and short analysis time (<5 min) were some of the advantages of the proposed method. In addition, good analytical performance (R > 0.999, % RSD <4.5% for intra-day precision and <5.8% for inter-day precision, and recoveries in the 92-105% range) was obtained.
在本研究中，使用联苯基功能化的核壳柱，观察到苯扎氯铵（BAK）基于混合模式反相（RP）/亲水相互作用液相色谱（HILIC）的保留行为。尽管文献中报道了混合功能化相中极性化合物呈U形保留，但在本研究中，该行为取决于具有混合官能团（铵基、苄基和烷基链）的分析物的化学结构以及色谱柱的高选择性。使用流动相中乙腈含量为65%至95%的条件，观察到四种BAK同系物呈现双峰保留。数据被拟合为多项式方程，可用于模拟和预测U形保留。研究了流动相中的盐浓度（50和100 mM）、盐的阴离子（甲酸根和乙酸根）和阳离子（铵根和三乙铵）、pH（4和5），以了解它们对两种保留模式的影响。两种保留模式均涉及显著的静电相互作用，尤其是当乙腈含量高于90%时。当盐和三乙胺浓度、pH以及盐的性质改变时，在较宽的乙腈百分比范围内发现了四种同系物保留因子之间的线性关系。当增加烷基链长度时，同系物保留的差异在RP模式中更为显著，这是由于主要的疏水相互作用。pH降低和盐浓度增加导致两种模式下的保留均降低。当乙酸根阴离子被甲酸根阴离子取代时，观察到保留降低。根据所用流动相不同，多项式方程的顺序不同，这证实了流动相在与分析物和固定相相互作用中的重要作用。选择了一种流动相（85%乙腈、pH 4和100 mM甲酸铵）用于测定含有不同活性药物成分和辅料的皮肤、耳部和眼部制剂中的BAK。低进样量（500 μL）和短分析时间（<5分钟）是该方法的一些优点。此外，还获得了良好的分析性能（R>0.999，日内精密度的%RSD<4.5%，日间精密度的%RSD<5.8%，回收率在92 - 105%范围内）。
