Investigation of the Retention Mechanism of Cephalosporins by Zwitterionic Hydrophilic Interaction Liquid Chromatography

Abstract

adverse effects on human health, such as resistance development and allergic reactions [1][2][3][4]. There is no doubt that the importance of cephalosporin antibiotics in medicine requires the development of high performance separation methods for their analysis [5,6]. Literature survey revealed that numerous chromatographic methods have been developed for the analysis of cephalosporins including isocratic or gradient techniques coupled with various methods of detection [7][8][9][10][11][12][13][14]. These compounds are highly polar molecules and represent perfect candidates to study the retention mechanism in hydrophilic interaction liquid chromatography (HILIC). Up to now research on the retention of cephalosporins in HILIC has been focused only on the use of plain silica columns [15,16]. The zwitterionic hydrophilic interaction liquid chromatography used in this study is a unique form of HILIC which involves the use of stationary phases with zwitterionic functional groups covalently bonded at a silica or polymeric substrate.HILIC chromatography is an alternative chromatographic technique using polar stationary phases and low aqueous/high organic mobile phases in order to achieve effective retention for polar analytes. It appears to be complementary to reversed-phase LC as the order of elution is reversed to reversed-phase LC and thus it provides reasonable retention for polar compounds [17,18]. When developing robust methods in HILIC, it is important to understand the dominant mechanism that governs retention which is related to the physicochemical characteristics of the analytes and to the analytical column substrate. The basic types of HILIC columns include plain silica, neutral, polar chemically bonded, ion-exchange and zwitterionic stationary phases [19,20]. The retention mechanism in HILIC chromatography is complex involving hydrophilic partition, absorption and electrostatic interactions and further studies are needed to elucidate this [21][22][23]. AbstractThe importance of cephalosporin antibiotics in medicine requires the development of high performance separation methods for their analysis. In this paper cefazolin, cefuroxime, cefoxitin, cefaclor and ceftriaxone were used as model compounds to investigate the retention mechanism under zwitterionic HILIC conditions. The analysis was performed using a ZIC ® -HILIC analytical column (150.0 × 2.1 mm i.d., particle size 3.5 μm, 200 Å) with isocratic elution. The mobile phase was a mixture of acetonitrile and ammonium acetate or formate buffer. To get a better insight into the retention mechanism on ZIC ® -HILIC columns the main factors influencing the retention and selectivity of the analytes have been thoroughly investigated. It is shown that the type of the salt and its concentration along with the volume fraction of water in the mobile phase and pH of the aqueous buffer greatly affect the retention of the tested cephalosporins due to their structures and physicochemical properties. The method was applied successfully to the analysis of injection dosage...

Quantification of oligopeptide‐20 and oligopeptide‐24 in cosmetic creams using hydrophilic interaction liquid chromatography with electrospray ionization mass spectrometry

“…For this reason, a ZIC®‐HILIC column whose functional group consists of a strongly acidic sulfonic acid group and a strongly basic quaternary ammonium group separated by a short alkyl spacer was chosen. In this zwitterionic stationary phase, the electrostatic forces of each charge are partly counterbalanced by the proximity of ions with opposite charge, and the negatively charged sulfonic acid groups might be responsible for weak, but important, electrostatic interactions, since it is more approachable to interact with the analytes . The chromatographic parameters examined are: the effect of the salt type and concentration, pH value and acetonitrile percentage regarding retention and peak shape with the one‐factor‐a‐time method.…”

Insights into the Mechanism of Separation of Bisphosphonates by Zwitterionic Hydrophilic Interaction Liquid Chromatography: Application to the Quantitation of Risedronate in Pharmaceuticals

“…In this zwitterionic stationary phase, the electrostatic forces of each charge were partly counterbalanced by the proximity of an ion with opposite charge. Though the accessibility to the positively charged quaternary ammonium groups was limited, the negatively charged sulfonic acid groups might be responsible for weak, but important, electrostatic interactions [25]. The studied bisphosphonates were retained adequately in this analytical column through hydrophilic interactions, even if they had the same charge with the sulfonic acid groups of the stationary phase.…”

Quantification of three beta‐lactam antibiotics in breast milk and human plasma by hydrophilic interaction liquid chromatography/positive‐ion electrospray ionization mass spectrometry

“…[35] Chromatographic conditions were optimized so as to achieve adequate separation of the analytes from matrix interferences and to reach the optimum sensitivity in ESI/MS detection for each analyte. The active layer of this stationary phase is covalently attached to wide-pore silica gel and it contains both strongly acidic sulfonic acid groups and strongly basic quaternary ammonium groups separated by a short alkyl spacer.…”

“…Previous findings support that the separation mechanism of cephalosporins in ZIC®-HILIC comprises both hydrophilic partition and secondary electrostatic interactions, and that the enhancement of the electrostatic interactions, by reducing the salt concentration, seems to improve the peak shape of cephalosporins. [35] Chromatographic conditions were optimized so as to achieve adequate separation of the analytes from matrix interferences and to reach the optimum sensitivity in ESI/MS detection for each analyte. Various combinations of acetonitrile (organic modifier) and aqueous ammonium acetate buffer solution with changed content of each component were thoroughly investigated to identify the optimal mobile phase composition.…”