The use of cephalosporins during breast feeding raises several issues, including the risk of drug exposure through breast milk for the infant. In this paper, a hydrophilic interaction liquid chromatography/positive ion electrospray mass spectrometric assay (HILIC/ESI-MS) was developed for the quantitation of cefuroxime, cefoxitin, and cefazolin in breast milk and human plasma. The assay was based on the use of small sample size, 25 μL of biological samples, following acetonitrile precipitation of proteins and filtration that enabled injection into the HILIC/ESI-MS system. All analytes and the internal standard, alfuzosin, were separated by 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 composed of a 6% 12.5 mM ammonium acetate water solution in acetonitrile and pumped at a flow rate of 0.25 mL min . The assay was linear over a concentration range of 0.2 to 5 µg mL and 0.4 to 20 µg mL for all the analytes in breast milk and in human plasma, respectively. Intermediate precision was found to be less than 4.2% over the tested concentration ranges. A run time of less than 12 min for each sample made it possible to analyze a large number of biological samples per day. The method is the first reported application of HILIC in the analysis of antibiotics in breast milk and human plasma and it can be used to support a wide range of clinical studies. Copyright © 2016 John Wiley & Sons, Ltd.

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...