Applications of hydrophilic interation chromatography in the analysis of drugs and metabolites

• Main Author: Bawazeer, Saud
• Published: University of Strathclyde 2012
• Subjects: 610
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.576420

Hydrophilic interaction liquid chromatography (HILIC) has grown in popularity during the last ten years. It is somewhere between normal phase and reverse phase liquid chromatography in terms of its mode of action. HILIC involves the separation of polar analytes through their partitioning into a water rich layer that is formed on the surface of the stationary phase. However, there are also additional mechanisms involved in HILIC including ion exchange interactions. There are an increasing number of HILIC columns on the market. A number of different factors can be set to control the separation of compounds in HILIC mode including column temperature, mobile phase composition, pH and buffer type and concentration. In the current study a cyanopropyl (CN) HPLC column was used for the separation of some basic compounds in HILIC mode. Good separation of a test mix of basic compounds was obtained with a mobile phase consisting of acetonitrile/water (95:5) containing 3.25mM ammonium acetate. The retention times of the basic compounds decreased with increased ionic strength or with increased water content in the mobile phase. The effect of the size of the basic centre, pH, and buffer concentration was also studied using a series of model bases. The utility of the column for impurity profiling of two basic drugs was tested. The CN column showed reasonable stability although there appeared to be loss of stationary phase with time. The retention properties of a silica gel column and a type C silica (silicon hydride) column for bases, sugars and polar acids were compared in HILIC mode with formic acid or ammonium acetate as aqueous phase modifiers. Seven different HILIC stationary phases were characterised with regard to their retention properties for 100 metabolite standards. In addition three of these phases were evaluated with regard to their ability to profile metabolites in urine. Finally a HILIC method was developed in order to determine the level of nicotine exposure in companion animals resulting from passive smoking.