High-Throughput Liquid Chromatography/Mass Spectrometry Method for the Determination of the Chromatographic Hydrophobicity Index

A fast gradient reversed-phase liquid chromatography (LC) method, using an acetonitrile gradient was developed to determine the chromatographic hydrophobicity index (CHI), as reported by Valco et al. (Anal. Chem. 1997, 69, 2022-2029).The analytical method provides retention times, based on UV detection at two different wavelengths, which then are converted into CHI values after calibration with a set of test compounds. The CHI of each compound is measured at three different pH values, 2.0, 7.4, and 10.5; so using an 8-min gradient at each pH value one compound can be analyzed in ~24 min. The aim of this work is to improve the throughput of the CHI screening using a LC/MS approach, so the application of the LC/MS technique is an extension of the LC/UV approach previously reported by Vaico et al. This approach allows contemporary injection of N compounds into the LC/MS system, the retention time of each compound can be then retracted from the selected ion recording chromatograms. The throughput of the existing screening method could be increased by N times, where N is the number of compounds injected, so only three runs are needed to determine the CHI at three different pH values for a set of N compounds. The highest value of N depends on the total number of channels that can be monitored simultaneously; in the present work, 32 channels were used. This LC/MS method has been tested for a number of commercial products analyzed as mixtures, and data obtained were compared with those coming from the classical LC/UV approach. In the same way, the method was tested for a number of compounds associated with two GlaxoWellcome projects in the antibacterial area. Data reported show that the LC/MS method can be successfully applied for analyzing compounds in mixtures and for compounds with poor UV absorption, which cannot be analyzed with the standard LC/UV method.