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 similar to 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 Valco et al. This approach allows contemporary injection of N compounds into the LC/MS system, the retention time of each compound can be then extracted 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.