HPLC method development for the evaluation of pheromones from the dwarf African clawed frog Hymenochirus

This research project is an ongoing project in collaboration with colleagues in the Department of Biology at the University of the Pacific. The main purpose of this project was to separate and identify the female-attracting substance secreted by male frog by developing chromatography methods for the male and female aqueous extracts. Three methods were used to concentrate the samples: sample lyophilization,..solid phase extraction (SPE), and direct sample loading. Different parameters such as the type and concentration of the phase modifier, wavelength for UV-detection, composition of the mobile phase, flow rate, and gradient elution were studied to achieve the required separation. The optimal method was determined as: direct sample loading, 0.01% (v v) formic acid in in mobile phase A (water) and mobile phase B (acctonitrile), 200 nm, 0.60 mL/min, and gradient method. The chromatograms of male and female skin gland water samples were compared and the fractions specific to male frog were collected and lyophilized for bio-activity testing and mass spectrometry analysis. Three different mass spectrometer systems, JEOL LCMate (ES+), Micromass Q-Tof Ultima™ Global (ES-). Voyager-DE™ STR Biospectromelry™ Workstation (MALDI-TOF), and Varian Mercury 300 MHz FT-NMR were utilized to investigate the structure of the fractions collected through HPLC. Only Micromass Q-Tof Ultima™ Global (ES+) gave some potential results. After analysis, the proposed protonated molecular ion peak was determined to be at m/z 779 by analyzing the abundance and relationship among the peaks at higher m/z values. The detail structure was inconclusive. v