Comprehensive Two-Dimensional HPLC and Informative Data Processing for Pharmaceuticals and Lipids

Kakkonto, traditional Chinese drug, is believed to be effective against cold, headache, stiff shoulders, etc. It consists of natural plants and contains many compounds such as ephedrine, glycyrrhizic acid, and cinnamic acid. Nexera-e, comprehensive two-dimensional liquid chromatograph, is useful to separate such a complex matrix. In general, different separation modes are selected for 1st and 2nd dimensions for comprehensive two-dimensional chromatography to obtain high resolution that can not be achieved by each individual separation mode. We tried comprehensive separation using a semi-micro reversed-phase column and neutral pH mobile phase for 1st dimension and an ultra-high speed reversed phase column and acidic pH mobile phase for 2nd dimension. Herbal medicine contains many relatively polar compounds, so pH is an important parameter to modify separation selectivity. We performed differential analysis for commercial Kakkonto products and determined glycyrrhizic acid contained with photodiode array detector (PDA).
In lipidomics, phospholipids are the attractive targets of LC/MS analysis since lipids are important and essential components of biological membranes. However, conventional HPLC system by a single separation mode performs poorly on biological lipid sample, because it contains various kinds of lipids with common moieties that govern their behavior on column. Nexera-e was capable of characterizing phospholipids both quantitatively and qualitatively when coupled with triple quadrupole and iontrap-TOF type of mass spectrometer (MS/MS and IT-TOF) respectively. Reliable identification of lipid species was performed by acquiring m/z values of related parent and fragment ions at high accuracy with the IT-TOF and matching the data to commercially available data-base.