PLS-STOCSY identified three signals of the unknown metabolite at 3.2 ppm, 3.52 ppm and 4.08 ppm. To elucidate the structure a 2D-NMR experiment was performed using a sample of group 2. The special experiment, which is called HSQC, allows elucidating the direct conectivities of proton and carbon atoms. Therefore the carbon atoms directly connected to the proton atoms, which had been identified by PLS-STOCSY, could be identified.
2D-NMR spectrum (HSQC) of a sample of group 2, which allows identifying directly connected proton atoms and carbon atoms. The protons previously identified are marked by green circles. The directly connected carbon atoms are marked by green squares.
Due to the chemical shifts of the carbon atoms, which are connected to the previously identified proton atoms, the unknown metabolite could be identified as choline, which is an important endogenous metabolite.