Fast Detection Of Choline Containing Compounds In Complex Biomixture By NMR

Choline containing compounds(CCCs)are a class of small molecular metabolites with important biological functions.Choline is the precursors of neurotransmitter acetylcholine and phosphatidylcholine and thought to be related to human brain development and memory.Phosphatidylcholine is an important component of cell membrane and plays an important role in cell morphology maintenance,material transport and signal transmission.Choline is a necessary nutrient for human,part of choline can be synthesized by the human body,but most of them come from daily diet,it's also one of the ingredients of food additives.Abnormal CCCs content is considered to be related to the carcinogenesis process and cancer development.CCCs are widely exist in milk and liver.Therefore,investigation of CCCs is important for food research and clinical diagnosis.Mass spectrometry,enzyme colorimetric method and Ray's salt spectrophotometry are normally used in vitro detection of CCCs,but the complex separation process hindered their application.PET and fluorescence are common methods for in vivo CCCs detection,but the radioactive or fluorescence probe may have cytotoxic problems.NMR spectroscopy is a nondestructive method suitable for the investigate of CCCs in complex biomixture.However,the application of normally used 1H and 31P NMR spectroscopy is impeded by broad line width and serious overlap in them.Nowadays,1H-14N HSQC NMR spectroscopy has been developed for the detection of CCCs in milk and tissues.it's high selectivity makes it an ideal tools for CCCs detection,but the long prescan delay used for the signal recovery reduces the sensitivity of the experiment.Herein we applied SOFAST-HMQC to investigate the CCCs in dairy product to further enhance the sensitivity of the detection.The main experimental parameters of 1H-14N two-dimensional correlation experiments was optimized by standard sample,through a series of experiments and simulation.Those parameters that are series for the 2D experiment,including prescan delay,polarization transfer time,shaped pulse has all been obtained in both SOFAST-HMQC and HSQC.The sensitivity of CCCs detection using SOFAST-HMQC was compared with that of the previously published HSQC under the same conditions.The unit time sensitivity of CCCs detection using SOFAST-HMQC is 1.47-2.05 times higher than that of the HSQC method,the sensitivity enhancement may because of the optimized flip angle excitation used in the HMQC type experiment.Finally,the 1H-14N SOFAST-HMQC has been used for the detection of CCCs in dairy products.It is found that the fast detection experiment can selective detect the CCCs in those complex mixture by effective eliminating the broad high level background signals,and the signal intensities of the CCCs obtained by it are 1.32-3.00 folds higher than that obtained by HSQC.The results also show that the choline content in those milk powder samples are relative higher than that of the milk,whereas,the phosphocholine content are much lower,this may because of the addition of the choline in milk powder.