Research On The Determination Of Nicotinamide Mononucleotide In Complex Samples By Molecularly Imprinted Solid Phase Extraction Combined With UHPLC-MS

Nicotinamide mononucleotide（NMN）is an endogenous substance in the human body,one of the five precursors of nicotinamide adenine dinucleotide（NAD）.It is also a precursor with better pharmacokinetic and pharmacological properties which participates in the synthesis of intracellular NAD.The aging process of the human body is accompanied by the decrease of the NAD content in the body,a large number of studies have shown that the decrease of the NAD level in the body will lead to some age-related degenerative diseases and metabolic diseases.Taking NMN can effectively increase the content of NAD in the body to prevent and treat some diseases related to the decrease of NAD levels.Therefore,NMN has become an important issue in the fields of medicine and health care in recent year.Because NMN is an endogenous substance in the organisms,the analysis and detection of NMN will inevitably encounter two key problems:one is how to eliminate the matrix effect of the sample or reduce the matrix effect on the analysis accuracy,and the other is how to eliminate the matrix effect and analyze low content samples at the same time.In this study,molecularly imprinted solid-phase extraction technology and UHPLC-MS detection technology were combined to realize the detection of NMN in complex samples,which provided theoretical guidance and technical support for the medical research of NMN and the development of health food.The main content of study can be divided into the following three aspects:（1）Nicotinamide mononucleotide molecularly imprinted polymer microspheres were prepared by precipitation polymerization with NMN as the template molecule andα-methacrylic acid as the functional monomer,which has good specific adsorption capacity for NMN.The isothermal adsorption process is more in line with the Freundlich model,and the imprinted sites are inhomogeneous;the dynamic adsorption process is more in line with the quasi-second-order kinetic equation,which belongs to chemical adsorption.In addition,the results of selective adsorption experiments show that the separation factor is up to 15.68,and the relative separation factor is up to 2.83,showing a strong specific adsorption capacity for NMN.The molecularly imprinted polymer show high application performance in solid phase extraction of NMN from broccoli extract.（2）Using silica gel as the carrier,the surface of the silica gel was gradually modified,then the functional monomerα-methacrylic acid was grafted to the surface of the modified silica gel.Nicotinamide mononucleotide surface molecularly imprinted polymer were prepared by surface molecular imprinting method,it has a good specific adsorption effect on NMN,and the imprinting factor reach 2.06.Its isotherm adsorption process is more in line with the Freundlich model and has two types of binding sites;The dynamic adsorption process is more in line with the quasi-second-order kinetic equation,which belongs to chemical adsorption process.In addition,the results of selective adsorption experiments show that the separation factor is up to 25.52,the relative separation factor is up to 1.70,which has a strong specific adsorption capacity for NMN.The surface molecularly imprinted material not only has good selectivity,good thermal stability,but also has good reuse performance.（3）The nicotinamide mononucleotide molecularly imprinted solid phase extraction（MISPE）column was prepared by using the prepared nicotinamide mononucleotide surface molecularly imprinted polymer as the filler for solid phase extraction.The recovery rate reach93.32%,showing excellent practical application performance in the study of solid phase extraction of actual samples;the established MISPE-UHPLC-MS method has a good linear correlation in the concentration range of 0.25～80 ug/m L（R~2>0.99）,the detection limit of the method reaching 0.85 ng/m L,and this method can effectively eliminate the matrix effect in the detection process of UHPLC-MS,and the range of the matrix effect is improved from43.19%～130.87%to 89.72%～112.39%,and the linear correlation has also been improved,and the linear correlation coefficient R~2 has increased from 0.9469 to 0.9921,which greatly improves the sensitivity and accuracy of the analytical results;while eliminates the matrix effect,the MISPE-UHPLC-MS can also achieve the Quantitative analysis of trace NMN in orchids,the NMN content in broccoli samples is 50.5 ug/100g.This not only has reference and guiding significance for the preparation,optimization and application of NMN molecularly imprinted polymers,but also provides theoretical guidance and technical support for NMN medical research and health food development,it has very important research significance and practical value.