Study On The Detection Of 13 Free Amino Acids In Tea Based On Double-templated Molecularly Imprinting

Amino acids are involved in the formation of tea aroma,which are one of the important components of tea and an important indicator that determines the quality of tea.Tea contains a variety of free amino acids,and the difference in amino acid composition and content also determines the difference in aroma and taste of different types of tea.However,due to the complex matrix of tea samples,the interference with the test results is large.The fast and selective sample preparation technology is of great significance for the detection of amino acid content in complex matrices.Molecular imprinting technology can selectively identify and adsorb the target objects with similar molecular structure to the template.The prepared MIPs have the characteristics of predestination,specificity and practicability,and can be used as solid phase extraction materials to specifically identify the target objects and achieve efficient separation and enrichment.The magnetic imprinted solid phase extraction based on magnetic separation technology and molecular imprinting technology can achieve efficient separation and mass enrichment of target compounds in complex sample matrix.This research is mainly divided into three parts:(1)In the modes of electrospray ion source,positive ion scan(ESI+)and multi-reaction monitoring(MRM),ion source parameters were optimized,including collision gas:12 psi;Air curtain air:45 psi;Atomized gas:30 psi;Auxiliary gas:50 psi;Ion source temperature:550?;Ionization pressure:5500 V,the optimal mass spectrometry parameters were determined.The liquid chromatography conditions were optimized,such as column:150 mm C₁₈8 column;Column temperature:25?;Mobile phase composition:A₁ is 0.1%formic acid water of 10 mmol/L ammonium acetate,B₁ is acetonitrile and gradient elution procedure.A liquid chromatography tandem mass spectrometry method with simultaneous detection of 13 amino acids with high selectivity,high sensitivity,and high accuracy was established.The detection limit of 13 amino acids was less than 12?g/L,and the quantification limit was less than 39.6?g/L.The RSD was less than 4.95%,and the correlation coefficient was greater than 0.9981.The established13 kinds of amino acid instrument detection methods have the advantages of high selectivity,high sensitivity,and high detection efficiency,and the detection time is within15 min.(2)Molecular imprinting technology was used to optimize the ratio of double template molecule to functional monomer and crosslinking agent to 1:6:8,and the molecularly imprinted polymer was synthesized.The polymer materials were characterized by FT-IR and SEM,and the results showed that the polymer was polymerized successfully and showed a microsphericity,uniform dispersion and abundant surface folds.Scatchard model was used to analyze the polymer binding site and the apparent maximum adsorption amount.The binding constant of the arginine molecularly imprinted polymer binding site was 370.37?g/mL,the corresponding apparent maximum adsorption amount was 13327.39?g/g,and the binding constant of the phenylalanine molecularly imprinted polymer binding site was 39.52?g/mL,and the corresponding apparent maximum adsorption amount was 825.49?g/g.By optimizing the use conditions of DMIP-SPE,the optimal sample volume was 10 mL,the eluent was 1 mL pure water and the eluent was 3 mL 1%formic acid solution.The DMIP-SPE-LC-MS/MS method in composite tea was established.Under the three standard multiples(0.5 times,1 times and1.5 times)of burdock tea and chrysanthemum tea,the recoveries of 13 amino acids in burdock tea ranged from 71.0%to 118.2%,and the relative standard deviation(n=5)was less than 4.8%.The recovery rate of chrysanthemum tea ranged from 70.6%to 119.0%,and the relative standard deviation(n=5)was less than 4.7%.(3)First,Fe₃O₄ magnetic nanoparticles were prepared by coprecipitation method,then SiO₂ was prepared to package Fe₃O₄ magnetic nanoparticles by hydrolysis method,and amino groups were modified.Finally,Fe₃O₄@SiO₂-NH₂@MIP and Fe₃O₄@SiO₂-NH₂@NIP were successfully prepared using arginine and phenylalanine as template molecules.The magnetic molecularly imprinted nanospheres(MMIPs)were characterized by FT-IR,TEM and SEM.The results showed that the pores of the magnetic nanoparticles were enlarged and distributed evenly after the modification of amino group.Scatchard model was used to analyze the binding sites and the apparent maximum adsorption amount of the polymer.The binding constants were 476.19?g/mL and 40.16?g/mL respectively,and the corresponding maximum apparent adsorption amounts were18689.05?g/g and 978.88?g/g respectively.The magnetic dispersion solid phase extraction(MDSPE)conditions were optimized,and the optimal extraction time was 15min,the elution solvent was 2 mL 1%formic acid solution and elution was 3 min.The method of MDSPE-LC-MS/MS in burdock tea and chrysanthemum tea was established based on magnetic separation technology.Under the three standard multiples(0.5 times,1times and 1.5 times)of burdock tea and chrysanthemum tea,the recovery range of burdock tea was 73.1%¹18.8%,and the relative standard deviation(n=5)was less than4.9%.The recovery of chrysanthemum tea matrix ranged from 72.9%to 119.6%,and the relative standard deviation(n=5)was less than 4.8%.The method can save the pretreatment time and speed up the sample processing efficiency.