Preparation Of Chiral Synephrine Purity Reference Material By Molecularly Imprinted Polymerization And Its Chiral Separation

Synephrine is one of the important natural products of citrus,which has anti-oxidant,bacteriostatic,vasoconstrictive and pharmacological effects of increasing blood pressure.Since the United States issued a ban on ephedrine in dietary supplements in 2004,Synephrine is widely used in medicine,food,beverage,feed and cosmetics industries as a substitute because of its structure and pharmacological effects similar to ephedrine.In this study,a molecularly imprinted polymer of synephrine was synthesised,which was used to extract Synephrine from Fructus Aurantii,then Synephrine was purified by anti-solvent recrystallization and the certified reference material candidate was prepared,determination of chiral resolution of the prepared synephrine reference material was carried out,this provides new idea for the extraction,purification and chiral separation of Synephrine..Synephrine was used as a template molecule,acrylic acid and hydroxyethyl acrylate were bifunctional monomers,azobisisobutyronitrile was used as an initiator,and ethylene glycol dimethacrylate was used as a crosslinking agent.Synephrine was prepared by precipitation polymerization molecularly imprinted polymer.The static adsorption method was used to explore the specific adsorption capacity of Synephrine molecularly imprinted polymers under different template molecules,functional monomers,and initiator ratios.The prepared imprinted polymeric materials were characterized by scanning electron microscopy,and the synthesized molecularly imprinted polymers were used as solid phase extraction filler for extracting synephrine.Extraction ratio of Synephrine was used as reference target,and the types and ratios of eluent were optimized.The purity of the extracted synephrine was determined according to the preparation method of the national standard substance.The H-NMR and infrared spectroscopy were used to preliminary characterize the extracted synephrine.After the characterization,the anti-solvent recrystallization method was utilized to further purification of Synephrine reference materials until the purity is more than 99%.The values of the synephrine purity reference materials are determined by mass balance method and nuclear magnetic quantification method.The moisture was measured by Karl Fischer Coulomb titration.The content of inorganic elements is determined by microwave digestion-inductively coupled plasma mass spectrometry.ICP-MS)and Microwave Digestion-Inductively Coupled Plasma Emission Spectroscopy(Microwave-assisted ICP-OES);the content of organic volatile impurities is measured by headspace gas chromatography,and then the homogeneity and stability studies of the Synephrine purity reference material were performed using high performance liquid chromatography Test,and finally evaluate the uncertainty introduced during the development of the Synephrine purity reference material was evaluated.The prepared synephrine purity reference material is a racemate and has no optical rotation.Due to the different physiological and pharmacological activities of the R and S synephrine,in this study uses high-performance liquid chromatography was used to perform chiral resolution,the effects of chiral col?mn types,mobile phase types and ratios,col?mn temperature,and flow rate on the separation of the enantiomeric synephrine were investigated.The main results of this research were as follow:1.Synephrine was used as a template molecule,acrylic acid and hydroxyethyl acrylate were bifunctional monomers,azobisisobutyronitrile was used as an initiator,and ethylene glycol dimethacrylate was used as a crosslinking agent.The experimental results show that when the amount of template molecules to functional monomers,initiators,and cross-linking agents is 1: 4: 2: 20 in 10 mL of acetonitrile.The polymer morphology and specific recognition are good,and the adsorption effect is the best.The maxim?m adsorption amount is 228.82 ?mol / g.The molecularly imprinted solid-phase extraction technology(MISPE)was used to extract and purify the synephrine from the crude powder of Fructus Aurantii,acetonitrile: ethyl acetate = 6: 4(V / V)as the eluent,and methanol: acetic acid = 8: 2(V / V)was the eluent.The mass fraction of synephrine in the synephrine extract was increased from 1.93% to 93.34%,and the extraction rate was 73.90%.2.The anti-solvent recrystallization method was used to further purify the extracted Synephrine.The purity of synephrine was rised from 93.34% to 99.78%,and the extraction rate was 73.84%.The purity meets the purity requirements of China's secondary reference materials.Based on the purification conditions explored,many repeated experiments were performed to obtain a large n?mber of purified product which provides idea for the subsequent development of similar certified reference materials.at same time qualitative research was performed by nuclear magnetic resonance spectroscopy and infrared spectroscopy.Then reference materials were determined by quantitative nuclear magnetic resonance method and mass balance method.During assignment Synephrine,detection wavelength,sample concentration,and flow ratio were optimized,and determination method of Synephrine by HPLC was established.The conditions for quantitative analysis of Synephrine by HPLC are as follows: Col?mn: Waters SCX col?mn(4.6 mm × 250 mm,5 ?m);mobile phase: acetonitrile: water(containing 50 mmol potassi?m dihydrogen phosphate)= 10: 90;flow rate : 1 mL / min;col?mn temperature: 25 ?;injection vol?me: 10 ?L;detection wavelength: 275 nm,isocratic elution.The results shown that mass fraction of main component is 99.78%,the contents of moisture,organic volatiles and inorganic impurities were respectively 0.2501%,0.015%,0.00302%,the value purity value was 99.52%.Synephrine purity reference material has good stability and homogeneity within six months,all indicators meet the requirements of China's secondary reference materials,and have already declared the national secondary reference materials.3.The high performance liquid chromatography was used to the racemic synephrine,and Chiralpak ZWIX(+)chiral col?mn,most suitable for the separation of the racemic synephrine was selected,then the optim?m of mobile phase,col?mn temperature,flow rate and detection wavelength were carried out.the optimal conditions: the mobile phase was 2 amino-1-butanol: tetrahydrofuran : methanol : water=0.1:49:49:2(vol?me ratio),and the flow rate was 0.5 mL/min at a col?mn temperature of 25 ? and a detection wavelength of 275 nm.The enantiomeric resolution of synephrine obtained under this condition was 4.41.This method is simple,fast,and has high resolution,and is the preferred method for the resolution of synephrine racemate.