Modified QuEChERS Method With Magnetic Solid-phase Extraction For Estrogen Mimics In Meat Samples Prior To Pre-column Derivatization And UHPLC-MS/MS

In this research,a traditional pretreatment method for food samples,which was called QuEChERS and known as quick,easy,cheap,effective,rugged and safe,was modified by using magnetic solid-phased extaction?MSPE?to replace the clean-up procedure by column SPE.This QuEChERS-MSPE method was successfully applied to extract 11 estrogens and its mimics?including estrone?E1?,estradiol?E2?,estriol?E3?,17?-ethinylestradiol?EE2?,2-methoxyestradiol?2ME2?,estradiol benzoate ?E2B?,estradiol-17-valerate?E2V?,diethylstilbestrol?DES?,dienestrol?DE?,hexestrol?HEX?,bisphenol A?BPA??from pork and chicken meat samples which had complex matrices.To be specific,C₁₈/NH₂-mixed functionalized mesoporous magnetic nanoparticles?MNPs?were synthesized and were regarded as the sorbent in MSPE.With the aids of an extra magnetic field,the pre-concentration and purification of target analytes could be easier,faster and more powerful.Then,the processed samples were analyzed by UHPLC-MS/MS after pre-column derivatization.The whole experimental procedures are as follows.Part 1.C₁₈/NH₂-Fe₃O₄@SiO₂ mesoporous MNPs were synthesized and a MSPE method based on the MNPs was set up.First,different methods to synthesis C₁₈/NH₂-Fe₃O₄@SiO₂ mesoporous MNPs were investigated in this study,including selection of basic condition in producing magnetic core by solvothermal method,molecular weight of polymer,one-step or two-step synthesis,anhydrous toluene condition and so on.Different magnetic materials synthesized by different methods showed various extraction capacities,and the best one was adopted.Then,the prepared MNPs were characterized by several measures,such as dynamic light scattering?DLS?,transmission electron microscope?TEM?,Brunner-Emmet-Teller measurements?BET?,Fourier transform infrared spectrum?FT-IR?,thermo-gravimetric analysis?TGA?,Vibrating Sample Magnetometer?VSM?,etc.The results suggested that the MNPs owned a magnetic core with diameter of 330 nm,SiO₂ shell of 10 nm and functional groups shell of 20 nm.Additional,it is full of mesoporous of 2.5 nm and the pore volume reached 0.216 cm³/g,which increased its specific surface area to268.10 m2/g and promoted the efficiency of MSPE.Last,some parameters which could influence the MSPE efficiency were tested systematically,involving pH and salty concentration of sample solution,volume of sample solution and desorption solvent,extraction and desorption time,desorption solvent type.Finally,a most remarkable MSPE condition was choiced.Part 2.A QuEChERS extraction method which could be applied well to meat samples was established.A prior enzymatic hydrolysis step was carried out to extraction and was optimized.Then,the influencing factors in QuEChERS method were investigated.5 mL ACN was use to extract analytes and anhydrous NaCl,trisodium citrate dihydrate,sodium citrate dibasic sesquihydrate,anhydrous MgSO₄were added to remove water residue with a ratio of 2/2/1/8,while extraction time was set as 20 min,then the complete procedure was repeated,extracts were collected.In this study,7 typical estrogen mimics?E1,E2,E3,EE2,DES,DIEN,BPA?were chosen to optimize QuEChERS-MSPE conditions.Part 3.A UHPLC-MS/MS analysis method was established to detect and quantify 11 estrogens and its mimics in animal muscle tissue of 2 species after QuEChERS-MSPE extraction.First,dansyl chloride was introduced to samples to initiate derivatization,and the products were detected in positive mode MRM by ESI-MS which could cause higher response.Method validation was investigated in matrices and internal sample calibration method was used for quantitative.Under the reasonable condition,all analytes showed good linearity with acceptable correlation coefficients?R²?above 0.99.Method detection limit?MDL?and method quantitation limit?MQL?were 0.02³.00 and 0.07¹0.00 ng/g,respectively.Besides,the established method also suggested ideal recovery and precision.The result showed that this QuEChERS-MSPE-UHPLC-MS/MS method was a safe,accurate,effective,sensitive scheme to determine the analytes of very low concentration in food sample with complex matrix.