Establishment And Preliminary Clinical Application Of New Methods For Methotrexate Detection

ObjectiveTo improve the sensitivity and selectivity of methotrexate（MTX）detection,a new electrochemical detection method based on stearyltrimethylammoniumbromide（STAB）was constructed.In order to further improve the specificity of MTX detection,new selective recognition material-magnetic molecularly imprinted polymers（MMIPs）were prepared.The identification material was used as a pre-treatment material to achieve efficient adsorption,selective recognition and rapid magnetic separation of MTX in patient plasma.The pretreatment method was combined with high performance liquid chromatography to realize the direct detection of MTX drug concentration in plasma,which provided a new method support for the clinical detection and monitoring of MTX drug concentration.Methods1.A novel acetylene black（AB）sensor modified with STAB was fabricated and applied to the determination of MTX in serum.The sensor was characterized by cyclic voltammetry（CV）and electrochemical impedance spectroscopy（EIS）.CV was used to optimize several factors affecting the performance of the sensor,such as the concentration and modification time of STAB,the amount of mineral oil,MTX enrichment time,buffer p H,and scan rate.At the same time,we also determined whether the selectivity,repeatability and stability of the sensor met the requirements.Finally,the sensor was used to detect MTX concentration in clinical sample under optimal conditions,and the results were compared with those obtained by high-performance liquid chromatograph-ultraviolet（HPLC-UV）.2.Fe₃O₄nanoparticles（NPs）were synthesized by hydrothermal method.Then Fe₃O₄@SiO₂was prepared by wrapping SiO₂thin film on the Fe₃O₄NPs surface with tetraethyl orthosilicate（TEOS）.Next,MMIPs were prepared by sol-gel method with Fe₃O₄@SiO₂as the reaction substrate,MTX as the template molecule,（3-aminopropyl）triethoxysilane（APTES）as the functional monomer and TEOS as the cross-linking agent.Morphology analysis,infrared spectrum analysis（FT-IR）,X-ray diffraction analysis（XRD）and X-ray energy spectrum analysis（XPS）were used to characterize the materials.The adsorption performance of MMIPs on MTX was evaluated by static,dynamic and selective adsorption experiments.3.The extraction conditions were optimized with 0.02mg/m L MTX-methanol solution as the treatment object,MMIPs as the adsorbent,water-methanol solution as the leacheate,and methanol-acetic acid solution as the eluent.A magnetic solid phase extraction（MSPE）technique for MTX detection in plasma samples was established and combined with HPLC-UV for rapid detection of MTX blood concentration in clinical samples.Results1.STAB modification of ABPE can significantly improve the adsorption and catalytic performance of the sensor to MTX.Under the optimal conditions,the prepared sensor showed favorable performance in the determination of MTX with the linear range of 0.005μM to 7.0μM and the detection limit of 3.07 n M.Furthermore,the fabricated sensor exhibited good stability and repeatability,which was of importance in the electrochemical detection.In addition,the sensor was less affected by interferences and had better selectivity.Finally,the sensor was applied to the detection of MTX in real sample of plasma,and the standard recovery was 4.38%-7.58%.The detection results were in good agreement with the HPLC-UV.2.Fe₃O₄NPs were successfully synthesized,and a layer of SiO₂film was further wrapped on the surface of Fe₃O₄NPs to obtain Fe₃O₄@SiO₂with core-shell structure.MMIPs were prepared by molecular imprinting technology.Transmission electron microscope,FT-IR,XPS,XRD and other characterization results showed that MMIPs had been successfully prepared and had high magnetic response.The adsorption isothermal curve showed that MMIPs adsorbed the template molecule at 39.56 mg/g,imprinted factor at 9.40 and balanced for 60 min.The poor adsorption capacity of the material to the interfering material indicated that the material had good anti-interference ability and selectivity to the adsorption of MTX.3.Under the optimal MSPE conditions,the MMIPs dosage was 100 mg,the sample loading time was 120 min,the leacheate was water-methanol（8:2,v/v）,the eluent was methanol-acetic acid（4:1,v/v）,and the elution time was 60 min.MTX was linear in the range of 0.00005-0.25 mg/m L,and the detection limit was 12.51 ng/m L.The MSPE-HPLC-UV method established in this experiment had a good accuracy in the detection of MTX,and was in good agreement with the results of drug concentration analyzer.ConclusionsIn this study,a novel MTX electrochemical sensor based on STAB had been successfully prepared,which improved the sensitivity of MTX detection.On this basis,a new type of MTX adsorption material,MMIPs,was prepared.A high performance liquid detection method based on magnetic molecularly imprinted solid phase extraction technology was established,which provided a new idea for the selective determination of MTX blood drug concentration.It has been proved that both of the two methods can realize the direct detection of MTX plasma drug concentration,which provides new method support for the detection and monitoring of MTX drug concentration.