磺酸功能化共价有机骨架固相微萃取纤维的制备及其在小鼠脑部神经递质分析中的应用

Preparation of sulfonic acid functionalized covalent organic framework solid phase microextraction fibers and their application in the analysis of neurotransmitters in the mouse brain

神经递质(NTs)是细胞间交流的基本化学物质之一,研究表明,其含量的异常变化与多种神经性疾病相关。因此,建立精准的分析方法对神经递质的检测具有重要意义。本研究以三醛基间苯三酚和1,4-二氨基-2-硝基苯为共价有机骨架单体,经过衍生化后,在室温下制备了一种结晶性好、化学/热稳定性好、疏水性强、介孔结构均匀的磺酸功能化共价有机骨架材料(COF-SO_(3)H)。然后,将其涂敷在不锈钢纤维上制备了具有强阳离子交换作用的新型涂层,利用扫描电镜、红外光谱、N 2吸附-脱附等表征手段对COF-SO_(3)H纤维的形貌、比表面积等性质进行表征,并比较了COF-SO_(3)H纤维与HLB、C18、MCX、Amino、PXC 5种萃取纤维对神经递质的萃取效率,考察了相关参数对COF-SO_(3)H萃取目标物的影响。结果表明,COF-SO_(3)H具有良好的晶型,介孔结构分布均匀,与其他涂层材料相比,COF-SO_(3)H纤维对神经递质表现出优异的萃取性能。最佳的萃取参数:脱附溶剂为甲酸-甲醇-水(0.5∶49.5∶50,v/v/v)、萃取时间和脱附时间均为15 min。在优化条件下,将COF-SO_(3)H固相微萃取纤维与高效液相色谱-串联质谱法(UPLC-MS/MS)结合,建立了检测小鼠脑部神经递质的分析方法。7种目标神经递质在线性范围内具有良好的线性关系(相关系数r 2均大于0.99),单胺类神经递质和氨基酸类神经递质的定量限(S/N≥5)分别为0.003~0.005μg/mL和3~5μg/mL,一日内连续制备4组同一质量浓度的样品进行分析,其4次检测的RSD低于20%,7种目标神经递质在小鼠脑匀浆中3个水平下的精密度(0.80%~9.70%)和准确度(2.08%~17.72%)良好,绝对基质效应为82.22%~117.92%,表明复杂基质对目标分析物的准确测定影响较小。所建立的方法可以成功用于小鼠脑部目标神经递质的分离分析。

Neurotransmitters(NTs)are essential for intercellular communication and primarily include monoamine,amino acid,and cholinergic NTs.These molecules play important roles in the body’s stress response,motor coordination,neuronal communication,and homeostatic functions.Previous studies have shown that abnormal changes in NT levels are associated with various neurological disorders.Therefore,the development of accurate analytical methods for NT detection will enhance the current understanding on complex neuropathophysiology by providing functional knowledge and techniques for early diagnosis,thereby facilitating the development of new therapeutic options for the related diseases.The solid phase microextraction(SPME)technique combines sample preparation,separation,and enrichment in a single step and is minimally invasive,low cost,solvent free,and high throughput.SPME has been successfully applied to the in vivo analysis of target analytes in animal,human,and plant tissues.The coating material plays a significant role in the development of in vivo SPME methods and must meet various analytical requirements,including a suitable geometry for the SPME device,high extraction capacity,excellent selectivity,and wide extraction coverage for the target analytes.Covalent organic frameworks(COFs)are porous crystalline polymers constructed from organic framework units through strong covalent bonds;these materials are characterized with a low density,large specific surface area,permanent porosity,excellent chemical/thermal stability,and easy functionalization.In this study,a sulfonic acid-functionalized COF material(COF-SO_(3)H)with good crystallinity,excellent chemical/thermal stability,strong hydrophobicity,a uniform mesoporous structure,and narrow pore size distribution was prepared using 2,4,6-triformylphloroglucinol and 1,4-diamino-2-nitrobenzene as monomers.Then,the COF-SO_(3)H was coated onto the surface of stainless-steel fibers and used for in vivo enrichment of NTs.The structural properties of COF-SO_(3)H were characterized using various techniques,such as scanning electron microscopy(SEM),Fourier transform-infrared spectroscopy(FT-IR),and X-ray diffraction(XRD),all of which showed that COF-SO_(3)H had a good crystalline structure and uniform mesopore distribution with a specific surface area of 46.17 m 2/g.Compared with the SPME fibers of HLB,C18,MCX,amino,and PXC columns,the prepared COF-SO_(3)H fibers showed better extraction efficiency for the target NTs.Next,the factors affecting SPME efficiency were optimized.The optimal desorption solvent was formic acid\|methanol\|water(0.5∶49.5∶50,v/v/v),and the optimal extraction and desorption times were 15 min.A method for the in vivo analysis of NTs in the brains of mice was established by combining the COF-SO_(3)H fibers with ultra performance liquid chromatography\|tandem mass spectrometry(UPLC-MS/MS)under optimal conditions.The NTs were separated on an Acquity UPLC BEH-C18 analytical column(100 mm×2.1 mm,1.7μm)with 0.1%formic acid aqueous solution(A)and acetonitrile(B)as the mobile phases.The flow rate was set to 0.2 mL/min,and the gradient elution procedure was as follows:0-4 min,5%B-6%B;4-7 min,6%B-5%B;7-11 min,5%B.Under optimal conditions,the method showed good linearity(r 2>0.99).The limits of quantification(S/N≥5)were in the range of 0.003-0.005μg/mL and 3-5μg/mL for monoamine and amino acid NTs,respectively,with RSDs of less than 20%.The method showed good precision(0.80%-9.70%)and accuracy(2.08%-17.72%),with absolute matrix effects in the range of 82.22%-117.92%.These values reflect the good purification and enrichment abilities of the proposed fibers for the target analytes.Finally,the established SPME method was combined with UPLC-MS/MS and successfully applied to quantify target NTs in the brains of mice.The proposed strategy provides a practical method for the in vivo detection and quantitative analysis of NTs and expands the applications of functionalized COF materials for the analysis of various targets.