Abstract: Microcystins (MCs) are cyclic heptapeptide toxins that cause aquatic pollution. Through bioaccumulation, they can induce irreversible cellular and organ damage and significantly increase the risk of liver cancer, posing a serious threat to human health. However, interference from complex matrices in environmental water samples, as well as the trace concentrations of MCs, necessitates the development of efficient pretreatment techniques. Capillary microextraction (CME) is a miniaturized sample preparation technique derived from in-tube solid-phase microextraction (SPME). By integrating extraction and concentration into a single step, CME offers distinct advantages, including low sample consumption, rapid equilibration, and high extraction efficiency. The performance of CME is critically dependent on the properties of its stationary phase, which governs analyte retention and selectivity. Traditional organic polymer monolithic columns are widely used due to their simple preparation and the availability of diverse functional monomers. Nevertheless, their fabrication process often suffers from issues such as particle agglomeration and heterogeneous pore distribution. These structural irregularities lead to limited specific surface area, poor mechanical stability, and ultimately, compromised extraction efficiency. With their high specific surface area, uniform pore size distribution, and highly tunable surface properties, metal organic frameworks (MOFs) are ideal adsorbents that effectively overcome the limitations in porosity and specific surface area associated with traditional polymer-based materials. This study employed an ionic liquid-mediated ionothermal reaction to rapidly fabricate a metal-organic framework HKUST-1-functionalized polymer monolith column (HKUST-1@poly(DVB-co-AM-co-GMA)), which was used as a capillary microextraction (CME) medium to enrich MCs in water. In detail, the HKUST-1@poly(DVB-co-AM-co-GMA) functionalized monolithic column was prepared using glycidyl methacrylate (GMA) and acrylamide (AM) as bifunctional monomers, divinylbenzene (DVB) as the crosslinking agent, a mixture of 1-hexyl-3-methylimidazolium tetrafluoroborate ([HMIM][BF₄]) and cyclohexanol as dual porogens, and HKUST-1 as the dopant. The morphology, elemental composition, specific surface area and pore size distribution of the monolithic material were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS) and nitrogen adsorption-desorption isotherms. The results indicate that the incorporation of the metal-organic framework HKUST-1 significantly increases the proportion of mesopores and micropores in the monolithic column material. This enhancement increases the specific surface area and overcomes the low porosity, small surface area, and swelling susceptibility characteristic of traditional organic polymer monolithic columns. Consequently, the extraction efficiency of the proposed MOF hybrid monolith for enriching MCs is significantly improved. Meanwhile, the effects of extraction conditions (sample pH, sample flow rate) and elution conditions (formic acid concentration in eluent, elution flow rate) on enrichment efficiency were systematically investigated. Under optimized extraction conditions, the HKUST-1-functionalized monolith-based CME coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to enrich and analyze three MC isomers in environmental water samples. Over the concentration range of 0.01-10 µg/L, the three MCs exhibited good linearity (R²≥0.995 5) between their concentrations and the peak areas of corresponding quantified ions. The limits of detection (LODs) and quantification (LOQs) were 0.28-0.60 ng/L and 0.94-1.99 ng/L respectively, both significantly below the WHO maximum permissible concentration for MC-LR in water. The established analytical method yielded recovery rates ranging from 82.2% to 111.1% for three environmental water samples, with relative standard deviations (RSD) ≤7.9% (n=3), exhibiting satisfactory precision and accuracy. Compared to other reported methods, the developed method requires less sample solvent (1.0 mL), achieves a lower limit of detection, and completes enrichment within 30 minutes. In conclusion, we demonstrate that the hyphenated analytical method efficiently detects ultra-trace MCs in environmental water, due to the specific coordination, hydrogen bond and π-π interactions afforded by the HKUST-1 functionalized monolith. This method shows promising value for monitoring trace environmental contaminants and advancing miniaturized pretreatment techniques.

Key words: microcystins (MCs), metal organic frameworks (MOFs), monolithic columns, capillary microextraction (CME), liquid chromatography-tandem mass spectrometry (LC-MS/MS)