H‐Bonded Supramolecular Crystals‐Coated Solid‐Phase Microextraction enables the Matrix Interference‐Free Detection of the COVID‐19 Antiviral Drugs

Since the outbreak of COVID‐19, many antiviral drugs have been used to treat infected patients. Excessive drug concentrations may not only damage patients' organs, but also lead to increased mortality. The lack of data makes it difficult for them to be treated with these drugs under proper and safe dosage. However, due to the complexity of fluid matrixand structures of the antiviral drugs, it is still difficult to detect the drugs in biofluids. Herein, solid‐phase microextraction (SPME) is applied for the detection of biological matrix, which has the advantages of simplicity, efficiency and environmental protection. With abundant hydrogen bond sites and appropriate molecular volume, biocompatible graded‐pore HOF10x series materials are selected as the fiber coating for exploration. Consequently, HOF101 could achieve high enrichment effect of drugs due to the commensurate mesopores and hydrogen bond non‐covalent interaction. Moreover, the excellent extraction capacity of the HOF101 fiber is verified to be consistent in various interfering conditions. The obtained limits of detections (LODs) are 0.24–0.66 ng L−1, and the recoveries of the biofluids in three concentration levels ranged from 80.30% to 120.1%. It shows the low LODs and satisfactory recoveries of this analytical method, which is expected to be applied in medical systems for drug monitoring.


Introduction
Since the Outbreak of the severe acute respiratory coronavirus 2 (COVID- 19) in December 2019, the pandemic has caused a huge pretreatment of solid phase extraction, and then analysis by liquid-mass [10][11][12][13] .Due to the limitation of drug monitoring, there are few clinical test data on these drugs at present, and it is difficult to find the optimal dose. [14]It is particularly important to track and adjust the dosage of drugs used by patients after infection with COVID-19, as blindly conforming to the previous dosage may lead to subtherapeutic or toxic effects. [15,16]eanwhile, since DDI may exist between different drugs, leading to unpredictable drug levels in the body, dose adjustments should be made according to individuals. [17]Therapeutic drug monitoring (TDM) is crucial for the effectiveness and safety of treatment. [18]Therefore, it is of vital importance to establish a facile and sensitive method for antiviral drug detection.
Developing and validating sensitive and precise methods for bio-samples is of great significance for pharmacokinetic monitoring and efficacy study of drugs.[21][22] In general, those conventional methods are costly and time-wasting, requiring a large amount of organic solvent and may lead to loss or cross-contamination of analytes at low concentrations.Solidphase microextraction (SPME) is a kind of green sample pretreatment method, which integrates sampling, extraction, enrichment and desorption into one step.SPME is an efficient, lowcost, and easily automated method.Thus, SPME has been wildly applicated in environment, food and life sciences. [23,24]SPME coupled with liquid chromatography-tandem mass spectrometry (SPME-LC-MS/MS) has been employed for bioanalysis of complex samples.Moreover, biocompatible SPME (bio-SPME) fiber can be directly applied to complex biological samples without any pre-treatment step, which makes it suitable for in vivo and nondestructive sampling. [25]Simple operation, high sensitivity, and minimally invasive probing make bio-SPME a promising sample processing method to meet the current demand for TDM of COVID-19 patients.
The material of the SPME coating is the key factor for extraction efficiency and consequently the sensitivity and stability of the method. [26]Due to the large molecular volume of these antiviral drugs, their varying concentrations in human biological fluids, and the complex composition of their biological substrates, the choice of coatings is of paramount importance.To date, there is no SPME method established for detecting anti-COVID-19 drugs in human body fluids, which is a huge challenge.
Up to now, many extended frame materials are used to be SPME coating, including metal-organic frameworks (MOF), covalent-organic frameworks (COFs), etc. [27] Porous frame materials feature great potential as SPME coating material because of large specific surface area, low density, easy functionalization, and modification. [28]Among them, hydrogen-bonded organic frameworks (HOFs) are a novel and promising class of porous frame materials.HOFs are self-assembled from organic monomers with electron donors and acceptors by intermolecular hydrogen-bonding interactions. [29]With the advantage of weak, reversible and flexible hydrogen bond connections, HOFs can be synthesized environmentally friendly, under rather mild conditions.HOFs feature easy purification and regeneration, high porosity and flexibility, and workable solution processability.Compared with MOFs or COFs, the inherently weaker bonding nature of hydrogen bonds makes HOFs relatively harder to stabilize the frameworks and porosities, but much more easily to be purified and regenerated by simple recrystallization. [30]Moreover, due to their metal-free characteristics, HOFs are more biocompatible with organisms than traditional metal-based porous materials, thus exhibiting great perspective for their biomedical applications, such as in vivo SPME, drug delivery, enzyme encapsulation, photodynamic therapy, etc. [31,32] Herein, based on the molecular volume and abundant hydrogen bond sites of these three drugs, three kinds of HOFs (HOF100, HOF101, and HOF102) with the same functional group but different pore sizes were synthesized as extraction materials. [33]The large molecular volume of the antiviral drugs requires that the coating material has large pores, and the abundant N and O elements can form hydrogen bond interactions with the abundant carboxylic acid groups on the surface of HOF to promote adsorption.Notably, the obtained HOF101 exhibited hierarchical pores (1.8 and 2.4 nm) as well as H-bond donor and acceptor, providing efficient diffusion pathways and adsorption sites.By coupling SPME to LC-MS/MS, the self-made HOF101 SPME fiber showed superior enrichment capabilities for the antiviral drugs.These results are in accord with the diffusion ability of their hierarchical pores.Furthermore, the extraction performance of HOF101 fiber was shown to be unaffected under different pH values and the existence of endogenous substances.Therefore, the HOF101 fiber is expected to be used to develop a simple, environmentally friendly and sensitive analytical method for antiviral drugs (Scheme 1).

Synthesis and Characterization of HOF100, HOF101, HOF102
Herein, we used a series of isostructural HOF family, including HOF100, HOF101, and HOF102.H 4 TCPy, H 4 TBAPy, and H 4 TNAPy have been used to construct stable HOFs, corresponding HOF100, HOF101, and HOF102, respectively (Figure 1).The monomers have one pyrene center, which is a fused benzene ring molecule with planar conformation and large - conjugated system. [34]The HOFs materials can be easily made by adding acetone into the DMF solution of dissolved monomers.
Scanning electron microscopy (SEM) images showed that the HOF101 exhibited a rod-like shape (Figure 3a,b).Similarly, HOF100 and HOF102 also exhibit rod-like structure    (Figure S2, Supporting Information).Electron microscopy characterization of HOF101 was conducted to study more about the nanostructure.Measured from high-resolution TEM images (Figure 3c-e), the lattice width of HOF101 was 2.07 nm, which is in correspondence with the (001) crystal plane of HOF101.The transmission electron microscopy (TEM) image (Figure 3f) and the corresponding elemental mapping showed that the C, O elements were uniformly distributed in HOF101 and (Figure 3g,h).The TEM images of HOF100 and HOF102 were showed in Figure S1 (Supporting Information).In addition, according to thermogravimetric analysis (TGA) under nitrogen atmosphere (Figure S2, Supporting Information), the thermal decomposition temperatures of HOFs were all above 400 °C.The HOF family showed excellent thermal stability.Additionally, Fourier transform infrared (FT-IR) spectra were used for the monomers and HOFs.As shown in Figure S3 (Supporting Information), there is no significant difference between the spectra of the monomers and HOFs.However, because of the formation of hydrogen bonds, the peaks of the carbonyl group shifted slightly toward the low wave number, which also proves the successful synthesis of HOFs. [36]

High-Efficient Extraction of the Antiviral Drugs
The extraction efficiency of antiviral drugs from human biofluids by HOF101 fiber was studied by SPME and HPLC-MS/MS.As the thriving pretreatment technique combining separation, enrichment and purification, SPME was reported to be successfully applied for sample pretreatment techniques in several biological analysis studies. [37,38]Because of the surface hydroxyl functional groups of HOFs, the HOFs exhibited excellent water affinity, which was confirmed as the water contact angle of HOF100, HOF101, and HOF102 was 28°, 0°, and 0°.(Figure S4, Supporting Information).In this way, HOFs have excellent hydrophilicity and are expected to demonstrate high extraction efficiencies for polar analytes.In terms of the large molecule volume and high boiling point of these antiviral drugs, the drugs were extracted by the direct immersion mode from the sample matrix, instead of headspace mode.
As shown in Figure S5 (Supporting Information), the thickness of the self-made HOF101 fiber was 30 μm.The extraction efficiencies of HOFs fibers toward the antiviral drugs were compared with commercial SPME fibers.Commercial polydimethylsiloxane (PDMS), polyacrylate (PA), PDMS/divinylbenzene (PDMS/DVB), and DVB/carboxyl/PDMS (DVB/CAR/PDMS) were selected.Comparing the enrichment factors of the HOFs fiber with that of the commercial fibers, it could be concluded that HOFs showed exceptional extraction efficiencies (Figure 4).What's more, the enrichment factors of HOF101 toward Arb, Rem, and Rit were 3554, 1655, and 5271, respectively.While HOF100 and HOF102 fibers remained only 0.43-0.63 and 0.55-0.67fold extraction efficiencies toward the antiviral drugs compared to HOF101 fiber.These satisfactory enrichment efficiencies of HOF101 fiber could be attributed to the more appropriate mesopores of HOF101 compared to HOF100 and HOF102.In addition, HOF101 can form abundant hydrogen bonds with antiviral drug molecules, which further promotes the adsorption effect (Figure 5).Moreover, the conjugation interaction between HOF101 and the antiviral drugs was also beneficial for adsorption.These results fully demonstrated the proper and outstanding pore structure formed by the HOF101.
The self-made HOF101 fiber was successfully applied in the detection of the antiviral drugs in biofluids.Parameters during the SPME process, namely extraction time, desorption time and desorption solvent were investigated, in order to provide optimization conditions for subsequent experiments, and further improve the performance of the method.As shown in Figure S6 (Supporting Information), the optimal extraction time was 40 min, the optimal desorption time was 20 min, and the optimal desorption solvent was MeOH.

Adsorption Mechanism
To study the absorption mechanism of HOFs and the analytes, the TEM images and the corresponding elemental mapping were taken after the HOF101 absorbed the antiviral drugs.Figure S7 (Supporting Information) showed that the N, P, S elements were uniformly distributed in HOF101, which means the analytes were uniformly adsorbed.In addition, the FT-IR spectra of pristine HOF101 and HOF101 after absorbing antiviral drugs were shown in Figure S8 (Supporting Information).There was no obvious difference between these spectra.We speculate that it was because the functional groups of the analytes and the HOFs were similar, and the amount of the analytes were significantly lower than that of the HOFs.Moreover, the N 1s X-ray photoelectron  spectroscopy (XPS) spectra of HOF101 after absorbing the target drugs and HOF101 were also acquired to further confirm the adsorption (Figure S9, Supporting Information).Compared with the N 1s spectrum peak of HOF101, the fitting result of the N 1s spectra after adsorption of the analytes by the HOF101 were consistent with the molecular structure of the analytes, [39] further proving the successful adsorption of the HOF101 to the analytes.
To thoroughly understand the pore-dependent absorption mechanism in these isostructural HOFs, the host-guest interaction was analyzed using computational simulation (Figure 5; Figure S10, Supporting Information). [35]Theoretically, owing to the large molecular volume of the Rit and the small pore size of HOF100, it can be seen that the Rit cannot diffuse into the HOF100 channel.Although HOF102 has the largest pore size, the number of hydrogen bonds formed with analytes by calculation was much less than that formed by HOF101.Therefore, through calculation of simulation and practical effects, HOF101 had the best adsorption effect, because its pore size structure well matched the analytes, and more hydrogen bonds could be formed to achieve better adsorption.

Matrix Substance Resistance of the HOF101 Fiber
Coating selectivity is an important factor in SPME, which enables the absorbent to extract target analytes directly from biological samples without other pretreatments.Herein, the anti-interference performance of the HOF101 fiber to different substrates was investigated. [40]o investigate the selectivity and the anti-interference ability, HOF101 fiber was employed to extract the antiviral drugs under different pH values (3 and 9) , [40] a salt solution (PBS) or a mixture of endogenous substances (including proteins, carbohydrates, organic acids, and amino acids, 36 times the total concentration of the target analytes).The antiviral drug solution was adjusted to different pH values by concentrated HCl or NaOH solutions.The extraction capabilities of the HOF101 fiber toward the antiviral drugs in acid or alkaline conditions were shown in Figure 6a,b.The extraction efficiencies of the HOF101 fiber were negligibly affected by the change in pH values.
As for the interference of substances in real bio-fluids, the extraction efficiencies of the HOF101 fiber toward antiviral drugs in the environment of the interfering matrix were satisfactorily consistent with those in standard deionized water solutions (Figure 6c).These optimal results of the stability of extraction efficiencies were guaranteed by the unique adsorption mechanism of hydrogen bond and host-guest interaction.With the large pore structure and strong hydrothermal stability, HOF101 fiber could directly and efficiently extract target antiviral drugs from untreated biological samples, which greatly contributed to simplifying the sample pretreatment procedure and improving the sensitivity of the analytical method.The results demonstrated the stability of HOF101 fiber in acid-base environment and further proved its potential application in the human matrix environment.

Method Evaluation and Application in Real Sample
As concluded in Table 1, excellent enrichment and extraction performances toward all target antiviral drugs were obtained.The HOF101 fiber was used for the quantification of the antiviral drugs (Arb, Rem, and Rit) using the direct immersion SPME method.The method exhibited a linear range from 5 to 20 000 ng L −1 , with regressive coefficient in the range of 0.9928-0.9992.Limits of detection (LODs) fell within the range of 0.24 to 0.59 ng L −1 .Single-fiber reproducibility and fiber-to-fiber reproducibility in the form of relative standard deviations (RSDs), range from 7.8% to 8.8%, 1.2% to 6.7%, respectively.In conclusion, based on the unique H-bond interaction and size selectivity of the HOF101 fiber, a highly sensitive and selective analytical method was successfully developed for the antiviral drugs.Subsequently, in order to investigate the application of this method in real biofluid samples, extractions of the antiviral drugs were investigated in artificial urine and human serum samples.The recoveries of antiviral drugs were approximately 82.7%-120.1% (Table 2 and 3), which proved the practicability of the method.

Conclusion
In conclusion, we successfully synthesized a series of isostructural HOF materials (HOF100, HOF101, and HOF102) with different pore size and satisfactory stabilities.Since the pore sizes of HOF101 matched the molecular volume of the antiviral drugs and the HOF101/drugs formed abundant hydrogen bonds, the HOF101 fiber showed the highest extraction efficiency of the antiviral drugs.The HOF101 SPME fiber showed satisfying results including low LODs, wide linear range and qualified recoveries, thus can be adopted for efficient extraction of three antiviral drugs from the serum and urine.Moreover, the extraction performance was proved to be excellent and consistent under various interfering conditions, including different pH values and matrix, which make it possible to directly extract from biofluids.Overall, we have developed a highly sensitive method using the bio-HOF101 fiber to analyze antiviral drugs, providing an accurate and feasible method for TDM in the current healthcare system.

Experimental Section
Chemical Reagents and Materials: The details including chemical reagents, materials and the synthesis of HOFs were presented in the supporting information .
SPME Fiber Coating Fabrication: SPME fibers with different HOFs were fabricated via a facile dip-coating method.The 400 μm-sized stainless steel (SS) wires were cut into 4 cm pieces and cleaned in acetone, ethanol (EtOH) and methanol (MeOH) by ultra-sonication, each for 30 min.The pretreated SS wire was first dipped into a PDMS cyclohexane solution (0.5 g mL −1 ) to get a thin layer of PDMS.Then, the wire was immersed in different HOF powders to obtain fiber with a yellow layer of HOF coating.Residual solvents were removed at 80 °C for 1 h.The procedure was then repeated for two times.At the end of the preparation, the fiber would be dipped into the glue again, so that the fiber surface has an extra protective layer of glue to avoid the material from falling off into the sample.All self-made fibers were shaken in methanol solution for 10 min before use to remove uncemented material.SPME Procedures: All SPME analytical experiments of the antiviral drugs were performed in direct immersion mode (Scheme 1).The physical properties of the antiviral drugs were listed in Table S1 (Supporting Information).The standard solution was prepared by dissolving target analytes in phosphate buffer saline (PBS) solutions.Then the self-made SPME fiber was immersed into the solution for extraction, with mechanical vibration at 500 r min −1 .At certain intervals, the fiber was washed by running deionized water for 10 s, wiped gently, then immersed into a 250 μL glass vial with 100 μL solvents for desorption.The desorption solvents were MeOH, acetonitrile (ACN) or EtOH.After a certain desorption time, the fiber was taken out and the eluent was stored at −20 °C until analysis.Details about the instrument analysis are described in the Supporting Information.Positive ion mode was used for the transitions monitor; more details were listed in Table S2 (Supporting Information).

Scheme 1 .
Scheme 1. Preparation of the HOF101 fiber and the scheme of extraction and detection processes.

Figure 2 .
Figure 2. PXRD patterns of a) HOF100, b) HOF101, and c) HOF102.Nitrogen adsorption and desorption isotherms (The inner illustration showed the pore size distribution based on density functional theory) of d) HOF100 e) HOF101, and f) HOF102.

Figure 3 .
Figure 3. SEM images of a, b) HOF101.c, d) High-resolution TEM images of HOF101.e) The corresponding topological structure of HOF101.f) TEM image of HOF101 and g, h) corresponding elemental mappings for HOF101.

Figure 5 .
Figure 5. a) Molecular dimensions of Arb, Rem, and Rit.C blue, H white, N deep blue, O red, P gray, S yellow, Br pink spheres.b) The energy-favorable conformation when Arb, Rem, and Rit were diffused into the pore of HOF101, respectively.C pink, H white, N blue, O red, P origin, S yellow, Br deep red spheres.

Figure 6 .
Figure 6.Normalized extraction efficiency of the HOF101 fiber to antiviral drugs under a, b) pH interfering (statistics were calibrated by the value obtained in solution with pH = 7.4, n = 4), c) matrix effect (statistics were calibrated by the values obtained in matrix-free solution, n = 4).and d) PBS solvent effect (statistics were calibrated by the values obtained in PBS-free solution, n = 4).

Table 1 .
The method evaluation results of antiviral drugs by HOF101 fiber coupling to HPLC-MS/MS.

Table 3 .
Recoveries of antiviral drugs for human serum samples (n = 3).