Preparation of a metal organic framework (MOF)-based monolithic column for the solid-phase extraction (SPE) of diosmetin from traditional Chinese medicine with determination by high-performance liquid chromatography (HPLC)

Abstract A metal organic framework (MOF)-based monolithic column was prepared by in situ polymerization using modified MIL-101-NH2 and glycidyl methacrylate (GMA) as monomers. The monolithic column was used as a solid phase extraction (SPE) adsorbent for on-line enrichment and preliminary purification of diosmetin in traditional Chinese medicine. After being characterized by scanning electron microscopy (SEM) and nitrogen adsorption-desorption, the advantages of the monolithic column with MIL-101-NH2 added were reflected in the pore structure and specific surface area. The monolithic column for SPE had a linear relationship from 0.13 µg/mL to 5.0 × 102 µg/mL. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.04 µg/mL and 0.13 µg/mL, respectively. The recovery was from 96% to 104%, and the intra-day and inter-day relative standard deviations (RSDs) were below 1.1%. This method was suitable for the determination of diosmetin in complex samples.


Introduction
Diosmetin (3 0 , 5, 7-trihydroxy-4 0 -methoxyflavone) is a flavonoid obtained from plants, [1] which is abundant in citrus fruit and also present in Rosaceae and Cruciferous medicinal species. [2] Its structure is shown in Figure S1. Diosmetin has anti-inflammatory and antibacterial pharmacological properties. [3,4] It inhibits the proliferation of tumor cells and induces tumor cells apoptosis [5] and is also an antioxidant; [6] so diosmetin has their high mechanical and chemical stability. The combination of the polymer alleviates the fragility and difficulty in recycling and handling of the MOFs. The incorporation of MOFs increases the specific surface area of the polymer and adsorption capacity for the target.
Using modified UiO-66-NH 2 and N-methylolacrylamide as monomers, Zhang et al. prepared a MOF-polymer composite monolithic column for online enrichment and purification of aristolochic acid-I in medicinal plants. [20] The results showed that the addition of UiO-66-NH 2 increased the specific surface area, the permeability of the monolith, and the capacity for aristolochic acid-I.
MIL-101(Cr) is an ideal MOF for SPE due to its extremely high specific surface area and thermal and water stability. The MIL-101-NH 2 , which was synthesized and modified in this study, was employed with glycidyl methacrylate as monomers to prepare a monolithic column for the solid-phase extraction of diosmetin. Moreover, a method was established for the online enrichment and purification of diosmetin in traditional Chinese medicine.

Instrumentation
All analyses were performed using a Thermo Unitmate 3000 system (Thermo Scientific, USA), which included a solvent delivery pump, pump mixer, autosampler, and absorbance detector. A C18 analytical column (Athena, 200 mm Â 4.6 mm i.d.) was employed for the separations.

Synthesis of MIL-101-NH 2
MIL-101-NH 2 was prepared and purified according to the literature with slight modifications. [21][22][23] 1.8 g of 2-aminoterephthalic acid, 4.0 g of CrNO 3 Á9H 2 O, and 0.9 g NaOH were sequentially added to 75 mL deionized water and sonicated for 30 minutes to obtain a uniform dispersion. The mixture was transferred to a Teflon-lined autoclave and heated at 150 C for 12 h. After cooling to room temperature, the mixture was centrifuged at 10,000 r/min for 10 minutes. The obtained green precipitate was washed three times with DMF and ethanol and 75 mL of absolute ethanol were added into a Teflon lined autoclave to purify the green precipitate at 100 C for 12 h. The resulting product was separated by centrifugation and dried in a vacuum oven at 100 C for 12 h.
Modification of MIL-101-NH 2 MIL-101-NH 2 was modified according to the following procedure. 2.3 g of HATU, 0.5 mL of methacrylic acid, and 1.4 mL of triethylamine were added to 30.0 mL of DMF. The mixture was sonicated at room temperature for 5 minutes until clear, and 0.5 g MIL-101-NH 2 were added and evenly dispersed in solution. After reaction for 10 hours in a 30 C water bath, the resulting precipitate was washed three times with DMF and absolute ethanol. The modified MOF was obtained after drying in an oven. The preparation and modification of MIL-101-NH 2 are shown in Figure S2.
Preparation of the MOF-polymer monolithic column 2 mg of modified MIL-101-NH 2 , 3 mg of BPO, 0.25 mL of GMA, 0.30 mL of TMPTMA, 0.90 mL of PEG200, and 0.80 mL of N-propanol were sequentially added to a clean centrifuge tube, vortexed for 2 minutes, and sonicated for 1 h, so that the BPO was completely dissolved and the modified MIL-101-NH 2 was uniformly dispersed. Next, 30 mL of DMA were added and the solution was poured into a clean empty column and reacted for 3.5 h at 30 C in a water bath. The monolithic column was removed and rinsed with methanol at a flow rate of 1.0 mL/min for 30 minutes to remove the remaining monomers and pore-forming agents.
A polymer monolithic column without the modified MIL-101-NH 2 was prepared by the same procedure for comparison.
The morphology and structure were identified using scanning electron microscopy (SEM).
The specific surface area and pore size analyzer was used to characterize the specific surface area of the prepared materials.
Standard and sample preparation 5 mg of diosmetin were dissolved in 5 mL methanol with sonication.
The traditional Chinese medicine materials were crushed and passed through 40 mesh sieves. 10 g of Lonicerae japonicae Caulis, Taraxaci Herba, and Lobelia chinensis Herba powder were separately added to 200 mL ethanol-water (80:20, v/v) and refluxed at 70 C for 70 min. The extracts were filtered and concentrated to 10 mL.
10 g of Trichosanthis pericarpium powder were added to 200 mL ethanol-water (70:30, v/v) and refluxed at 70 C for 70 min, and the extract was filtered and concentrated to 10 mL.
The above solutions were passed through a 0.45 mm filter membrane and stored at 4 C.

On-line SPE coupled to HPLC
The MOF-based monolithic column was connected to the HPLC and used as the adsorbent for SPE. Using different proportions of acetonitrile-water as the enrichment mobile phase, the diosmetin standard was injected into the monolithic column for enrichment and a C18 column was employed to elute the analyte using different concentrations of acetonitrile-water. The column temperature was 30 C, and the absorption detection wavelength was 340 nm.

Results and discussion
Characterization of MIL-101-NH 2 The characterization of MIL-101-NH 2 is shown in Figure 1. The SEM image ( Figure 1A) shows that the microstructure of MIL-101-NH 2 was a polyhedron with a relatively regular size. The XRD pattern ( Figure 1B) indicated that the synthesized MIL-101-NH 2 had a stable crystal structure. The above results are consistent with the literature. [21,22] Moreover, the XRD patterns of modified MIL-101-NH 2 and MIL-101-NH 2 were consistent, indicating that the derivatization did not change the crystal structure of MOF.
The nitrogen adsorption-desorption isotherm was measured to characterize the porosity and specific surface area of the MOF ( Figure 1C). The specific surface area of MIL-101-NH 2 was 1580.3378 m 2 /g, which was similar to the literature value. [23] After modification, the specific surface area of the MOF material was 984.0103 m 2 /g.
The infrared spectra of Cr(NO 3 ) 3 Á9H 2 O, 2-aminoterephthalic acid, MIL-101-NH 2 , and modified MIL-101-NH 2 are shown in Figure 1D. The peaks of the synthesized MIL-101-NH 2 between 3400 cm À1 and 3500 cm À1 are the symmetric and asymmetric stretching vibration peaks of the N-H group. The absorption peaks at 1640 cm À1 also showed the presence of the -NH 2 group. The peaks at 1380 cm À1 and 1500 cm À1 are the symmetric and asymmetric stretches of the carboxyl functional groups. The peak at 1260 cm À1 was due to the stretching vibration of the C-N bond. These results show that MIL-101-NH 2 was successfully synthesized. The infrared spectrum of modified MIL-101-NH 2 had one additional absorption peak than the MIL-101-NH 2 at 1680 cm À1 due to the C ¼ C stretching vibration, showing that the MIL-101-NH 2 was successfully modified.

Optimization of the composition of the monolith
When the MIL-101-NH 2 was modified, a carbon-carbon double bond was introduced. Hence, this material may be used as a monomer to react with --cross-linking agent to prepare the monolithic column. As shown in Table 1, several monolithic columns were prepared with different compositions. The influence of the quantities of monomers (GMA and modified MIL-101-NH 2 ), cross-linking agent (TMPTMA), and porogens (PEG 200 and N-propanol) upon the performance of the monolithic columns were investigated. The SEM images in Figure S3 demonstrate that the pore size of the monolithic column decreased when the quantities of GMA or TMPTMA increased (M1, M3-M6), resulting in an increase in pressure and a decrease in permeability. The composition proportion and total porogen content determined the pore structure of the monolithic column. When the total quantities of PEG200 and N-propanol or the percentage of PEG200 were reduced (M1, M7, M8, M10), the pore structure became more dense and the permeability decreased, which increased the back pressure of the monolith. When no MIL-101-NH 2 was added or the quantity of MIL-101-NH 2 increased (M2, M9), the porosity and the permeability of the monolithic column decreased and the back pressure increased. When only modified MIL-101-NH 2 was used as the monomer, the monolith had a poor mechanical strength and was unsuitable as an SPE adsorbent.
Following comprehensive consideration of the permeability and mechanical strength, the monolithic column M1 was selected for subsequent experiments.
The pore structures of the monolithic columns were characterized by nitrogen adsorption-desorption experiments and mercury intrusion and the results are shown in Figure 2. The nitrogen adsorption-desorption isotherms in Figure 2A exhibited type III isotherms, which show that both monolithic columns contain macroporous structures. The specific surface areas of the monolithic column with and without incorporated modified MOF were 21.2293 m 2 /g and 10.4861 m 2 /g, respectively, which shows that the addition of MOF material increased the specific surface area of the monolith.
The macroscopic pore size distributions of the monolithic columns obtained by mercury intrusion are shown in Figure 2B. The total intrusion and porosity of the monolithic column with and without introduced modified MOF were 2.6062 mL/g and 70.8234% and 2.2778 mL/g and 68.4048%, respectively. These results demonstrate that the incorporation of the MOF increased the porosity of the monolithic column.

Influence of the mobile phase composition upon the enrichment and elution
Diosmetin is a flavonoid and soluble in organic solvents such as methanol, ethanol, acetonitrile, ethyl acetate, and ether. Methanol-water and acetonitrile-water systems are often used as the mobile phase for online SPE-HPLC. When methanol was used in the mobile phase, the elution ability was relatively weak and diosmetin was highly retained. Therefore, acetonitrile, which is less viscous and provides lower retention, was selected to be the organic solvent in the mobile phase. Figure 3A,a shows the relationship between the content of acetonitrile in the mobile phase and the recovery of diosmetin during the enrichment. The recovery of diosmetin was highest when the acetonitrile content was 0%. Therefore, 100% water was selected to be the enriched mobile phase.
The influence of acetonitrile content in the elution mobile phase upon the recovery of diosmetin is shown in Figure 3A,b. When the acetonitrile content was 70%, the recovery of diosmetin was highest. Therefore, 70:30 (v/v) acetonitrile-water was employed as the elution mobile phase.
Influence of the flow rate upon the enrichment and elution Figure 3B shows the influence of the flow rate upon the recovery of diosmetin during enrichment and elution. When the flow rate was 1.0 mL/ min, both recoveries of diosmetin were the highest, so 1.0 mL/min was selected to be the optimum value to enrich and elute diosmetin.

Influence of the mobile phase pH upon the elution and enrichment
In order to determine the optimal pH for the enrichment and elution of diosmetin, various ratios of phosphoric acid and triethylamine were added to adjust the pH of the mobile phase to 2.5, 4.0, 5.5, 7.0, and 8.5. Figure  3C shows when the pH of the mobile phase was 8.5, the recoveries of diosmetin for both enrichment and elution were low. Moreover, the chromatographic peak severely tailed with bifurcation. When the pH of the elution mobile phase was 2.5, the recovery of diosmetin was low. After a comprehensive consideration, a pH of 7 was deemed to be optimum for the enrichment and elution mobile phases.  The back pressure is an important index to evaluate the performance of monolithic columns. Figure 4A shows that the back pressures of both monolithic columns increased and decreased with the acetonitrile content of the mobile phase. Moreover, the back pressure of the monolithic column with added MOF was lower than for monolith without MOF. Hence, the addition of the MOF reduced the back pressure of the monolithic column.
The adsorption capacity of the monolithic column for diosmetin was investigated using 100% water and 70:30 (v/v) acetonitrile-water as the enrichment and elution mobile phases. Figure 4B shows that the adsorption of diosmetin increased and reached a plateau. The breakthrough volumes of the monolithic columns with and without incorporated MOF were 19.43 mg/g and 14.93 mg/g, respectively. These results demonstrate that the MOF increased the adsorption capacity of the monolithic column for diosmetin.
The performance of the monolithic column was investigated by the enrichment factor (EF). This parameter is the ratio of the difference in solution concentration before and after SPE. The enrichment factor of the monolithic column for diosmetin was determined to be 45.

Actual application of the online SPE-HPLC method
Four traditional Chinese medicines, namely Taraxaci Herba, Lobelia chinensis Herba, Trichosanthis Pericarpium, and Lonicerae japonicae Caulis, were selected to be samples to investigate the practical application of the MOF-based monolithic column.
The optimization studies for the enrichment of diosmetin indicated that the recovery of diosmetin was high when the acetonitrile content in the enrichment mobile phase was less than 15%. This information was employed to optimize the mobile phase for the enrichment and purification of diosmetin in traditional Chinese medicine. Figure 5 shows that more impurities were removed as the acetonitrile content in the enriched mobile phase increased (A1, B1, C1, and D1). Correspondingly, fewer impurities were accompanied by diosmetin during elution from the monolith (A2, B2, C2, and D2). Moreover, the quantity of eluted diosmetin did not vary significantly. Hence, 15:85 (v/v) acetonitrile-water was selected to be the enriched mobile phase for the samples.
From the optimization of the elution condition, the recovery diosmetin was highest when the acetonitrile content was 70%. However, diosmetin and impurities in the samples were not well separated when 70:30 (v/v) acetonitrile-water was employed as the elution mobile phase. When the acetonitrile content was reduced to 60%, diosmetin and the impurities were well separated with little change in the recovery of diosmetin. Therefore, 60:40 (v/v) acetonitrile-water was selected to be the mobile phase for the samples. Figure 6 demonstrates that the quantity of diosmetin in the samples enriched on the composite monolithic column increased with the injection volume, indicating that this method is suitable for the determination of diosmetin in traditional Chinese medicine. Validation 2 mg of diosmetin were dissolved in 2 mL of methanol with sonicated to obtain a reference solution of 1.0 mg/mL. This stock solution was diluted with methanol to prepare 5.0 Â 10 2 mg/mL, 4.0 Â 10 2 mg/mL, 3.0 Â 10 2 mg/ mL, 2.0 Â 10 2 mg/mL, 1.0 Â 10 2 mg/mL, 5.0 Â 10 1 mg/mL, 2.5 Â 10 1 mg/mL, 1.0 Â 10 1 mg/mL, 2.5 mg/mL, 0.50 mg/mL, and 0.13 mg/mL solutions. Each concentration was injected in triplicate.
A calibration relationship was obtained with peak area as the ordinate and the concentration of the solution as the abscissa equal to y ¼ 1444.8x À 2.3838 with r equal to 0.9993. The results showed that the peak area of diosmetin had a linear relationship with concentration from 0.13 mg/mL to 5.0 Â 10 2 mg/mL. The limit of detection (based upon a signal-to-noise ratio of 3) and the limit of quantification (based upon a signal-to-noise ratio of 10) were 0.04 mg/mL and 0.13 mg/mL, respectively.
The precision of the method was evaluated by the relative standard deviation of a 0.05 mg/mL diosmetin. The standard was injected 6 times in a day and once per day for 6 consecutive days, respectively. The concentration was calculated using the peak area. The intra-day and inter-day relative standard deviations for diosmetin were 0.42% and 0.67%, respectively.
The accuracy of the method was evaluated by the recovery for standard addition as shown in Table 2. The average recoveries of diosmetin in Taraxaci Herba, Trichosanthis Pericarpium, Lobelia chinensis Herba, and Lonicerae japonicae Caulis were 96.7%, 103.7%, 100.5%, and 96.3%, respectively. The diosmetin content in these traditional Chinese medicines were 59 mg/kg, 9 mg/kg, 18 mg/kg, and 20 mg/kg, respectively.
In order to characterize the repeatability of the developed method, six columns were prepared under the same conditions on the same day. The intra-day relative standard deviation of the concentration of diosmetin was 0.81%. One monolithic column was prepared using the same conditions every day for 6 consecutive days. The inter-day relative standard deviation of the concentration of diosmetin was 1.01%. Moreover, the retention time and peak area of diosmetin were nearly unchanged after the monolith was used as the SPE absorbent 100 times, showing that the reported composite monolithic column had favorable stability.
The results were compared with those obtained using the reported SPE-HPLC method as shown in Figure 7. The MOF-based monolithic column was suitable for the purification of diosmetin in traditional Chinese medicine.
For the separation and analysis of diosmetin, a comparison of the developed protocol and comparable methods are shown in Table 3. The reported SPE-HPLC method offers a long linear range with relatively high sensitivity and a suitable correlation coefficient.

Conclusion
In this study, a new MOF-based monolithic column was prepared and employed as the adsorbent for on-line solid-phase extraction of diosmetin together with HPLC. The specific surface area and the porosity of the monolithic column increased and the back pressure was reduced in the   [25] 0.065-26.0 0.9997 0.0117 0.0353 -HPLC-UV-DAD [9] 0.019-1.24 0.9990 0.0015 0.0195 95-108 HPLC-UV [26] 0.223-35.7 0.9998 0.046 0.115 95-105 LC-DAD-MS [27] 0.085-0.303 0.9997 0.019 -97-102 RP-LC [28] 11-220 0.9996 0.156 0.48 95-104 CE-DAD [29] 2.5-100 0.9980 0.25 0.84 88-97 presence of the MOF. Most importantly, the adsorption capacity of the monolith for diosmetin increased. The composition of the monolithic column and chromatographic conditions were optimized to achieve the required separation, sensitivity, and specificity for on-line SPE of diosmetin. Under the optimum conditions, diosmetin was enriched on the monolithic column with high recovery values while removing most impurities from the traditional Chinese medicine, allowing for the protection of the analytical column. The established SPE-HPLC method was simple with a long linear range. In addition, the precision, accuracy, and repeatability were favorable for practical analysis.
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