The optimum [HO₃S(CH₂)₄mim]HSO₄ concentration in aqueous solution for ILMASEH was determined by carrying out extractions and hydrolyses with [HO₃S(CH₂)₄mim]HSO₄ solutions of different concentrations (from 0.25 to 2.5 M). In the experiments, 1.0 g of sample was soaked in 30 mL of [HO₃S(CH₂)₄mim]HSO₄ and then extracted under a microwave-irradiation power of 120 W and an irradiation time of 15 min. The results in Figure 1a show that the yield increased in the [HO₃S(CH₂)₄mim]HSO₄ concentration range 0.25–1.5 M. Upon further increases, however, a slight decrease in the yield was observed. The 1.5 M [HO₃S(CH₂)₄mim]HSO₄ solution was therefore selected as the optimum ionic-liquid concentration. We speculate that the degradation or isomerization of aglycones often increases at high ionic liquid concentrations, which could lead to a decrease in the calculated yield of glycosides.

Optimization of the microwave power used during ILMASEH is very important for ensuring efficient extraction and hydrolysis, and the effect of this variable was examined. In the experiments, 1.0 g of sample was soaked in 30 mL of 1.5 M [HO₃S(CH₂)₄mim]HSO₄ and then extracted under irradiation for 15 min. The yields obtained at different powers (120, 230, 385, 540 and 700 W) gradually decreased as the microwave power increased from 120 to 700 W. The ILMASEH time was maintained constant at 10 min throughout the experiments. It was found that as the microwave power increased, the yields decreased consistently (Figure 1b). This indicated that high-power microwave irradiation might result in carbonization of the raw materials as a result of internal overheating, isomerization of flavonol aglycones, and energy consumption. This phenomenon was also observed for ionic-liquid microwave-assisted extraction of lignans by Ma et al. [17]. The minimum microwave power tested (120 W) was therefore the optimum power for maximum yield.

In the experiments, 1.0 g of sample was soaked in 30 mL of 1.5 M [HO₃S(CH₂)₄mim] HSO₄ and then extracted under a microwave-irradiation power of 120 W. The influence of the microwave-irradiation time on the yields of flavonol aglycones was examined over the range of 2–60 min, and the results are shown in Figure 1c. The results show that when the irradiation time was increased from 2 to 15 min, the yields of the three flavonol aglycones increased significantly. The yields of the three flavonol aglycones were low during the first 15 min indicating that more time was needed for microwave irradiation to disrupt the cell walls, aid the release of the flavonol glycosides into the solvent, and effectively hydrolyze the flavonol glycosides. On prolonged application of microwave irradiation, for more than 15 min, the yields of the three flavonol aglycones from Ginkgo foliage decreased, which indicated that the higher temperature probably caused degradation or isomerization of the flavonol aglycones; similar results were obtained in the extraction of alkaloids from Camptotheca acuminate [23] and of saponins from Ganoderma atrum [30]. The application of microwaves for 15 min was therefore selected for all subsequent experiments.

The solid–liquid ratio is a crucial factor and was also studied to optimize the yields. Large solvent volumes could make the procedure difficult and lead to unnecessary waste, but small volumes may lead to incomplete extraction and hydrolysis. A series of experiments was carried out with different solid–liquid ratios (1:10, 1:15, 1:20, 1:25, 1:30 and 1:40 w/v) in 1.5 M [HO₃S(CH₂)₄mim]HSO₄ under a microwave-irradiation power of 120 W and an irradiation time of 15 min to evaluate the effect of the solid–liquid ratio. Figure 1d shows that the yields of the three flavonol aglycones increased with increasing solvent volume for solid–liquid ratios up to 1:30. Higher solvent volumes, however, did not significantly improve the yields of the flavonol aglycones. A solid–liquid ratio of 1:30 was therefore chosen as the optimum solid-liquid ratio.

Based on the above experiments, the optimum ILMASEH conditions were found to be as follows: 1.5 M [HO₃S(CH₂)₄mim]HSO₄ as the extraction solvent, a solid-liquid ratio of 1:30 (w/v), a microwave power of 120 W, and an irradiation time of 15 min.

In the present study, pharmacopoeia of the People’s Republic of China sample preparation method (PPRCM), European pharmacopoeia sample preparation method (EPM), United States pharmacopeia and national formulary sample preparation method (USP-NFM), acidic ethanol microwave-assisted sample preparation method (AEMM), 1-butyl-3-methylimidazolium bromide ([Bmim]Br) microwave-assisted sample preparation method (BMM) and acidic [Bmim]Br microwave-assisted sample preparation method (ABMM) techniques were compared with respect to their yields of total flavonol glycosides from Ginkgo foliage. The total flavonol glycosides yields obtained using these six methods are summarized in Table 1. The total flavonol glycosides yields obtained using ILMASEH and ABMM were higher than those obtained using AEMM, PPRCM, EPM, and USP-NFM. In ILMASEH and ABMM, the flavonol glycosides in Ginkgo foliage could absorb sufficient microwave energy to be quickly transferred into the extraction solvent and hydrolyzed; the extraction time was significantly reduced and the yields increased considerably. This showed that compared with traditional methods, ILMASEH used only a small amount of ionic liquid and could obtain higher yields in shorter extraction times (15 min compared with 6.50 h for PPRCM, 2.75 h for EPM, and 2.25 h for USP-NFM), indicating that the [HO₃S(CH₂)₄mim] HSO₄ ionic-liquid was a dual catalyst–solvent and that ILMASEH was a more rapid and efficient method.

The stabilities of the aglycones under the experimentally derived optimum conditions were assessed by subjecting standards of quercetin, kaempferol, and isorhamnetin to microwave irradiation for 15 min at a microwave power of 120 W. The recoveries of the aglycones were assumed to be indicative of the stability of the aglycones under the ILMASEH conditions used (Table 2). The average complete recovery under the ILMASEH operating conditions varied from 98.0% to 99.3% with no changes in retention times observed for the aglycones. Degradation is therefore insignificant under the selected optimum conditions. For the standards in 1.5 M [HO₃S(CH₂)₄mim]HSO₄ solution stored for 7 days, the average recoveries of quercetin, kaempferol and isorhamnetin were 97.0%, 92.7% and 91.0%, respectively.

Under the optimized conditions detailed above, three samples of Ginkgo foliage, which had been spiked with quercetin, kaempferol and isorhamnetin were extracted and the recoveries were analyzed; the results are shown in Table 3. The recoveries of quercetin, kaempferol, and isorhamnetin from Ginkgo foliage were 99.49%, 99.75% and 100.29%, respectively.

To determine the repeatability of the novel method, five samples of the same weight (0.5 g) were processed under the optimum conditions. The mean yields of quercetin, kaempferol and isorhamnetin obtained under the optimized conditions showed good repeatability with calculated RSD values of 3.3%, 4.2% and 3.9%, respectively. This shows that the proposed ILMASEH method has an acceptable level of repeatability.

The results suggested that quercetin, kaempferol and isorhamnetin were stable in the ionic-liquid solutions and in the extracts. These method validation studies indicate that the proposed method is credible.

Ginkgo foliage was purchased from the Tancheng Medicinal Materials Market (Shandong, China). The dried samples were powdered to a homogeneous size and then sieved (60–80 mesh) prior to use. Quercetin, kaempferol, and isorhamnetin standards were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). HPLC-grade methanol (J&K Chemical Ltd. Beijing, China) and ultrapure water prepared using a Milli-Q purification system (Millipore, Bedford, MA, USA) were used as the HPLC mobile phase. Ionic liquids, [HO₃S(CH₂)₄mim]HSO₄ and [Bmim]Br, were obtained from Chengjie Co., Ltd. (Shanghai, China) and used without further purification. Some physicochemical properties of the ionic liquids studied are listed in Table 4. All other chemicals used in this study were of at least analytical grade and were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All solutions and samples prepared for chromatographic analysis were filtered through a 0.45-μm microporous membrane (Guangfu, Tianjin, China) before being injected onto the HPLC column.

The HPLC system consisted of a Waters 717 autosampler, a 1525 binary pump, a 717 automatic column temperature control compartment and a 2487 UV-detector (Waters, Milford, MA, USA). Chromatographic separation was performed on a Kromasil 100-5C₁₈ reversed-phase column (4.6 mm × 250 mm, 5 μm, AkzoNobel, Bohus, Sweden).

Stock solutions of quercetin, kaempferol, and isorhamnetin were prepared in methanol at a concentration of 30 μg/mL. Working standard solutions were prepared by serial dilution of the stock solutions in methanol and then stored at 1–4 °C in darkness until the HPLC analysis.

The extracts were directly injected into the liquid chromatograph. The mobile phase was composed of methanol: 1% aqueous acetic acid solution (52.5:47.5, v/v). The flow rate was 1 mL/min and the injection volume was 10 μL. The column temperature was maintained at 25 °C. The UV detection wavelength was 360 nm, where quercetin, kaempferol, and isorhamnetin have optimum absorbances. Under these conditions, the quercetin, kaempferol and isorhamnetin were separated sufficiently (Figure 2). Quercetin, kaempferol, and isorhamnetin were identified by comparing their retention times with the corresponding peaks in the standard solutions.

The corresponding calibration curves for each compound are Y_quercetin = 258971x − 34818 (r = 0.9991), Y_kaempferol = 254792x + 8635.7 (r = 0.9993), and Y_isorhamnetin = 100039x + 34462 (r = 0.9995). Good linearities were found for quercetin, kaempferol, and isorhamnetin in the range of 2.5–30 μg/mL.

The experimental setup is shown schematically in Figure 3. A household microwave oven (Glanz, Shunde, China) was used for ILMASEH with at a microwave irradiation frequency of 2450 MHz. The maximum output power of the oven was 700 W. In order to examine the effect of the microwave power, five power levels, 100% (700 W), 77% (540 W), 55% (385 W), 33% (230 W), and 17% (120 W) were studied. The whole system was run at atmospheric pressure.

The dimensions of the interior cavity of the oven were 215 × 350 × 330 mm³. The microwave oven was modified by drilling a hole in the top. A round-bottomed flask of a capacity of 50 mL was placed in the oven and connected to a reflux condenser through the hole. After placing the reflux condenser in the oven, the hole around the neck of the flask was covered with polytetrafluoroethylene (PTFE) to prevent microwave leakage.

A dried sample (0.5 g) was mixed with 15 mL of ionic-liquid solution in a 50 mL flask and then the suspension was irradiated with microwaves. The optimum concentration of the selected ionic liquid, microwave power, irradiation time, and solid–liquid ratio was systematically studied in this work. The extracts obtained were rapidly cooled to room temperature using a cold bath and filtered through a 0.45-μm PTFE microporous membrane (Guangfu, Tianjin, China) for subsequent HPLC analysis.

The flavonol glycoside concentrations were determined from the peak areas of quercetin, kaempferol and isorhamnetin using the following equation:

where C is the aglycone concentration determined from the standard curve, F_V is the final volume (mL), F is a conversion factor (2.504 for quercetin, 2.588 for kaempferol, and 2.437 for isorhamnetin) [31], and W is the sample weight (g). So,

Powdered Ginkgo foliage was weighed (1.0 g) and transferred to a Soxhlet extractor. Chloroform (100 mL) was added and the mixture was heated under reflux for 2 h. The chloroform solution was discarded, 100 mL of methanol was added, and the mixture was heated under reflux for 4 h. The liquid extract was decanted and concentrated under a vacuum to dryness. A methanol/25% HCl (4:1) solution (25 mL) was added and the mixture was refluxed for 0.5 h [32]. An aliquot of the sample was filtered (PTFE, 0.45 μm, Guangfu, Tianjin China) prior to HPLC analysis.

Powdered Ginkgo foliage was weighed (2.5 g) and transferred to a 250 mL boiling flask. Acetone solution (60% v/v, 50 mL) was added and the mixture was heated under reflux for 70 min. The liquid extract was decanted and filtered, and 40 mL of 60% v/v acetone solution were added to the remaining solid. The mixture was heated under reflux for an additional 70 min. The sample was cooled and filtered into a 100 mL volumetric flask containing the initial extract. The final extract (50 mL) was transferred to a 250 mL round-bottomed flask. The contents were evaporated under a vacuum until the steady flow of condensed solvent ceased. The contents were transferred to a 50-mL volumetric flask, 4.4 mL of hydrochloric acid were added, and the sample was diluted to volume with methanol. The flask contents were centrifuged for approximately 5 min. Aliquots (10 mL) of supernatant were transferred to 10-mL glass vials and the vials were sealed with aluminum caps. The vials were submerged in a boiling-water bath for 25 min [33]. An aliquot of the sample was filtered (PTFE, 0.45 μm, Guangfu, Tianjin, China) prior to HPLC analysis.

Powdered Ginkgo foliage was weighed (1.0 g) and transferred to a 250 mL boiling flask. Ethanol (50 mL), water (20 mL), and hydrochloric acid (8 mL) were added and the mixture was refluxed for approximately 135 min. The sample was cooled to room temperature and filtered into a 100 mL volumetric flask. The sample was diluted to volume with water and mixed well by inversion [34]. An aliquot of the sample was filtered (PTFE, 0.45 μm, Guangfu, Tianjin, China) prior to HPLC analysis.

Acidic ethanol microwave-assisted sample preparation method (AEMM): Powdered Ginkgo foliage was weighed (1.0 g) and transferred to a 250 mL boiling flask. Ethanol (50 mL), water (20 mL), and hydrochloric acid (8 mL) were added, and the mixture was subjected to microwave irradiation for 15 min at 120 W. The subsequent steps were the same as those in USP-NFM.

[Bmim] Br microwave-assisted sample preparation method (BMM): [Bmim] Br was used as the solvent in the sample preparation. The experiments were operated under the optimum conditions, except for the solvent type. The subsequent steps were the same as those in ILMASEH.

Acidic [Bmim] Br microwave-assisted sample preparation method (ABMM): A 1.5 M [Bmim] Br acidic solution (pH 0.5, adjusted with 6.0 mol/L of hydrochloric acid) was used as the solvent in the sample preparation. The experiments were operated under the optimum conditions, except for the solvent type. The subsequent steps were the same as those in ILMASEH.