# Green chemistry strategies for Flavonol extraction from onion: Exploring natural deep eutectic solvents

Ana González-De-Peredo, Alicia Maroto, Antony Memboeuf

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Ana González-De-Peredo, Alicia Maroto, Antony Memboeuf. Green chemistry strategies for Flavonol extraction from onion: Exploring natural deep eutectic solvents. Microchemical Journal, 2025, 215, pp.114445. 10.1016/j.microc.2025.114445. hal-05168398

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# Green chemistry strategies for Flavonol extraction from onion: Exploring natural deep eutectic solvents

Ana V. González-de-Peredo, Alicia Maroto, Antony Memboeuf*

Univ Brest, CNRS, UMR 6521 CEMCA, Brest, France

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

### Keywords:

- Box-Behnken design

- Flavonols

- Green chemistry

- Mass spectrometry

- Natural deep eutectic solvents

- Onions

## ABSTRACT

The increasing interest in bioactive compounds from fruits and vegetables has driven the need for efficient and environmentally friendly extraction methods. Traditional solvents such as methanol or acetonitrile, although effective, pose significant environmental concerns. This study explores the use of choline chloride-based natural deep eutectic solvents (NaDES) for the extraction of flavonols from onions, compounds known for their health benefits. Various NaDES combinations (levulinic acid, tartaric acid, 1,3-propanediol, and urea) and molar ratios (1:1, 1:2, 1:3, 1:4, 2:1, 2:3) were tested. The choline chloride:levulinic acid mixture at a 1:2 ratio showed the highest extraction efficiency for five major onion flavonols: quercetin 4'-glucoside, quercetin 3-glucoside, iso-rhamnetin 3,4'-diglucoside, isorhamnetin 4'-glucoside, and quercetin 3,4'-diglucoside, as determined by liquid chromatography-mass spectrometry. To optimize extraction conditions, a Box-Behnken design was applied to the choline chloride:levulinic acid NaDES system, with the variables studied being water content (25-65 % v/v), extraction temperature (20-40 °C), and extraction time (2-20 min). The optimized NaDES-based method was compared with traditional extraction techniques using methanol and green solvents such as ethanol, ethanol/water, and water. The results indicated that the NaDES system yielded comparable or higher amounts of flavonols (4.83 ± 0.05 mg g⁻¹), demonstrated good intermediate precision (RSD < 10 %), and significantly reduced the extraction time. This approach improves flavonol extraction and offers a more sustainable and environmentally friendly alternative, positioning NaDES as a promising and green method for future extraction processes.

## 1. Introduction

In the field of food chemistry, there has been a growing concern regarding using green extraction techniques to isolate bioactive compounds from food matrices without causing environmental harm. In this context, non-conventional extraction methods have gained significant attention due to their efficiency and sustainability [1,2]. Numerous studies have highlighted the advantages of techniques such as ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and pressurized fluid extraction (PFE) for extracting bioactive

compounds from various matrices, including strawberries [3], corn [4], or citrus fruits [5]. These methods are generally associated with lower solvent consumption and improved extraction yields [6]. In addition to the type of technique used, an emerging alternative that is gaining importance is the use of environmentally friendly solvents.

Deep Eutectic Solvents (DES), often used as a good alternative to conventional solvents, are defined as mixtures of two or more components, typically a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD) [7]. These two components interact through hydrogen bonds to form a macromolecular mixture with a significantly lower

<footnote>* Corresponding author.</footnote>

<footnote>E-mail addresses: ana.velascogonzalezdeperedo@univ-brest.fr (A.V. González-de-Peredo), alicia.maroto@univ-brest.fr (A. Maroto), antony.memboeuf@univ-brest.fr (A. Memboeuf).</footnote>
melting point than the individual components [8,9]. When these eutectic mixtures are derived from naturally abundant molecules, they are called Natural Deep Eutectic Solvents (NaDES). Common natural components used in NaDES formulations include choline, sugars, organic acids, amino acids, and natural alcohols [10]. From a green chemistry perspective, NaDES exhibits ideal properties, including low volatility, biodegradability, and stability at room temperature [11].

Flavonoids are natural compounds with variable phenolic structures, found in fruits, vegetables, roots, stems, or flowers [12]. These compounds have demonstrated numerous health benefits, including the prevention of cardiovascular and degenerative diseases [13,14]. Onions are one of the most widely consumed vegetables globally, not only for their culinary applications but also as a primary dietary source of quercetin [15]. Specifically, onions are rich in flavonols such as quercetin 3,4'-diglucoside and quercetin 4'-glucoside [16,17]. In the literature, most studies on flavonol extraction from onions have relied on solvents like methanol [17–19], which is both environmentally toxic and hazardous to researchers. Therefore, this study proposes NaDES as a sustainable alternative for flavonol extraction from the onion matrix.

To our knowledge, NaDES has been applied twice (in 2019) to extract phenolic compounds from onions [20,21]. However, neither of these studies included individual flavonoid analysis using advanced analytical techniques such as liquid chromatography (HPLC) or mass spectrometry (MS), relying instead on spectrophotometric assays such as the Folin-Ciocalteu assay, ABTS, DPPH, and FRAP. This is particularly relevant since recent publications have highlighted limitations in spectrophotometric methods for phenolic quantification and antioxidant activity assessment [22]. The unique physicochemical properties of NaDES, including their viscosity and potential interactions with assay reagents, may interfere with spectrophotometric readings, leading to discrepancies between the results of these assays and those obtained from more accurate chromatographic techniques such as HPLC-MS [22].

To overcome these limitations, this research identifies and quantifies flavonols in onion samples using ultrasound-assisted extraction (UAE) with NaDES, combined with liquid chromatography and mass spectrometry. Although the UAE is known for its efficiency, several factors can influence the extraction process, including temperature, extraction time, and solvent composition [23–25]. In the case of NaDES, the water content plays a critical role in reducing solvent viscosity, one of the main limitations of NaDES, thereby enhancing mass transfer and improving extraction efficiency [2,26]. To optimize these parameters and maximize extraction yields, this study employs the Design of Experiments (DOE). DOE is a systematic methodology used to evaluate the effects of multiple independent variables and their interactions on one or more dependent variables, reducing the number of experimental trials required [27].

This study aims to provide a comprehensive understanding of the potential of NaDES combined with UAE for the extraction of flavonols from onions. To achieve this, different combinations of HBD were tested in combination with choline chloride as HBA. The results were compared with methanol, a conventional solvent, as well as ethanol and water, which are also considered green chemistry solvents. A Box-Behnken Design (BBD) was employed to optimize the green extraction method and evaluate the effects of key factors, including extraction time, temperature, water percentage, and the solid-to-liquid ratio, on flavonol yield. Furthermore, unlike commonly used global profiling or spectrophotometric techniques, accurately identified flavonols via UPLC-MS were used as response variables for evaluation.

## 2. Materials and methods

### 2.1. Chemicals

The reagents for the preparation of NaDES (L-(+)-Tartaric acid, 1,3-Propanediol, Choline chloride, Levulinic acid, and Urea ACS reagent) were sourced from Merck (Horsham, France). Mixtures of acetonitrile,

methanol (HPLC grade, Honeywell, Guyancourt, France), and Milli-Q water, obtained from a Milli-Q water purification system (Merck Millipore, Darmstadt, Germany), were employed as extraction solvents and LC mobile phases. For mass spectrometry calibration, a sodium iodide (NaI) solution (ACS reagent grade with a purity >99.5 %) with a concentration of 10 ng µL⁻¹ was prepared in a 90:10 isopropanol: water mixture. The NaI stock solution was purchased from Sigma-Aldrich (Steinheim, Germany), and the isopropanol from Honeywell (Guyancourt, France). Quercetin 3-glucoside, purchased from Sigma-Aldrich (Steinheim, Germany), served as the commercial standard for quantifying the extracted compounds.

### 2.2. Sample preparation

The plant matrix used in this study was the Rose de Bretagne onion, of French origin, obtained from a local supermarket in Brest, France, and grown by Le Vili (Mespaul, France). Prior to flavonol extraction, the onion samples were pretreated by removing the outer layers and freeze-drying the bulbs using a BenchTop Pro freeze dryer (Virtsp SciENTIFIC, Gardiner, USA). After freeze-drying, the bulbs were ground into a fine powder for subsequent analyses.

### 2.3. Preparation of NaDES and evaluation of ChCl-based NaDES

To prepare NaDES, an HBA and an HBD are typically combined to form strong hydrogen bonds, resulting in a low-temperature transition mixture [28]. Choline chloride (ChCl) quaternary salt is the most used HBA. In contrast, amines, sugars, polyalcohols, and carboxylic acids can be used as HBD [29], obtaining in each case NaDES with different physicochemical characteristics [10]. In this work, to prepare the NaDES, which were later used as extraction solvents, ChCl was combined with the following HBDs in different molar ratios ranging from 2:1 to 1:4 (ChCl: HBD): 1,3-propanediol, urea, levulinic acid, and tartaric acid.

In general, the parameters to be considered in the synthesis of NaDES are mixing time, mixing speed, and mixing temperature [7]. Specifically, in this work, the HBA: HBD mixture was agitated in a magnetic stirrer at 80 °C and 500 rpm for 120 min. The stability of the NaDES was also studied over 48 h. The results are shown in Table 1.

1,3-Propanediol and levulinic acid formed liquids suitable for use as extraction solvents in various ratio combinations, while tartaric acid and urea formed solids in most ratios, with the resulting liquids being highly viscous, as shown in Table 1. It has already been reported that the chemical structures of HBDs play a crucial role in the formation and stability of NaDES, as the melting point is influenced by the hydrogen bonds formed between the HBD and HBA [26,30]. Furthermore, some authors have noted that a clear and less viscous NaDES, without precipitation, was synthesized when HBDs were in liquid form [31,32]. In our study, of the four HBDs tested, 1,3-propanediol and levulinic acid were commercially available in liquid form, whereas urea and tartaric acid were in solid form.

To reduce viscosity when using urea and tartaric acid as HBD, a percentage of water was added to the mixture, as there is evidence that water also participates in the molecular structure when forming NaDES. Different percentages of water (ranging from 5 to 50 % w/w) were added only to the combinations that initially formed a liquid or semi-liquid solvent (NaDES-1.3, NaDES-1.4, NaDES-2.2, NaDES-2.3, NaDES-2.4, NaDES-3.2, NaDES-4.1, and NaDES-4.5). After testing, the lowest water content, which resulted in a liquid NaDES easily manageable with picpettes for all combinations, was selected. The selected percentage was 25 % w/w water. This led to a reduction in viscosity for the NaDES combinations containing urea and tartaric acid (in agreement with earlier reports [33]). The NaDES selected for extraction, all of them containing 25 % water, are highlighted in the table with a yellow colour.
<caption>**Table 1**</caption>

Testing and evaluation of different molar ratios of ChCl: HBD before adding water. NaDES that formed a stable liquid phase upon the addition of 25 % water (v/v), making them suitable for use as extraction solvents, are highlighted in yellow.

<table><thead><tr><th>Name</th><th>HBDs</th><th>Molar Ratio (ChCl: HBD)</th><th>Stability at 48 h*</th></tr></thead><tbody><tr><td>NaDES-1.1</td><td>1,3-propanediol</td><td>1:1</td><td>Solid</td></tr><tr><td>NaDES-1.2</td><td>1,3-propanediol</td><td>1:2</td><td>Solid</td></tr><tr><td>NaDES-1.3</td><td>1,3-propanediol</td><td>1:3</td><td>Liquid</td></tr><tr><td>NaDES-1.4</td><td>1,3-propanediol</td><td>1:4</td><td>Liquid</td></tr><tr><td>NaDES-1.5</td><td>1,3-propanediol</td><td>2:1</td><td>Solid</td></tr><tr><td>NaDES-1.6</td><td>1,3-propanediol</td><td>2:3</td><td>Solid</td></tr><tr><td>NaDES-2.1</td><td>Levulinic acid</td><td>1:1</td><td>Solid</td></tr><tr><td>NaDES-2.2</td><td>Levulinic acid</td><td>1:2</td><td>Liquid</td></tr><tr><td>NaDES-2.3</td><td>Levulinic acid</td><td>1:3</td><td>Liquid</td></tr><tr><td>NaDES-2.4</td><td>Levulinic acid</td><td>1:4</td><td>Liquid</td></tr><tr><td>NaDES-2.5</td><td>Levulinic acid</td><td>2:1</td><td>Solid</td></tr><tr><td>NaDES-2.6</td><td>Levulinic acid</td><td>2:3</td><td>Solid</td></tr><tr><td>NaDES-3.1</td><td>Urea</td><td>1:1</td><td>Solid</td></tr><tr><td>NaDES-3.2</td><td>Urea</td><td>1:2</td><td>Liquid viscous</td></tr><tr><td>NaDES-3.3</td><td>Urea</td><td>1:3</td><td>Solid</td></tr><tr><td>NaDES-3.4</td><td>Urea</td><td>1:4</td><td>Solid</td></tr><tr><td>NaDES-3.5</td><td>Urea</td><td>2:1</td><td>Solid</td></tr><tr><td>NaDES-3.6</td><td>Urea</td><td>2:3</td><td>Solid</td></tr><tr><td>NaDES-4.1</td><td>Tartaric acid</td><td>1:1</td><td>Liquid very viscous</td></tr><tr><td>NaDES-4.2</td><td>Tartaric acid</td><td>1:2</td><td>Solid</td></tr><tr><td>NaDES-4.3</td><td>Tartaric acid</td><td>1:3</td><td>Solid</td></tr><tr><td>NaDES-4.4</td><td>Tartaric acid</td><td>1:4</td><td>Solid</td></tr><tr><td>NaDES-4.5</td><td>Tartaric acid</td><td>2:1</td><td>Liquid very viscous</td></tr><tr><td>NaDES-4.6</td><td>Tartaric acid</td><td>2:3</td><td>Solid</td></tr></tbody></table>

## 2.4. Ultrasound-assisted extraction technique

Ultrasound-Assisted Extraction (UAE) was used for the flavonols extraction. Technique employed. More precisely, an Elmasonic S15H ultrasonic bath (Elma Schmidbauer GmbH, Singen, Germany), working at a maximum output power of 280 W and a frequency of 37 kHz, was used. The extraction process was the following: a quantity of freeze-dried onion sample, ranging approximately from 0.05 g to 0.2 g, was weighed and mixed with 5 mL of the extraction solvent in a 10 mL Falcon tube to achieve the desired sample mass-to-solvent volume ratio, which varied depending on the experimental conditions. The tube was then placed in the ultrasonic bath, which was set to a specific temperature and extraction time. To ensure temperature control throughout the process, a Lauda chiller (France) was used.

Once the compounds of interest were extracted, the supernatant was separated from the solid onion residue by centrifuging the sample at 1164 x g for 15 min. 2.5 mL of this supernatant was transferred to a 5 mL volumetric flask, which was then filled to the mark with water. The final extracts obtained were stored in the freezer until further analysis by LC-MS/MS.

## 2.5. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Once the flavonols were extracted, they were analyzed using high-performance liquid chromatography in combination with mass spectrometry (LC-MS). While LC-MS in scan mode was used for general profiling and quantification of flavonols, MS/MS experiments were selectively conducted to confirm the identity of key flavonols.

An ACQUITY UPLC® H-Class liquid chromatography system (Waters Corp., Manchester, UK) coupled with a Quattro Ultima Platinum triple quadrupole Mass Spectrometer (Waters Corp. /Micromass Inc., Manchester, UK) was used as an LC-MS system. Chromatographic separations were performed using a C18 BEH column (2.1 mm × 100 mm, 1.7 µm) (Waters Corp., Manchester, UK), along with a C18 guard column

cartridge (3.5 µm, Waters Corp., Manchester, UK) at a column temperature of 50 °C. A binary solvent system was used, consisting of water (phase A) and acetonitrile (phase B), with a flow rate set to 0.35 mL min⁻¹. The gradient employed to achieve optimal resolution of target compounds in a shorter analysis time was the following: 0 min, 10 % B; 2 min, 20 % B; 3 min, 30 % B; 3.5 min, 30 % B; 4 min, 31 % B; 5 min, 31 % B; 8 min, 32.5 % B; 9 min, 50 % B; 10 min, 10 % B. The injection volume was 10 µL. All extract samples were filtered using a 0.22 µm syringe filter and diluted with ultrapure water (Milli-Q, Merck Millipore, Darmstadt, Germany) up to 10 times.

Mass spectrometric analysis was conducted in negative ion mode using an electrospray ionization triple quadrupole mass spectrometer (ESI-QqQ, Quattro Ultima Pt, Waters Co./Micromass Inc., Manchester, UK). Positive mode was also tested, resulting in poorer ionization, due to the greater tendency of the identified flavonols to lose a proton. The source temperature was maintained at 150 °C, and nitrogen was used as the desolvation gas, flowing at 860 L h⁻¹ with a temperature of 450 °C. The electrospray voltages were applied at 2 kV for the capillary and 46 V for the sample cone. Argon was used as collision gas at a pressure of 1.21 × 10⁻³ mbar. The collision energy was chosen depending on the flavonol being analyzed, aiming to generate useful fragment ions while retaining around 20–30 % of the precursor ions intensity to prevent excessive fragmentation. Data acquisition and processing were carried out using MassLynx 4.2 (Waters Corp., Milford, MA, USA). Detailed mass spectrometry calibration parameters, including LM and HM resolutions, are provided in the Supplementary Material Table S1.

Quercetin-3-glucoside, the only available standard, was identified by direct comparison of its retention time, exact mass, and MS/MS spectrum with those of the pure compound. The remaining flavonols, for which no standards were available, were identified using the same analytical criteria, supported by literature data and databases such as Phenol-Explorer [34]. In onions, these compounds predominantly originate from aglycones such as quercetin, kaempferol, isorhamnetin, and myricetin, which are commonly glycosylated with sugars like glucose, rhamnose, or galactose. A precursor ion scanning strategy using LC-MS/MS enabled the detection of ions that produce characteristic aglycone fragments (e.g., *m/z* 303 for quercetin), facilitating the identification of flavonols and their glycosylation profiles in the samples.

Flavonol peak areas were quantified by LC-MS using quercetin 3-glucoside as the reference compound, with the concentrations expressed as quercetin 3-glucoside equivalents (mg eq. Q3Glu g⁻¹ dry sample). A calibration curve was established in the concentration range of 0.1–5 mg g⁻¹, also acquired by LC-MS, and fitted to a second-order polynomial eq. (Y = -934,200 ×² + 9,000,000 × - 338,420), yielding a coefficient of determination (R²) of 0.9948.

## 2.6. Statistical analysis

Analysis of variance (ANOVA) followed by multiple comparisons test was performed using R software [35] and RStudio [36]. When significant differences were detected by ANOVA, pairwise t-tests with pooled standard deviation (SD) were performed. To account for multiple comparisons and reduce the risk of Type I errors, the Bonferroni correction was applied. Subsequently, a Principal Component Analysis (PCA) was conducted using the "mdatools" package [37]. PCA is an unsupervised statistical technique widely employed to reduce data dimensionality while retaining most of the variance. In this study, PCA was used to evaluate the similarity among the different NaDES and conventional solvents, such as methanol, ethanol, and water, in terms of their efficiency for flavonol extraction. Before the analysis, the data were pretreated using autoscaling to ensure that all variables contributed equally to the model regardless of their original scales.

Finally, regarding the Box-Behnken Design (BBD), Statgraphics Centurion version XVI software (Warrenton, VA, USA) was used to carry out the experimental design, data analysis, and model construction. The BBD analysis was conducted considering the values of area/g, without

<footnote>* The classifications of liquid, liquid viscous, and liquid very viscous are qualitative descriptions based on visual and tactile assessments of the solvent mixtures during preparation.</footnote>
normalizing to quercetin-3-glucoside, as the semi-quantification is performed relatively. This approach ensures greater accuracy in the optimization, with all values referenced to the grams of sample.

# 3. Results and discussion

## 3.1. Identification of flavonols

An initial identification of the flavonols present in the onion sample was performed to carry out the subsequent quantification and comparison of the obtained results. As mentioned in section 2.4, flavonols were analyzed using LC-MS/MS. First, a precursor ion scanning strategy was applied to detect the characteristic ions of the main flavonol aglycones, such as *m/z* 303 for quercetin, which allowed us to focus on the relevant compounds present in the sample. After identifying the precursor ions, these compounds were studied by CID MS/MS. The analysis was initially conducted on extracts obtained using methanol as the solvent, and then extended to extracts obtained with NaDES, considering the different behaviors these solvents may exhibit in ionization and the quadrupole analyzer. Specifically, five types of flavonols were identified in the onion sample extracted with methanol (Table 2), which have been previously reported in similar onion samples [37–40]. The results of the precursor ion scanning study and the MS/MS spectra can also be found in the Supplementary Material (Fig. S1 and Fig. S2, respectively).

Subsequently, these compounds were analyzed in the extracts obtained using NaDES. To compare NaDES and methanol extraction profiles, each solvent was first injected individually into the LC-MS. The total ion spectra of the different extraction solvents (MeOH and NaDES) are shown in Supplementary Material, Fig. S3a. From these spectra, it is evident that, since NaDES are mixtures of two components, such as sugars, salts, or organic acids, they generate characteristic signals with varying intensities, as observed in Fig. S3a. In contrast, methanol produces a much lighter signal. Therefore, when analyzing flavonols of interest, it is crucial to consider potential interference from these solvent components. For instance, when ChCl: tartaric acid was used as the NaDES, a highly intense peak at *m/z* 148.93 was observed, corresponding to the tartaric acid ion ([C₄H₆O₆ – H]⁻). This peak arises from NaDES formation and the deprotonation of tartaric acid under negative electrospray ionization conditions. Similarly, when a NaDES based on levulinic acid was used, two distinct peaks at *m/z* 169.05 and *m/z* 213.04 were observed, which were not present in other solvents. The ion at *m/z* 169.05 likely corresponds to the [M – H]⁻ ion of a levulinic acid-related species, while *m/z* 213.04 appears to be its formate adduct ([M + HCOO]⁻). Although the exact identities of these ions remain unclear, it is important to acknowledge their presence to avoid misinterpreting them as bioactive compounds from the onion matrix during mass spectrometry analysis. These signals may arise from clusters or complexes formed within the NaDES matrix under negative electrospray ionization conditions. From an experimental perspective, the high viscosity of NaDES posed challenges during LC-ESI-MS analysis, as it hindered the formation of a stable electrospray under conditions where methanol performed effectively. To overcome this, a higher chromatographic column temperature (50 °C) and an increased nitrogen desolvation gas temperature and flow rate (450 °C and 860 L h¹,

respectively) were necessary to achieve proper desolvation. Despite these challenges, all five previously identified flavonols were detected in every extract, regardless of the solvent used, as shown in the total ion spectra of the flavonol extracts obtained with different solvents (Supplementary Material, Fig. S3b). These compounds were confirmed by the MS spectra (Supplementary Material, Fig. S3 b) and their similar retention times (Supplementary Material, Fig. S4).

## 3.2. Extraction of flavonols

### 3.2.1. Preliminary MeOH solvent method

As indicated in section 2.2, UAE is the technique to extract flavonols from onion samples. UAE is a widely used extraction method for obtaining bioactive compounds from natural matrices and is commonly applied in combination with organic solvents such as methanol or acetonitrile [41–43]. In this study, to make a proper comparison between traditional solvents, like methanol, and the green NaDES solvents proposed as alternatives, an optimization of the UAE process using methanol as the extraction solvent was first carried out. Specifically, a Box-Behnken Design (BBD) response surface was used. A BBD response surface is a statistical experimental design used to model and optimize processes by evaluating the effects of multiple variables with a minimal number of experimental runs [44]. The factors studied and the range of each one were the following: 0, 25, and 50 % of water in methanol (X₁); 10, 25, and 40 g mL⁻¹ of sample mass-to-solvent volume ratio (X₂); 5, 15, and 25 min of extraction time (X₃). The response variables were each of the 5 flavonols identified in onions. The design was optimized for each variable (individual flavonol) to account for potential differences in behavior due to their distinct chemical structures. Subsequently, the desirability function approach was applied to achieve a global optimization. This method transforms multiple response variables into a single composite desirability score, ranging from 0 (completely undesirable) to 1 (fully desirable). By maximizing this overall desirability, a compromise solution is reached that balances the optimal conditions for all flavonols simultaneously [45]. An experimental matrix consisting of 16 treatments was conducted, including 4 repetitions at the center point for error calculation (Supplementary Material, Table S2). All experiments were conducted randomly.

Once the BBD matrix was constructed, ANOVA was performed for each flavonol. The results showed no lack of fit in any case (*p*-value >0.05), suggesting no significant evidence of lack of fit in any of the models constructed. Model validation was also carried out using the coefficients of determination, which were close to one for all flavonols (R²: 85.73–99.08). Fig. S5 in the Supplementary Material supports these findings by showing the experimental and predicted values based on the constructed polynomial model. The second-order polynomial equation for each response variable is also represented in the figure, further demonstrating the model’s accuracy.

In addition, Fig. 1 presents the significance of each factor and its interactions with the response variable using Pareto plots. Factors or interactions with *p*-values below 0.05 were considered significant at the 95 % confidence level and are indicated in the Pareto plots by horizontal bars extending beyond the vertical threshold line. Regarding Fig. 4, the ratio is the most influential variable in extracting all flavonols using

<table><caption>**Table 2**
LC ESI-QqToF based identification of flavonols on onion sample.</caption><thead><tr><th>Peak</th><th>Retention Time (min)</th><th>Compounds</th><th>Abbreviation</th><th>Ion Formulae</th><th>Measured m/z</th><th>Theoretical m/z</th><th>Product Ions</th></tr></thead><tbody><tr><td>1</td><td>1.95</td><td>Quercetin 3,4'-diglucoside</td><td>Q34diGlu</td><td>C<sub>27</sub>H<sub>29</sub>O<sub>17</sub></td><td>625.13</td><td>625.15</td><td>463.08 (C<sub>21</sub>H<sub>19</sub>O<sub>12</sub>)<br>301.15 (C<sub>15</sub>H<sub>9</sub>O<sub>7</sub>)</td></tr><tr><td>2</td><td>3.32</td><td>Isorhamnetin 3,4'-diglucoside</td><td>Iso34diGlu</td><td>C<sub>28</sub>H<sub>31</sub>O<sub>17</sub></td><td>639.08</td><td>639.16</td><td>477.71 (C<sub>22</sub>H<sub>21</sub>O<sub>12</sub>)<br>313.05 (C<sub>16</sub>H<sub>9</sub>O<sub>7</sub>)</td></tr><tr><td>3</td><td>4.2</td><td>Quercetin 3-glucoside</td><td>Q3Glu</td><td>C<sub>21</sub>H<sub>19</sub>O<sub>12</sub></td><td>463.05</td><td>463.09</td><td>301.08 (C<sub>15</sub>H<sub>9</sub>O<sub>7</sub>)</td></tr><tr><td>4</td><td>4.8</td><td>Quercetin 4'-glucoside</td><td>Q4Glu</td><td>C<sub>21</sub>H<sub>19</sub>O<sub>12</sub></td><td>463.00</td><td>463.09</td><td>301.06 (C<sub>15</sub>H<sub>9</sub>O<sub>7</sub>)</td></tr><tr><td>5</td><td>5.07</td><td>Isorhamnetin 4'-glucoside</td><td>Iso34diGlu</td><td>C<sub>22</sub>H<sub>21</sub>O<sub>12</sub></td><td>477.04</td><td>477.10</td><td>314.05 (C<sub>16</sub>H<sub>10</sub>O<sub>7</sub>)<br>299.00 (C<sub>15</sub>H<sub>7</sub>O<sub>7</sub>)</td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Pareto plots for each of the flavonols studied (response variables) in the MeOH BBD design: (a) Quercetin 3,4'-diglucoside; (b) Isorhamnetin 3,4'-diglucoside; (c) Quercetin 3-glucoside; (d) Quercetin 3'-glucoside; (e) Isorhamnetin 4'-glucoside. Blue bars indicate positive effects of the factor on the response variable, and grey bars indicate negative effects. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

methanol. Specifically, this factor exhibits a negative effect, meaning that lower ratios, i.e., a smaller amount of onion mass relative to the extraction solvent volume, enhance the extraction of the target flavonols from onions. In addition to the ratio, extraction time also appears to have a significant influence, showing a positive effect. In this case, longer extraction time leads to improved flavonol extraction.

Finally, following these trends, the experimental design enables the identification of optimal conditions for each factor to maximize the response variable. However, rather than focusing on the optimal values for each flavonol, as previously indicated, the desirability function was applied to determine intermediate conditions that enhance the extraction of all flavonols. Greater weights (3 instead of 1) were given in the software Statgraphics to quercetin-3-glucoside and isorhamnetin-3,4'-diglucoside, as they were the minor peaks in the analysis. Specifically, the optimal extraction conditions were determined to be 25 min of extraction time, a 10 mg mL⁻¹ sample-to-volume ratio, and 84 % water in methanol. These conditions yielded the highest flavonol extraction efficiency from the onion samples, as confirmed by the analysis of the experimental data. Specifically, after performing the extraction under optimal conditions, the following flavonol concentrations were obtained (mg g⁻¹ eq. Q3Glu ± standard deviation): 0.78 ± 0.07 for quercetin-3,4'-diglucoside, 0.10 ± 0.00 for isorhamnetin-3,4'-diglucoside, 0.08 ±

0.00 for quercetin-3-glucoside, 1.08 ± 0.01 for quercetin-4'-glucoside, and 0.24 ± 0.00 for isorhamnetin-4'-glucoside.

### 3.2.2. Application of the optimized MeOH-based method to various NaDES

After optimizing the methanol extraction method in the previous section, the optimal conditions were applied in combination with various types of NaDES to evaluate their behavior and performance. The extraction conditions, previously determined through the BBD, included 25 min of extraction, a 10 mg mL⁻¹ sample-to-volume ratio, and 84 % of water in methanol. This last characteristic was only used for methanol; for NaDES, the selected solvents already discussed and listed in Table 1 were employed with 25 % water.

Figs. 2a–f present the extracted amounts (mg g⁻¹) of each flavonol using different NaDES and methanol aqueous solutions, visualized through bar charts, where each bar represents the mean of three replicates and the error bars correspond to the standard deviation. Since all other parameters remained constant, this section focuses on the effect of the extraction solvent. In addition to comparing NaDES with methanol, the extraction efficiency of NaDES was also evaluated against ethanol, water, and a 50:50 EtOH/water mixture. Ethanol and water, as well as their combination, are considered “green” solvents due to their low toxicity to both the environment and individuals, making them a safer
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Comparison of flavonol content (mg g⁻¹) extracted using different NaDES, 84:16 MeOH/water, EtOH, and 50:50 EtOH/water as extraction solvents. Effect in the extraction of: (a) quercetin 3,4'-diglucoside; (b) isorhamnetin 3,4'-diglucoside; (c) quercetin 3-glucoside; (d) quercetin 4'-glucoside; (e) isorhamnetin 4'-glucoside; and (f) total flavonols. Different letters indicate significant differences according to pairwise t-tests.</figcaption></figure>

and more sustainable alternative to conventional solvents [46]. This comparison is particularly relevant when assessing the potential of NaDES.

In addition to Fig. 2, the principal component analysis (PCA) biplot illustrates the distribution of different solvent types used in flavonoid extraction and their relationship with the extracted compounds (Fig. 3). The first two principal components explain a substantial portion of the variance (PC1 = 85.9 %, PC2 = 7.2 %), indicating that most of the data variability is effectively captured within these two components. Notably, PC1 separates solvents based on their extraction performance: solvents with negative scores on PC1, such as the levulinic acid-based NaDES (NaDES 2.2 and NaDES 2.3) and 84 % methanol, are

associated with higher flavonol extraction yields. Regarding NaDES 2.4, where the ratio between the hydrogen bond acceptor (ChCl) and hydrogen bond donor (levulinic acid) is the highest (1:4), the similarity in extraction decreases slightly. The PCA results suggest that a higher proportion of levulinic acid improves the extraction of monoglucosylated flavonols. The 50:50 EtOH/H₂O mixture exhibits greater similarity with the NaDES based on tartaric acid (NaDES 4.1 and NaDES 4.5). In contrast, pure anhydrous ethanol shows an extraction profile more closely related to NaDES-1.3 (ChCl:1,3-propanediol 1:3) and the urea-based NaDES (NaDES 3.2). The combination of HBD and HBA in NaDES influences the interactions between the target flavonols and the solvent, ultimately affecting solubility and extraction efficiency. Key
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** PCA biplot of flavonoid extraction using different solvent systems: NaDES-1.3: 1:3 ChCl:1,3-propanediol; NaDES-1.4: 1:4 ChCl:1,3-propanediol; NaDES-2.2: 1:2 ChCl:Levulinic acid; NaDES-2.3: 1:3 ChCl:Levulinic acid; NaDES-2.4: 1:4 ChCl:Levulinic acid; NaDES-3.2: 1:2 ChCl:urea; NaDES-4.1: 1:1 ChCl:Tartaric acid; NaDES-4.5: 2:1 ChCl:Tartaric acid.</figcaption></figure>

factors such as the HBD:HBA molar ratio, hydroxyl group positioning, and methylene chain length significantly impact the polarity and viscosity of ChCl-DESs, which, in turn, influence flavonol extraction yield and optimal process conditions [47]. Regarding total extracted flavonol content (sum of all flavonols, Fig. 2f), this study demonstrates that levulinic acid-based NaDES is particularly effective for flavonol extraction, providing higher efficiency compared to traditional solvents such as water or ethanol. Regarding MeOH/water solvent, although it still offers better extraction, it is important to note that the experiments were conducted under optimal MeOH/water conditions. Once the most suitable NaDES was identified, the next step was optimizing the UAE process to determine the ideal extraction conditions for flavonols using NaDES-2.2. No significant differences were observed in pairwise comparisons among the three tested ChCl:levulinic acid ratios regarding total area. However, 1:2 ChCl:levulinic acid ratio with 25 % water was selected, as it showed a slight improvement in extraction efficiency for certain flavonols.

### 3.2.3. UAE optimization using the best NaDES

Once the appropriate NaDES was selected, the second stage of the research focused on determining the optimal extraction conditions. One of the main challenges associated with NaDES is their high viscosity, which can limit their extraction efficiency. To address this issue, the extraction conditions were optimized by increasing the extraction temperature [48]. Since, in the case of the BBD using methanol as a solvent, the extraction was carried out at room temperature, the effect of temperature on the extraction process remained unclear. As mentioned earlier, an appropriate increase in extraction temperature can enhance solute solubility and solvent diffusivity while reducing solvent viscosity. However, excessively high temperatures can also lead to the degradation of phenolic compounds [49]. For this reason, before conducting the BBD optimization for extraction using ChCl:levulinic acid (1:2), a

preliminary study was performed to evaluate the effect of different extraction temperatures (20, 30, and 40 °C). Additionally, considering that degradation can be influenced by the combination of time and temperature [47], three different extraction times (5, 15, and 25 min) were tested at each temperature. The results obtained are presented in the Supplementary Material, Fig. S6. For these analyses, the percentage of water in the NaDES was kept fixed at 25 % and the liquid-to-solid ratio at 10 mg ml⁻¹.

A significant difference in flavonol extraction at 40 °C for 25 min was observed based on the results obtained. In general, under these extraction conditions, a decrease in the concentration of all studied flavonols is observed, likely due to the degradation of these compounds. Interestingly, this degradation does not occur at 40 °C when the extraction time is shorter (5 and 15 min), suggesting that the degradation is a result of the combined effect of both factors. These findings indicate that working within this extreme range should be avoided to prevent degradation during the BBD analysis. Specifically, based on these results and for the experimental design, the following factor ranges were selected: 25, 45, 65 % of water (X₁); 20, 30, 40 °C extraction temperature (X²); 2, 11, 20 min of extraction time (X₃). The liquid-to-solid ratio was kept constant at 10 mg g⁻¹, as this was the optimal value obtained in the BBD study for methanol. Lower ratios were not considered meaningful, as they would generate excessive noise in the LC-MS analysis due to the low concentration. Higher ratios, on the other hand, would result in increased sample and solvent consumption (economic and environmental feasibility of the process) [47], making a low ratio particularly interesting for optimizing extraction efficiency.

As described in Section 3.3, a three-factor experimental design was applied, consisting of 16 treatments including three replicates at the center point for error estimation (Supplementary Material, Table S3). All experiments were conducted in randomized order. In this second design, no lack of fit was detected for any of the response variables (individual
flavonols), and the coefficient of determination (R²) values ranged from 96.25 to 98.47, indicating a strong fit of the models. Fig. S7 (Supplementary Material) shows the experimental and predicted values for each response variable, derived from the second-order polynomial equations. Furthermore, Fig. 4 illustrates the significance of the main factors and their interactions using Pareto charts, following the same statistical criteria described earlier.

Based on the Pareto diagrams, the most influential variable in flavonol extraction is temperature, along with its quadratic effect. Both factors have a negative impact on extraction, meaning that as the temperature increases, the amount of flavonols extracted decreases. Moreover, this reduction becomes more pronounced at higher temperatures, following an inverted parabolic curve. The experimental design also allows for determining the optimal values of each factor to maximize flavonol extraction. In this case, optimization was performed using the desirability function. With a desirability of 84.51 %, the following optimal values were obtained: 12 min of extraction, 25 % water in the NaDES (1:2 choline chloride:levulinic acid), and an extraction temperature of 25 °C.

Notably, the lowest water percentage within the studied range was selected. However, lower percentages were avoided to prevent handling difficulties associated with an excessively viscous and dense NaDES. Previous studies have reported that while a moderate increase in water content in the NaDES reduces viscosity and enhances solvent diffusivity, favoring extraction, excessive water can negatively affect the extraction efficiency of choline chloride-based DESs (ChCl-DESs) for phenolic compounds [47,50]. This occurs because excess water disrupts the

hydrogen bonds between DES components, altering their molecular structure and weakening their interaction with the target compounds [51,52].

Regarding temperature, as previously mentioned, the optimal value remains low at 25 °C. Studies suggest that the temperature during phenolic compound extraction using DESs must be carefully regulated, as certain temperatures can enhance solute solubility and solvent diffusivity while reducing solvent viscosity. However, excessively high temperatures can lead to the degradation of phenolic compounds [53].

Finally, an intermediate value within the studied range was obtained concerning extraction time. Similar results have been reported in previous studies, showing an initial increase in extraction yield over time, followed by either a plateau where extraction remains constant [47] or, in some cases, a decrease due to degradation processes [10,49].

### 3.2.4. Precision of the developed NaDES method

As the final step in this study, the precision of the developed method was evaluated in terms of repeatability and intermediate precision. Specifically, four extractions were performed per day over three consecutive days. The resulting 12 extracts were then randomly analyzed in duplicate using the LC-MS system. This setup allowed for the assessment of the repeatability associated with the UAE extraction process, the repeatability of the LC-MS analytical system, and inter-day precision. The results were expressed as coefficients of variation (CV, %). A summary of the results is presented in Table 3, which also includes the quantified amounts of each flavonol obtained using the optimized method.

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 4.** Pareto plots for each of the flavonols studied (response variables) in the ChCl:ELvulinic acid BBD design: (a) Quercetin 3,4'-diglucoside; (b) Isorhamnetin 3,4'-diglucoside; (c) Quercetin 3-glucoside; (d) Quercetin 3'-glucoside; (e) Isorhamnetin 4'-glucoside. Blue bars indicate positive effects of the factor on the response variable, and grey bars indicate negative effects. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>
<caption>**Table 3**</caption>

Summary of the precision assessment of the extraction method: average amount of flavonols extracted per day (mean ± standard deviation, n = 4) and associated errors expressed as coefficient of variation.

<table><thead><tr><th></th><th>Q34diGlu</th><th>Iso34diGlu</th><th>Q3Glu</th><th>Q4Glu</th><th>Iso4Glu</th><th>Total area</th></tr></thead><tbody><tr><td>Day 1<br>(mg g<sup>-1</sup> eq. Q3Glu)</td><td>1.100 ± 0.061</td><td>0.671 ± 0.006</td><td>0.673 ± 0.005</td><td>1.643 ± 0.172</td><td>0.744 ± 0.007</td><td>4.832 ± 0.218</td></tr><tr><td>Day 2<br>(mg g<sup>-1</sup> eq. Q3Glu)</td><td>1.089 ± 0.061</td><td>0.675 ± 0.005</td><td>0.679 ± 0.006</td><td>1.722 ± 0.132</td><td>0.751 ± 0.008</td><td>4.915 ± 0.182</td></tr><tr><td>Day 3<br>(mg g<sup>-1</sup> eq. Q3Glu)</td><td>1.102 ± 0.065</td><td>0.675 ± 0.005</td><td>0.677 ± 0.005</td><td>1.654 ± 0.183</td><td>0.754 ± 0.025</td><td>4.862 ± 0.272</td></tr><tr><td>Mean (days 1–3)<br>(mg g<sup>-1</sup> eq. Q3Glu)</td><td>1.085 ± 0.057</td><td>0.671 ± 0.005</td><td>0.676 ± 0.005</td><td>1.645 ± 0.152</td><td>0.750 ± 0.015</td><td>4.827 ± 0.209</td></tr><tr><td>Error HPLC<br>(CV, %)</td><td>2.884</td><td>0.393</td><td>0.405</td><td>5.013</td><td>1.605</td><td>2.223</td></tr><tr><td>Error extraction<br>(CV, %)</td><td>5.672</td><td>0.820</td><td>0.749</td><td>9.730</td><td>1.751</td><td>4.604</td></tr><tr><td>Error inter-day<br>(CV, %)</td><td>5.162</td><td>0.802</td><td>0.774</td><td>9.113</td><td>1.975</td><td>4.283</td></tr></tbody></table>

As observed, all results fall within the acceptable precision limits (RSD < 10%) established by AOAC [54], confirming the good intermediate precision of the flavonol extraction method using a green solvent composed of 25 %:1:2 water:ChCl:levulinic acid.

### 3.2.5. Comparison between MeOH method and NaDES-2.2 method

In this work, two different methods for extracting flavonols from onion have been developed and optimized, each employing a distinct approach. The first method utilizes methanol as the extraction solvent, which is widely used for bioactive compounds in natural matrices due to its high extraction efficiency and well-documented reliability. The second approach is designed to align with the principles of green chemistry, promoting the use of NaDES. These solvents offer several advantages, including low volatility, biodegradability, and stability at room temperature. NaDES have been extensively studied for their application in analytical chemistry sample preparation, particularly for the extraction of pharmaceuticals, plant bioactive compounds, pesticides, and dyes from various matrices. To compare the sustainability of both extraction

methods, green chemistry evaluation metrics can be employed. Among them, AGREEprep [55] and the Sample Preparation Metric of Sustainability (SPMS) [56] provide a systematic assessment of the environmental impact of sample preparation techniques. AGREEprep evaluates sustainability based on ten key factors, including solvent selection and consumption, waste generation, energy usage, sample size, and overall method efficiency. Additionally, it considers the environmental footprint of the analytical instrumentation used post-extraction. On the other hand, SPMS provides an alternative evaluation framework by analyzing parameters across 4 main categories: sample amount, extractant properties, procedural aspects, and energy consumption and waste production. The results of applying these metrics to the developed methods are presented in Fig. 5, and the criteria on which each metric is based are explained in Tables S3 and S4 of the Supplementary Material. AGREEprep yields a final score on a scale from 0 to 1, while SPMS uses a scale from 0 to 10. In both cases, a higher score, resulting from the sum or integration of the relevant criteria, indicates a greener, more sustainable sample preparation method.

<figure><img src="image_13.png" /><figcaption>**Fig. 5.** Pictograms obtained using: (a) AGREEprep for the evaluation of the MeOH method; (b) SPMS for the evaluation of the MeOH method; (c) AGREEprep for the evaluation of the NaDES method; (d) SPMS for the evaluation of the NaDES method.</figcaption></figure>
Both methods employ a sample-to-solvent ratio of 10 mg mL⁻¹, effectively minimizing sample and solvent use while ensuring sufficient material for LC-MS analysis. Economically, this reduction translates into significant cost savings in research. Moreover, it supports key green chemistry principles by decreasing reagent consumption and waste generation (Fig. S8a,c, criterion 5 of Table S4; Fig. S8b,d, criterion 1 of Table S5). To assess the environmental impact, the E-factor was calculated using eq. (1), which represents the total waste produced per unit mass of product [57]:

$$E = \frac{\text{Total mass of waste from process}}{\text{Total mass of product}} \quad (1)$$

In our experiments, 5 mL of extract was obtained by mixing 5 mL of supernatant with 5 mL of water, leaving 2.5 mL of supernatant unused as waste. Since both solvents, NADES and methanol, are considered non-recyclable under our experimental conditions, the entire volume of residual solvent contributes to the waste. For methanol (density ≈ 0.791 g mL⁻¹), the 2.5 mL leftover corresponds to approximately 1.98 g of waste. Assuming the extract has a density close to 1 g mL⁻¹, the product mass was 5.0 g, resulting in an E-factor calculated of 0.39. To account for the potential toxicity of the waste, the Environmental Quotient (EQ) was calculated by multiplying the E-factor by a hazard factor Q [58]. For methanol, Q was set at 3.0, yielding an EA value of 1.188. In contrast, NADES solvents typically have lower toxicity (Q ≈ 1). Although the E-factor is similar due to comparable waste volumes, the EQ value for NADES is significantly lower, reflecting a greener environmental profile. This combined assessment of both waste quantity (E-factor) and hazard (EQ) provides a more comprehensive evaluation of the environmental impact of the extraction methods, highlighting the benefits of greener solvents even when waste volumes are comparable.

Regarding extraction time, the methanol-based method achieved optimal results after 25 min, whereas the NaDES-based method required only 12 min. This significant reduction not only shortens the overall analysis time, an important factor in improving laboratory efficiency, but also decreases energy consumption, which is a key parameter in green chemistry evaluations (Fig. S8a,c; Table S4, criterion 6; Fig. S8b,d; Table S5, criterion 3). Specifically, reducing the ultrasound-assisted extraction time from 25 to 12 min led to a measurable decrease in total energy consumption, from 193.25 Wh in the MeOH method to 190 Wh in the NaDES method, corresponding to an approximate 1.7 % reduction. While modest, this energy saving reinforces the overall sustainability of the NaDES method, especially when considered alongside its higher extraction efficiency and the use of environmentally friendly solvents.

Regarding extraction efficiency, the NaDES-based method extracted a total of 4.83 ± 0.21 mg g⁻¹ of flavonols, nearly doubling the amount obtained using methanol (2.28 ± 0.047 mg g⁻¹ eq. Q3Glu) and significantly outperforming other commonly used green solvents, such as ethanol and ethanol/water mixtures (Fig. 4). This substantial increase in yield highlights the strong potential of NaDES formulations not only as sustainable alternatives but also as highly effective extraction media for phenolic compounds in onions.

All these findings confirm that levulinic acid-based NaDES is a promising green solution for the analysis of flavonols in onion matrices. Given the relevance of flavonol profiling for varietal classification and for assessing the impact of agronomic and environmental factors on bioactive compound composition, the development of a cost-effective, sustainable, and easy-to-implement extraction method represents a meaningful contribution to the field of food chemistry. Other authors have also demonstrated the potential of NaDES for the extraction of polyphenolic compounds from onions, particularly using choline chloride/urea mixtures [20,21]. In both cases, microwave-assisted extraction was employed in combination with NaDES, reflecting a wider interest in the use of more efficient extraction techniques, an approach aligned with the present work, which applies ultrasound-assisted extraction. Notably, the method developed here achieves significantly shorter

extraction times and lower resource use. For instance, Chandra Bhushan T. Pal et al. (2019) applied 120 min of extraction with a liquid-to-solid ratio of 50:1 [20], while another study by the same authors reported 15.03 min of irradiation and a 54.97 mL g⁻¹ ratio [21]. In comparison, the UAE-based protocol described here considerably reduces extraction time (12 min), energy input, and solvent consumption (10 mg mL⁻¹), making it a more environmentally and economically sustainable alternative for the extraction of flavonols from onion matrices. In terms of flavonol identification, both referenced studies focus exclusively on myricetin, quercetin, and kaempferol, reporting yields of 6.185 mg g⁻¹ for quercetin, 0.349 mg g⁻¹ for kaempferol, and 0.105 mg g⁻¹ for myricetin. In contrast, the present study provides a broader characterization of the flavonol metabolomic profile, identifying a wider range of compounds. Moreover, while the previous studies were conducted using onion peel, the current analysis focuses on bulb tissue, which contributes additional and complementary information to the understanding of flavonoid distribution in onion varieties.

## 4. Conclusions

In this study, various HBDs—levulinic acid, tartaric acid, 1,3-propanediol, and urea—were tested at different molar ratios (1:1, 1:2, 1:3, 1:4, 2:1, and 2:3) in combination with choline chloride (ChCl) as the hydrogen bond acceptor to form Natural Deep Eutectic Solvents (NaDES). Overall, the results indicate that HBDs that are originally liquid, such as levulinic acid and 1,3-propanediol, exhibited a greater ability to form NaDES. Additionally, the presence of water at moderate concentrations effectively reduced the viscosity of NaDES, facilitating their formation and improving their handling. Based on these findings, the following NaDES formulations were selected for flavonol extraction from onions: 1:3 ChCl:1,3-propanediol, 1:4 ChCl:1,3-propanediol, 1:2 ChCl:levulinic acid, 1:3 ChCl:levulinic acid, 1:4 ChCl:levulinic acid, 1:2 ChCl:urea, 1:1 ChCl:tartaric acid, and 2:1 ChCl:tartaric acid. Five major flavonols were detected and used as response variables throughout the study, with LC-MS selected as the analytical technique due to its high efficiency, sensitivity, and resolution. Among the tested NaDES, 1:2 ChCl:levulinic acid demonstrated the highest efficiency in extracting flavonols from onions, outperforming the other NaDES and conventional green solvents such as ethanol and water. To further evaluate its performance, the optimized NaDES was compared with methanol, the most commonly used traditional solvent of extraction. For this objective, two experimental designs were carried out: a BBD for ultrasound-assisted extraction (UAE) using methanol as the extraction solvent, and a BBD for UAE using 1:2 ChCl:levulinic acid as the extraction solvent. In both cases, desirability functions were employed to determine the optimal conditions, balancing all response variables: the 5 flavonols identified by LC-MS. For methanol extraction, the optimal conditions were: 25 min extraction time, a 10 mg mL⁻¹ sample-to-solvent ratio, and 84 % water in methanol. For NaDES extraction, a fixed sample-to-solvent ratio (10 mg mL⁻¹) was maintained, while water content, extraction time, and temperature were optimized. Given the high viscosity of NaDES, temperature was included as an additional factor, as previous studies have highlighted its importance in improving extraction efficiency. The optimal conditions for NaDES extraction were found to be: 12 min extraction time, 25 % water, and 25 °C extraction temperature. The optimized NaDES method required a shorter extraction time compared to methanol, demonstrating a significant reduction in processing time and energy consumption. As for temperature, although higher temperatures were expected to improve extraction efficiency, excessive heat instead led to flavonol degradation, highlighting the importance of optimizing this factor. Finally, the NaDES-based extraction method was validated in terms of repeatability and intermediate precision for each flavonol, with all analytes showing relative standard deviations (RSDs) below 10 %. The developed UAE extraction method using NaDES proved to be a highly efficient and sustainable alternative, enabling the extraction and determination of flavonols in onion samples while
adhering to the Principles of Green Analytical Chemistry. The method significantly reduced analysis time and energy consumption, reinforcing its potential as a viable alternative to conventional extraction approaches as demonstrated by our final AGREEprep and SDMS green chemistry evaluation metrics.

### CRediT authorship contribution statement

**Ana V. González-de-Peredo:** Writing – original draft, Validation, Software, Methodology, Investigation, Formal analysis, Data curation. **Alicia Maroto:** Writing – review & editing, Visualization, Supervision, Conceptualization. **Antony Memboeuf:** Writing – review & editing, Visualization, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

### Funding sources

This project has received funding from the European Union's Horizon 2020 research and Innovation program under the Marie Skłodowska-Curie grant agreement no. 899546: "Integrated machine learning metabolomics using ion mobility mass spectrometry to unveil biomarkers in *Allium* samples".

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Gonzalez de Peredo reports was provided by European Union's Horizon 2020 research and Innovation program under the Marie Skłodowska-Curie grant agreement no. 899546: "Integrated machine learning metabolomics using ion mobility mass spectrometry to unveil biomarkers in Allium samples". If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Antony MEMBOEUF reports financial support was provided by European Union. Antony MEMBOEUF reports financial support was provided by Brittany Region. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgments

The authors are grateful to the University of Brest for providing the necessary facilities to carry out the research.

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.microc.2025.114445.

### Data availability

Data will be made available on request.

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