Optimization of High-Performance Liquid Chromatography (HPLC) Conditions for Isoflavones Using Deep Eutectic Solvents (DESs) as Mobile Phase Additives by the HCI Program

Abstract Isoflavone exhibits estrogen-like structure and activity and has various physiological and pharmacological functions, such as anticancer, antioxidant, and anti-inflammatory. In this study, several deep eutectic solvents (DESs) were synthesized and used as mobile phase additives to optimize the analytical conditions of five isoflavones. The DES composed of choline chloride:citric acid (molar ratio 1:1) was the most effective mobile phase additive with reduced peak tailing, improved peak shape, and increased number of theoretical plates. The effectiveness of the DES highlights its potential as a mobile phase additive. The optimal high-performance liquid chromatography (HPLC) conditions for the isoflavones were determined by the Intelligent HPLC simulator. The optimal conditions for gradient elution were water (A): acetonitrile (B) for 0 min, 25% B; 15 min, 25% B; 17 min, 35% B; and 50 min, 35% B. This approach provided excellent linearity (0.5 to 500 μg/mL, R2 ≥ 0.9991), limits of detection (0.024 to 0.109 μg/mL), limits of quantification (0.079 to 0.363 μg/mL), and precision (1.74 to 4.80%).

Deep eutectic solvents (DESs) have emerged to complement these issues and attracted attention (Abbott et al. 2003;Fu, Chen, and Qiu 2022;Liu et al. 2022;Xiao et al. 2022;Zhang et al. 2022).DESs have similar physical and chemical properties to ILs.Moreover, they have low toxicity, are inexpensive and easy to use (Espino et al. 2016;An and Row 2021;Santana-Mayor et al. 2021;He, Tang, and Row 2022).DES may be prepared as a combination of a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD) at specific molar ratios and temperatures.The mixture has a significantly lower melting point than the components (Hansen et al. 2021).
One application of DES is as a mobile phase additive in high-performance liquid chromatography (HPLC).According to the literature, DESs improve chromatographic properties.DES (choline chloride [ChCl]: glycerol, molar ratio 1:3) was added at 0.10% to narrow the peak shape of caffeic acid to improve the number of theoretical plates and efficiency (Li, Zhu, and Lei 2015).The addition of ChCl:ethylene glycol (molar ratio 1:2) at 0.20% for quercetin determination provided a lower tailing factor and a symmetrical peak shape (Gao et al. 2018).
Isoflavones are plant-derived flavonoids that exhibit the same structure and activity as estrogen (Messina 2016).Isoflavones have various pharmacological and physiological functions, such as anticancer, antioxidant, anti-inflammatory, anti-allergic, and neuroprotective (Wu et al. 2012;Gao et al. 2015).Isoflavones are present at low concentrations in vegetables, grains, and legumes and at high levels in soybeans (Gacek 2014).In soybeans, isoflavones are present as glycosides bound to sugar molecules.The fermentation or digestion of soy yields the aglycone isoflavones, in which sugar is released from isoflavone glycosides (Murphy, Barua, and Hauck 2002;Fayed 2015).The glycoside contains genistin (GNI), daidzin (DZI), and glycitin (GLI).The aglycone form includes genistein (GNE), daidzein (DZE), and glycitein (GLE) (Islam et al. 2014;Yatsu, Koester, and Bassani 2016).Isoflavone assays have been reported by HPLC using methanol-water or ACN-water (da Costa C esar et al. 2006).However, the chromatographic peaks of isoflavones include tailing and asymmetry, and hence improvement of the chromatography is a subject of investigation (Tsai et al. 2007;Sun et al. 2011;Payette et al. 2022).
The identification of the optimal HPLC conditions is difficult.The HCI program (Intelligent HPLC simulator, 1.02L B0017) developed by the High Purity Separation Lab (Inha University) is a mathematical tool to identify the optimal conditions.The HCI program may be applied to normal and reversed-phase chromatography by calculating the isocratic and gradient modes using a small number of experimental measurements.The program predicts the mobile phase composition, sample retention time, elution profile, column resolution, optimized analytical conditions, and economics (Jin, Lee, and Row 2006;Zheng and Kyungho 2007).
In this study, to improve the separation of five isoflavones, the optimum HPLC mobile phase additive was identified and compared with the standard additive acetic acid.The optimal conditions were identified using the HCI software.
Preparation of standards and HPLC analysis GNI, GNE, GLI, and GLE were dissolved in ethanol DZE in DMSO/ethanol (1:1) to prepare 10000.00lgÁmL À1 stock solutions.The standard solutions were diluted to between 0.50 and 500.00lgÁmL À1 .The mobile phase was water: ACN.The injection volume and column temperature were 20 lL and 30 C, respectively.The flow rate was 1.0 mLÁmin À1 and the detection wavelength was 254 nm.
Table 1.Empirical constants and regression coefficients for the isoflavones.

Preparation of mobile phase additives
All mobile phases were composed of water and ACN.Mobile phases were prepared by adding the reference acids, ChCl, and DESs at 0.1% in water.All mobile phases were magnetically stirred with the additives, passed through a 0.45 lm nylon membrane filter, and sonicated for 15 min.The isoflavones were determined using these mobile phases.The DES additives were evaluated using the number of theoretical plates, tailing factor, and resolution.
Calculation of the theoretical plate number, tailing factor, and resolution The number of theoretical plates (N) was determined by N ¼ 16ð t R W Þ 2 where t R is the retention time and W is the width of the corresponding peak.The tailing factor (Tf) was evaluated by T ¼ w 0:05h 2f where w 0:05h is the 5% of the peak height of the peak width and f is the width of the front when a vertical line is drawn from the top of the peak.The resolution (Rs) was determined by Rs where t R1 and t R2 are the retention times of the analytes and w 1 and w 2 are the corresponding baseline peak widths.

HCI program
The HCI program expresses the retention factor of each substance as a function of the mobile phase composition of a binary system (Shoenmakers, Billiet, and De Galan 1979;Lee et al. 1996) Hence, the logarithmic retention factor k is described by a quadratic equation involving the volume percent (F) of the organic solvent, where L, M, and N are empirical constants determined experimentally.
The retention volume in isocratic mode is expressed by the retention coefficient: where V r, n and k n are the retention volume and retention coefficient of the mobile phase and V 0 is the dead volume.
A material balance for the solute for the number of plates N provides: il e ÀaV where C N is the outlet concentration of the solute, C 0 is the initial concentration, and a is the equilibrium constant described by a ¼ 1 V m þKv s where V m and v s are the volumes of the mobile phase and the stationary phase.These relationships allow the determination of the concentration dissolution profile for each component.
The equilibrium constant (K) is correlated with the partition coefficient by k where e is the total porosity of the chromatography column (Jin and Row 2006;Zheng and Kyungho 2007;Li, Tian, and Row 2012).

Optimization of HPLC conditions with HCI
Water/ACN (v/v) with the DES additives was used as the mobile phase.Each of the isoflavones was determined while varying the ACN composition.The experimental retention times of the isoflavones were employed to determine the corresponding factors.The column size, mobile phase components, mobile phase mode, isoflavone concentration, and other relevant calculated factors were entered into HCI to obtain the optimal conditions.Further experiments were performed using these conditions.

DESs as mobile-phase additives
Different DESs, acids and ChCl were tested as mobile phase additives for the determination of isoflavones by HPLC. Figure 1 shows the number of theoretical plates obtained through a mobile phase using 75/25 water/ACN without additives and acetic acid (0.1%), trifluoroacetic acid (0.1%), formic acid (0.1%), citric acid (0.1%), ChCl (0.1%), and DES-1 (0.1%) to DES-8 (0.1%).Acids, ChCl, DES-1, DES-4, and DES-6 showed a similar or higher number of theoretical plates compared to the mobile phase without additives.All acids showed similar performance, but acetic acid was the best.Only DES-6 showed similar or higher number of theoretical plates compared to acetic acid.
Figure 2 shows the tailing factors (Tf) of acetic acid, trifluoroacetic acid, formic acid, citric acid, ChCl, and DES.The acids, ChCl, DES-2, DES-6, and DES-8 showed better tailing factors than the mobile phase without additives DES-6 provided a similar or higher tailing factor than acetic acid.
Figure 3 shows the chromatography of acetic acid, trifluoroacetic acid, formic acid, citric acid, ChCl, and DES as additives When acids, ChCl and DESs were used, the retention time was reduced and the peak shape improved compared to without additives.However, only DES-6 showed chromatographic properties similar to or better than the acids.
DES-1, DES-2, and DES-3 were employed to study the alkyl chain length of the ammonium cation.DES-3 and DES-4 were used to characterize ammonium salts.However, consistent results were not obtained for these parameters.
The influence of HBDs was investigated using DES-5 through DES-8.An acidic HBD improves peak tailing and retention time.However, further studies are needed to determine the mechanism.The quaternary ammonium salts (especially ChCl) used as HBAs of DES affect the retention time and number of theoretical plates.Acidic HBDs, such as citric acid, also improve the peak shape and tailing factor.

Molar ratio of components of DES
Five molar ratios  of DES (ChCl:citric acid) as mobile phase additives were investigated.Figures S1 and S2 show the numbers of theoretical plates and tailing factors.The number of theoretical plates for DES-6 was the highest with the best tailing factor.Hence, when ChCl and citric acid were reacted in a 1:1 molar ratio, the best mobile phase additive properties were obtained.

Mechanism of DESs as mobile phase additives
Cations and anions use coulombic attraction to form ion pairs and act as independent units in solution.The DES components act as ions and ion pairs in the mobile phase.
Here, independent ions have a larger role than ion pairs.The mechanism of DES as a mobile phase additive may be due to the binding between hydrogen bond receptors and donors (Tan et al. 2016).Peak tailing is improved by the end-capping operation, which eliminates the effects of silanol.
The DES cation competes with the polar analyte for free silanol groups on the stationary phase.These cations form ion pairs with anionic solutes.Chloride bound to citric acid by hydrogen bonding disrupts the water envelope around the analyte and increases its hydrophobicity (Li, Zhu, and Lei 2015).The isoflavones may be considered to be HBDs.Therefore, isoflavones competitively interact with alcoholic HBD (citric acid) and chloride.Hence, DES-6 (1:1 ChCl:citric acid) as an additive improves the chromatographic shape and removes tailing to increase the number of theoretical plates.

Optimization of HPLC condition with HCI program
The retention coefficients for each component with retention times analyzed under constant mobile phase conditions were determined by k ¼ where k is the retention factor, V R is the retention volume of the substance, and V m is the column dead volume.Table S2 lists the retention factors of the isoflavones based upon the mobile phase composition.The retention factor decreased with the concentration of ACN.The retention factor from the corresponding experimental value was entered into the HCI program, and the experimental constant and correlation coefficient were calculated.Table 1 summarizes the calculated constants and regression coefficients by the binary-polynomial equation.The retention factor based upon the mobile phase composition was determined using the corresponding model equation and the elution profiles were evaluated using plate theory.
The HCI program calculates based upon the retention factor.However, the influence of the retention factor based upon the DES volume was insignificant, so it was fixed at 0.1%, the most used concentration (Table S3).
The separation of DZE and GLE was the most difficult.Hence, the optimal conditions were calculated using HCI so that the resolution exceeded 1.5.The optimal isocratic condition is 72/28 water/ACN with 0.1% DES-6.The calculated resolution of DZE and GLE was 1.57.The GNE retention time was 35.07 min.This condition was adopted because it is suitable for the experimental range (20% to 35% ACN) for the retention model.Figure S3 compares the experimental results and the calculated elution profiles.
After applying the HCI conditions to the experiments, the results were compared (Table 2).The retention time error was used to characterize the match of the experimental and calculated retention times.Table 2 shows the retention time error was from 0.76% to 3.06%, showing good agreement.In addition, the resolution indicates good agreement between the calculated and the experimental values.Hence, the retention time and separation of the isoflavones are accurately predicted.However, the isocratic conditions have a long analysis time.The optimal gradient conditions were determined using HCI to reduce this parameter.
The focus is upon the separation of DZE and GLE, which are difficult to separate, and the reduction of the retention time of GNE, which is the last analyte to elute.The comparison of the calculated and experimental elution profiles shows a close match  (Figure 4).The retention time error for GLI and GNI was larger than under isocratic conditions, but the errors for DZE, GLE, and GNE was less than 3% (Table 2).

Method validation
The developed method was validated by the linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, and repeatability.The results are summarized in Table S4.Linearity was obtained for the analytes between 0.5 and 500 mg/mL with correlation coefficients (R 2 ) from 0.9991 to 0.9999.The LOD and LOQ based on the signal-to-noise ratios of 3 and 10 were from 0.024 to 0.109 lg/mL and 0.079 to 0.363 lg/mL, respectively.
Six measurements were employed to characterize the repeatability using the relative standard deviation (RSD).The precision of the method was assessed by characterization of inter-day and intra-day variability for one day and three consecutive days.The RSDs were from 1.74% to 4.80%.Hence, the developed procedure for isoflavones was precise and accurate, highlighting its suitability for practical analysis.

Conclusion
Several DES were synthesized as mobile phase additives for the optimal determination of isoflavones.Relevant parameters were compared with acetic acid, an additive previously reported for isoflavone determination.DES-6 (ChCl:citric acid, molar ratio 1:1, reaction temperature 80 C) was the best additive with superior performance to acetic acid.The optimum isocratic mobile phase composition using the HCI program was water/ACN (72/28, v/v) with 0.1% DES.The optimal gradient conditions were water/ACN (75/25, v/v) to (65/35, v/v).Under these conditions, the isoflavone peaks had the higher number of theoretical plates, a tailing factor close to 1, and a resolution of 1.5 or more.The optimum DES is environmentally friendly, reducing damage to the column, which suggests practical application as a mobile phase additive.

Figure 1 .
Figure 1.Numbers of theoretical plates of isoflavones for the DESs as mobile phase additives at 0.10% (v/v).

Figure 2 .
Figure 2. Tailing factors of isoflavones with the DESs as the mobile phase additives at 0.10% (v/v).