Authors:
Luis Alejandro Pérez-López Departamento de Química Analítica, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, México

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Norma Cavazos-Rocha Departamento de Química Analítica, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, México

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Cecilia Delgado-Montemayor Departamento de Química Analítica, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, México

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Noemí Waksman-Minsky Departamento de Química Analítica, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, México

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Marcelo Hernández-Salazar Centro de Investigación en Nutrición y Salud Pública, Facultad de Salud Pública y Nutrición, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, México

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Omar J. Portillo-Castillo Departamento de Química Analítica, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, México

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https://orcid.org/0000-0002-7690-2153
Open access

Abstract

The analysis of phenolic acids (PAs) is of great importance, because they are frequently present in natural products and their derivatives, and these compounds also have multiple beneficial effects to human health. This work is focusing on the separation of seven PAs (caffeic acid, coumaric acid, gallic acid, ferulic acid, protocatechuic acid, sinapic acid, and syringic acid), in a reversed-phase liquid chromatographic (RP-HPLC) isocratic method using a hydrophilic deep eutectic solvent (DES) as a mobile phase additive. The analysis was carried out with a diode array detector. The used DES was composed by choline chloride and glycerol, and it was characterized by infrared spectroscopy. The combination of choline chloride:glycerol (1:4) added at 0.25% to mobile phase composed of 0.15% formic acid aqueous solution and methanol (80:20), showed the best separation for target analytes. The new proposed method was validated, and results indicated that the proposed method is linear, selective for almost all analytes, provided high sensitivity with limit of detection ranges from 0.009 to 0.023 mg mL−1, and has satisfactory precision and accuracy, with values of relative standard deviation of 0.24–2.65% and recoveries of 97.97–109%, respectively. Additionally, this method was successfully applied to simultaneous determination of phenolic acids in three kinds of samples of powder to prepare lemon flavour drink enriched with black tea extract.

Abstract

The analysis of phenolic acids (PAs) is of great importance, because they are frequently present in natural products and their derivatives, and these compounds also have multiple beneficial effects to human health. This work is focusing on the separation of seven PAs (caffeic acid, coumaric acid, gallic acid, ferulic acid, protocatechuic acid, sinapic acid, and syringic acid), in a reversed-phase liquid chromatographic (RP-HPLC) isocratic method using a hydrophilic deep eutectic solvent (DES) as a mobile phase additive. The analysis was carried out with a diode array detector. The used DES was composed by choline chloride and glycerol, and it was characterized by infrared spectroscopy. The combination of choline chloride:glycerol (1:4) added at 0.25% to mobile phase composed of 0.15% formic acid aqueous solution and methanol (80:20), showed the best separation for target analytes. The new proposed method was validated, and results indicated that the proposed method is linear, selective for almost all analytes, provided high sensitivity with limit of detection ranges from 0.009 to 0.023 mg mL−1, and has satisfactory precision and accuracy, with values of relative standard deviation of 0.24–2.65% and recoveries of 97.97–109%, respectively. Additionally, this method was successfully applied to simultaneous determination of phenolic acids in three kinds of samples of powder to prepare lemon flavour drink enriched with black tea extract.

Introduction

Phenolic acids (PAs) are secondary metabolites widely distributed in the plant kingdom, so that are present in natural products and derivatives. They commonly appear as hydroxylated derivatives of either benzoic or cinnamic acid [1]. PAs have attracted considerable interest in the past few years because of their potential health benefits. As the polyphenols, PAs are powerful antioxidants and have been reported than exhibit antibacterial, antiviral, anti-carcinogenic, anti-inflammatory, vasodilatory actions, among others [2]. The analytical procedures used for PAs analysis are commonly based on high-performance liquid chromatography (HPLC) [3–6]; although others separation techniques such as gas chromatography [7, 8] and capillary electrophoresis [9, 10] are also used.

On the other side, in recent years, the scientific community has focused on developing more sustainable methodologies. For example, in Analytical Chemistry, this has resulted in reduction of the use of reagents, appropriate elimination of waste, the use of non-toxic reagents and the development of faster methodologies and technologies [11]. In this idea, one of the goals within analytical separations in terms of the use of solvents, is the replacement of the volatile, flammable, and toxic solvents [12]. In the search for environmentally friendly solvents, Deep Eutectic Solvents (DESs) have emerged as a cheap and ecological alternative to conventional ionic liquids in the analytical separations, such as when are used as mobile phase modifiers in liquid chromatography [13].

DESs are mixtures result from the combination of at least two substances, a hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD) at specific molar ratio and temperature, the resultant eutectic mixture has a melting point lower than the individual component melting point [14] In general, DESs are formed by mixing a quaternary ammonium salt (e.g., choline chloride) as a HBA with a wide range of HBDs that contain functional groups such as amides, carboxylic acids and alcohols [15]. The first DES was made by Abbott et al. [16], composed of choline chloride and urea in a 1:2 molar ratio which is hydrophilic.

Multiple works focused on the use of DESs as extraction media in different sample preparation procedures has been development [17–20]. Additionally, DESs have been used as component of the mobile phases in different modalities of liquid chromatography [21, 22]. When DESs are used as one of the main components of mobile phase, the high viscosity can generate considerable increase in the pressure of HPLC system and strong interactions can occur between stationary phase and the DESs, due to the large amount of DES than is used [23, 24]. However, when DES are used as mobile phase additives, until a certain extent, a significant increase in HPLC system pressure is avoided due to the low amount of DES used. But a significant improvement in the chromatographic performance of the target compounds is obtained, since mobile phase additives such as DESs can modify their retention properties, enhancement the efficiency, and selectivity [25, 26].

In a previous report, a DESs of choline chloride and glycerol (molar ratio 1:3) was used as additive of mobile phase at 0.1% v/v to optimize chromatographic behavior of caffeic acid; obtaining an increase in the theoretical plate number, shorten the retention time and modify the chromatogram shape [27]. In other work, the addition of DESs in the mobile phase for separation of four quaternary alkaloids (coptisine chloride, sanguinarine, berberine chloride, and chelerythrine) in a C18 column was studied; the composition of acetonitrile and 1.0% DES aqueous solution (choline chloride:ethylene glycol, 1:3 molar ratio) to pH 3.3 showed the best symmetrical peaks and significantly improved the separation [28]. In the same way, the influence of DESs as mobile phase additives at the chromatographic behavior of quercetin was evaluated. The smaller tailing factors and better peak shapes were obtained using a mobile phase with 0.20% of DES additive, which was composed of choline chloride and ethylene glycol 1:2 molar ratio [29]. The previous results indicate that the DESs have the potential to replace or be used in combination with traditional mobile phase additives for HPLC. The use of DESs as additives to mobile phases in the chromatography separations has not been completely explored, therefore is still challenge.

The main objective of this work was to evaluate the effect of addition of a hydrophilic DES in different concentrations to the mobile phase of a method previously developed within the working group for the separation of seven phenolic acids, including hydroxylated derivatives of either benzoic acid (gallic acid, protocatechuic acid, and syringic acid) and cinnamic acid (caffeic acid, coumaric acid, ferulic acid, and sinapic acid). Furthermore, once selected the best separation conditions for the phenolic acids in presence of DESs, the proposed method was validated and its applicability in the analysis of target analytes in powder samples to prepare beverages enriched with black tea extract was evaluated.

Experimental

Reagents

Caffeic acid (CaA, ≥ 98.0%), para-coumaric acid (CoA, ≥ 98.0%), gallic acid (GaA, ≥ 97.5%), trans-ferulic acid (FeA, ≥ 99.0%), protocatechuic acid (PrA, 99.8%), sinapic acid (SiA, ≥ 98.0%) and syringic acid (SyA, ≥ 95.0%) were purchased from Sigma Aldrich (St. Louis, MO, USA). Choline chloride (ChCl, ≥ 98%) and Glycerol (Gly, ≥ 99.5%) both were from Sigma Aldrich (St. Louis, MO, USA). Methanol (HPLC grade) was supplied for J. T. Baker (USA) and formic acid (98–100%) was obtained from Merck (Germany). Deionized water was used in all experiments and was obtained with an Elga Veolia II system (France).

Preparation of working solutions

Individual stock solutions of all phenolic acids were obtained by dissolving an appropriate amount of each compound to a concentration of 500 μg mL−1 in methanol and stored at 4 °C in the dark until use. The working standard solutions were prepared at 80:20 ratio (aqueous phase:organic phase) of each mobile phase tested.

Preparation and characterization of hydrophilic deep eutectic solvents

The hydrophilic DES were synthesized by procedures obtained from modifications made to previously reported methods and the selected components were ChCl and Gly. [30, 31]. For DESs formation, the two reagents were simply mixed in a glass vial on a magnetic stir plate, within a water bath to 800 rpm as stirring constant rate and 70 °C as reaction temperature. The preparation was investigated to different molar ratios of ChCl and Gly (1:1, 1:2, 1.3, and 1:4), mixing and stirring these two components for 1 h until a homogeneous and colourless liquid was obtained. The obtained DESs was stored in a dissector at room temperature until use.

ChCl, Gly and all synthesized DESs were characterized by infrared spectroscopy (IR) using a Fourier Transform Infrared Spectrometer FT-IR-ATR Frontier from Perkin Elmer (USA). The IR analysis were carried out placing a small amount of each individual substance and of the synthetized DESs on the spectrometer sample holder for the acquiring of spectra between 550 and 4,000 cm−1.

Preparation of mobile phases: addition of DESs

All tested mobile phases were composed for 0.15% formic acid aqueous solution and methanol as organic modifier at a proportion 80:20 v/v, respectively. The three different DESs (ChCl:Gly 1:2, 1:3 and 1:4) were added to aqueous component of the mobile phase to concentrations of 0.05%, 0.15% and 0.25% v/v. Therefore, nine mobile phases were prepared. Then, all mobile phases were filtered using a vacuum pump system and a 0.45 µm nylon filter from Millipore (Ireland) and sonicated by ultrasonic bath Bransonic 3,510 from Branson Ultrasonics (Danbury, CT, USA) for 15 min prior to use. To select the most suitable mobile phase for separation of the target analytes, we prepared working standard solutions at 10 μg mL−1 by diluting stock solutions in all nine mobile phases that were tested, all assays were performed by triplicate. The chromatographic resolution was calculated according to Equation (1), where t R1 and t R2 are the retention times of the analyte that elutes first and the analyte that elutes later, respectively; and W b1 and W b2 are the width of peak measured to the baselines for the first and second peak, respectively.
R s = 2 ( t R 2 t R 1 ) ( W b 1 + W b 2 )

Chromatographic conditions

The analysis was carried out on a Waters Alliance 2,695 chromatography system (Waters Co., MA, USA) equipped with a quaternary pump, autosampler, column oven and Waters 2,998 photo diode array detector (DAD). The instrument was controlled by use of Empower software installed with equipment for data collection and acquisition. Compounds were separated on a Kinetex C18 reverse phase column (150 x 3 mm, 2.6 µm) from Phenomenex (USA). The mobile phase consisted of solvent A (0.15% formic acid aqueous solution-0.25% DES 1:4) and solvent B (methanol) with isocratic elution profile. The flow rate was of 0.2 mL min−1, injection volume of 5 µL and the column was maintained at 35 °C. All analytical runs were carried out for 45 min and all injections were performed by triplicate. Detection was to 295 nm for all analytes in the optimization experiments, and the quantification was performed at 325 nm for CaA, 309 nm for CoA, 272 nm GaA, 322 for FeA, 259 nm for PrA, 323 nm for SiA and 275 nm for SyA.

Method validation

The proposed method was validated by evaluating parameters such as linearity, selectivity, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ). To evaluate the linearity, external standard calibration curves were constructed by triplicate analysis of standard solutions of all target analytes at concentration levels of 0.05, 1, 5, 15, 20 and 30 μg mL−1 for CaA, CoA, FeA, PrA and SyA; 0.1, 1, 5, 15, 20 and 30 μg mL−1 for SiA and 0.05, 0.1, 1, 5, 15 and 20 μg mL−1 for GaA. All standard solutions were prepared with the mobile phase composed for 0.15% formic acid aqueous solution_0.25% DES 1:4 and methanol at a ratio 80:20, respectively. The association between variables was established by least-squares regression analysis for the responses of each analyte vs. concentration and calibration curves were plotted, the equation of the line was obtained, and correlation and determination coefficients (R 2) were calculated.

The selectivity was assessed by the investigation of possible endogenous interferences in mobile phase, samples, and blank samples at the retention times of the chromatographic peaks of the target analytes. Chromatograms of blank samples, samples and standards were compared by matching retention times and UV-spectrums of the chromatographic peaks, the spectrums were obtained by DAD detector.

Precision of the method was estimated by intra-day (one day) and inter-day (three consecutive days) independent analysis for triplicate of three blank samples spiked at low, medium, and high known concentration level. The blank samples were spiked to three different concentrations of 0.5, 8 and 25 μg mL−1 for CaA, CoA, FeA, PrA, SiA and SyA. The relative standard deviation (% RSD) of the concentration found in the samples and was taken as a measure of precision.

To evaluate the accuracy, recovery assays were performed for target analytes after spiking blank samples with standard to the same concentration levels that evaluated for precision. Accuracy was expressed in terms of recovery percentage (% R).

The LOD and LOQ under previously established chromatographic conditions were determined at a signal-to-noise (S/N) ratio of 3 and 10, respectively, using the chromatogram obtained from the lowest concentration standard of all analytes.

Method applicability

In order to evaluate the applicability of the proposed method, three kinds of samples of powder to prepare lemon flavour drink enriched with black tea extract and one sample of powder to prepare lemon flavour drink without tea extract were analyzed, which are commercially available in Monterrey, Nuevo León, México. The samples were: ZUKO® powder to prepare lemon flavour drink enriched with black tea extract from Tresmontes Lucchetii S.A. de C.V. (México), NESTEA® powder to prepare lemon flavour drink enriched with black tea extract from NESTLE-Productora de Alimentos Mexicanos S.A. de C.V.(México) and TANG® powder to prepare lemon flavour drink enriched with black tea extract from Mondelez México S. de R.L. de C.V. (México). As a blank sample, ZUKO® powder to prepare lemon flavour drink without black tea extract from Tresmontes Lucchetii S.A. de C.V. (México) were used. In all cases, 0.2 g of all samples were weighed, diluted to 10 mL with the mobile phase composed of 0.15% formic acid aqueous solution-0.25% DES 1:4 and methanol at a ratio of 80:20. Then, the samples were stirred in a vortex and before injection to HPLC system were filtered using a 0.2 µm nylon acrodisc from PALL (USA) and placed in amber glass vials. All analysis were performed for triplicate.

Results and discussion

Preparation and characterization of hydrophilic deep eutectic solvents

In this work, the hydrophilic DESs were synthetized by mixing different molar ratios of ChCl as HBA and Gly as HBD [31], only these compounds were used for the synthesis of DES. ChCl and Gly were selected because they have been described as forming DESs readily biodegradable [32]. For ChCl molar ratio was always 1, while of the Gly ratios were 1, 2, 3, and 4; both compounds were mixed under heating (70 °C) and constant stirring (800 rpm). The selected procedure for DESs synthesis is facile, one-step and thermal-assisted. Crystal formation was observed in the DES prepared at 1:1 molar ratio one hour after preparation. DESs obtained at 1:2, 1:3, and 1:4 ratios always were transparent and not crystal formation was observed. These results are congruent with reported in previous works, since they report the formation of crystals in the 1:1 ratio [30], while in another work they report an adequate formation of DESs in the other evaluated proportions [33]. Therefore, due to the obtained results the DES 1:1 was discarded for the following experiments.

The IR analysis is used to study the interactions between molecules of the DES and presence of the functional groups in their structures. The hydrogen bonding is the main interaction within eutectic mixtures. To prove the presence of this interaction between the molecules of different studied systems, IR spectra of the ChCl, Gly and synthetized DESs were obtained and shown in Fig. 1. As can be seen in Fig. 1a, for choline chloride, a wide band appears between 3,200 and 3,550 cm−1 due to O-H stretching of hydroxyl group. In addition, a weak band at 3,050–2,950 cml-1 attributable to the sp3 C-H stretching of carbon chains can be observed. Two intense bands can be seen between 3,050 and 3,200 cm−1 and 1,500–1,450 cm−1, which can be attributed to the C-H stretching of the methyl groups of the trimethyl ammonium ion and to the vibration deformation of the same group, respectively. Similarly, a strong band appears at about 950 cm−1, which is due to C-N stretching of the molecule [34, 35]. As shown in Fig. 1a, for glycerol, the intense and wide band at 3,290 cm−1 can be assigned to stretching vibration of the O-H functional group. The bands that appear to 2,950-2,850 cm−1 is due to C-H stretching of the molecular skeleton, while the three bands that appear between 1,150 and 950 cm−1 are from the C-O bond stretching [36]. On the other hand, in Fig. 1b, the spectra obtained from the DESs can be observed. The most interesting for DES spectra are the band of O-H group, which was broad and intense. This band shifted to higher wavenumbers (3,300–3,350 cm−1) and intensity increased as the Gly ratio increased, which evidence the enhancement hydrogen bonding interaction in eutectic mixture [37]. Other interesting bands between 1,100 and 1,500 cm−1 in the fingerprint region of all spectra of DESs, that be assigned to C-O and C-C-O stretching's and C-O-H bending vibrations were observed [37, 38].

Fig. 1.
Fig. 1.

IR spectrum of a) choline chloride (yellow line) and glycerol (blue line), and b) IR spectrum of DES 1:2 (black line), DES 1:3 (red line) and DES 1:4 (blue line)

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01055

Addition effect of DESs in the mobile phase

The reversed-phase high-performance liquid chromatography (RP-HPLC) has been commonly the analytical technique applied for phenolic acids separation. The PAs were easy retained on reversed phase columns, when the acidic mobile phase was used, because they are not ionizable [639, 40].

Within the working group, an analysis method for the seven PHAs was previously developed, using a Kinetex C18 column, a mobile phase composed of 0.15% formic acid aqueous solution and methanol (80:20), using a flow rate of 0.2 mL min−1 by isocratic mode and the column at 35 °C. Under these conditions, adequate separation of analytes was obtained, with the following elution order and retention times: GaA (4.56 min), PrA (6.78 min), CaA (13.57 min), SyA (15.74 min), CoA (24.74min), FeA (31.15 min) and SiA (35.05 min). However, for working group it is of particular interest to evaluate the use of DESs as additives in mobile phase. Therefore, it was decided to add the three synthesized DESs (ChCl:Gly 1:2, 1:3 and 1:4) to the aqueous component of the mobile phase at concentrations of 0.05, 0.15 and 0.25%, testing nine different mobile phases. The other analysis conditions were kept constant during development of the entire work. All analytical runs were carried out for 45 min by the effect that the analytes could experience in retention times.

During addition of DES at different concentrations, an adequate separation of the analytes was observed, a good baseline was obtained and no significant broadening or tailing of the peaks was observed. In general, when retention times are compared with obtained with only addition of formic acid, the most important effect was that for CoA, FeA and SiA retention times were shorter, while that for GaA, PrA, CaA and SyA practically eluted at the same time, as can be see in Fig. 2 plots.

Fig. 2.
Fig. 2.

Retention times depending on DESs addition at different concentrations: a) DES 1:2, b) DES 1:2 and c) DES 1:4

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01055

With the DES 1:2, to concentration of 0.25% yielded the shortest retention times for CoA, FeA and SiA, and no difference was found with concentrations of 0.05 and 0.15%. However, the retention times of this analytes were shorter than those obtained only with formic acid, as can be seen in Fig. 2a. While with DES 1:3 the shortest retention times were for SyA, CoA, FeA and SiA, compared to those found with only formic acid. But no differences were found in all analytes retention times to evaluated concentrations of DES, as can be seen Fig. 2b. For DES 1:4, the analytes SyA, CoA, FeA and SiA eluted faster at concentrations of 0.15 and 0.25%. While the retention times with 0.05% were similar those obtained when only formic acid was used, as can be seen in Fig. 2c. Although there were no great differences in the retention times, it was observed a tendency that as the concentration of DESs in the mobile phase increased and glycerol proportion in the DES, retention times decreased. Even the retention time of the analyte that elutes last (SiA) was shortened between 1 and 2.5 min (as can be seen in Table 1), which translates into a shorter analysis method without the need of a mobile phase gradient.

Table 1.

Retention times and tailing factors of phenolic acids in different mobile phases

Compound 0.15% formic acid aqueous solution and methanol 0.15% formic acid aqueous solution-0.25% DES 1:4 and methanol
t R a (minb ± SDc) TFd (value ± SD) t R (min ± SD) TF (value ± SD)
GaA 4.56 ± 0.006 1.50 ± 0.007 4.44 ± 0.009 1.40 ± 0.006
PrA 6.78 ± 0.005 1.33 ± 0.005 6.52 ± 0.012 1.37 ± 0.005
CaA 13.57 ± 0.015 1.35 ± 0.009 12.81 ± 0.018 1.10 ± 0.008
SyA 15.74 ± 0.025 1.33 ± 0.011 14.82 ± 0.030 1.12 ± 0.0012
CoA 24.74 ± 0.031 1.90 ± 0.013 23.23 ± 0.037 1.70 ± 0.013
FeA 31.15 ± 0.034 1.66 ± 0.015 29.15 ± 0.036 1.50 ± 0.012
SiA 35.05 ± 0.039 1.42 ± 0.02 32.54 ± 0.035 1.33 ± 0.010

a t R = Retention time.

b minutes.

c SD = standard deviation of three determinations.

d TF = tailing factor.

In the same way as in this work, a previous study has reported that retention parameters for some undissociated phenolic acids were shortened with the increase of the concentration of ionic liquids in the mobile phase. The authors mention that this phenomenon may be due to strength of eluent increases (by increase of ionic liquid concentration); because repulsion forces appear between analytes and a pseudo-stationary phase obtained in the column surface by addition of ionic liquids; and finally, the low content of organic solvent that causes non wetting of the stationary phase by the mobile phase and steric repulsion [41]. This probably explains what was observed in our experiments, because as the concentration of DESs increased, the retention times decreased. In addition, we work under conditions in which the analytes are not ionized, with a low content of organic solvent (20% methanol) and it is probable that small amount of DESs is deposited on the stationary phase.

On the other hand, a study focused on the optimization of chromatographic behavior of caffeic acid, shorter retention times were obtained after addition of a DES based on choline chloride and glycerol. The authors explain that this behavior may be since the components of the mobile phase play an ion-pair role, mainly because they did not control the pH of the mobile phase, therefore the analyte was ionized [27]. This cannot explain what happened in our work, because we work at acidic pH, the phenolic acids are not ionized and therefore cannot form the ion-pair.

In general, in this work only differences were found in retention times of some analytes when using the DESs. To find the best separation conditions of the phenolic acids, due to the changes that occurred in the retention times, a comparison of the resolution for the adjacent peaks was performed.

Resolutions (R s ) were calculated for the analytes with close retention times, such as the pairs gallic acid/protocatechuic acid, caffeic acid/syringic acid and ferulic acid/sinapic acid. Basically, as can be seen in Fig. 3 plot, the R s values found in all evaluated concentrations of DES 1:3 and DES 1:4 were higher than those found only with the use formic acid, which is indicative of a better separation when DESs are used. With DES 1:2 similar values of resolution were found with respect to use of formic acid.

Fig. 3.
Fig. 3.

Resolutions values depending on DESs addition at different concentrations

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01055

For pair gallic acid/protocatechuic acid, in all conditions evaluated, R s higher than 2 were found, being the lowest values when using the DES 1:2. While that for pair caffeic acid/syringic acid, R s values were lower than 2, finding values between 1.4 and 1.8, and the highest values were with DES 1:4. Instead for pair ferulic acid/sinapic acid, values higher than 2 were found in all conditions, except for 0.25% of DES 1:2 (R s = 1.94).

Regarding the lowest resolutions found, as mentioned above, these were for the pair caffeic acid/syringic acid. For this pair, with DES 1:2 at 0.25% was the condition that showed the lowest R s (1.45), even slightly lower than the found when only formic acid is used (R s = 1.48). On the other hand, the highest R s value was 1.79 with the DES 1:4 at the same concentration, this R s is higher than obtained only with formic acid (R s = 1.52). The literature mentions that when performing HPLC analysis, the satisfactory quantitation can be achieved with 1.5 > R s > 1, depending upon peak symmetry; the complete separation of chromatographic peaks, could be reached when R s = 2 or higher [42]. Therefore, taking account the obtained results regarding the lowest R s values, the most suitable (R s = 1.79) for correct quantification of caffeic acid and syringic acid was obtained with 0.25% of DES 1:4. Obtaining a higher Rs with 0.25% of DES 1:4 compared to the use of only 0.15% formic acid, is due to the improvement in the chromatographic peak shape after DES addition. Since with only formic acid, tailing factor (TF) of 1.35 for CaA and 1.33 for SyA were obtained; while in presence of DES the values were 1.10 and 1.12, respectively. Typically, a 0.9 < TF < 1.2 is a desirable performance of chromatographic method [43, 44]. Hence, it was decided to select the addition DES 1:4 at 0.25% as optimal condition for phenolic acids separation and at the same time guarantee the correct quantification of analytes. Under these conditions, the phenolic acids analysis was reached around two min earlier than when only formic acid was used, which reduces analysis time without to apply gradient elution. Figure 4 shows two chromatograms obtained under the conditions selected as optimal with the use of DESs and another in which only formic acid was used as additive.

Fig. 4.
Fig. 4.

Representative chromatograms obtained for a standard solution at 10 μg mL−1: standard solution without DES (blue line) obtained with mobile phase of solvent A (0.15% formic acid aqueous solution) and solvent B (methanol), and standard solution with 0.25% DES 1:4 (red line) obtained with mobile phase of solvent A (0.15% formic acid aqueous solution-0.25% DES 1:4) and solvent B (methanol). Both chromatograms were analyzed at 80:20 proportion, flow rate 0.2 mL min−1 with isocratic elution, injection volume of 5 µL, the column was maintained at 35 °C and detection to 295 nm

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01055

As can be see in Table 1, when retention times obtained with the formic acid mobile phase and the mobile phase with DES as additive are compared, it is observed that all retention times are shorter with DES addition, and this effect is more notable for FeA and SiA. Regarding the TF values, the results indicate that they are smaller with the presence of DES in the mobile phase, which means that the chromatographic peak shape is improved for all compounds. Therefore, DES 1:4 upgrade the chromatographic behavior of the selected phenolic acids, which translates into greater performance of the method. Furthermore, it is important to emphasize that in this research the simultaneous analysis of up to seven phenolic acids in presence of DES was evaluated, while other previously reported works for this class of compounds, only focused on the analysis of individual compounds such as caffeic acid [27] or quercetin [29].

Method validation

Method validation is a one of principal aspect that is evaluated of the analytical procedures to increase the level of confidence. In this sense, the proposed method was validated based on the International Conference on Harmonization (ICH) guidelines in terms of linearity, selectivity, precision, accuracy, limit of detection and limit of quantitation [45].

The linearity was expressed as correlation coefficient (R 2), considering values higher than 0.99. Calibration curves were constructed, and a good linear relationship was observed between the peak areas and corresponding concentration of the whole range of tested PAs was found. The least-squares regression analysis results are summarized in Table 2, the developed method was linear for all analytes, since R 2 values higher than 0.99 were obtained.

Table 2.

Validation parameters of developed method for quantitative analysis of selected phenolic acids

Compound λ (nm) Range (µg mL−1)a Equation R 2 LOD (µg mL−1) LOQ (µg mL−1)
GaA 272 0.05–20 y = 100,798 × −9,721 0.999 0.009 0.030
PrA 259 0.05–30 y = 122,673 × −5,714 0.999 0.013 0.043
CaA 325 0.05–30 y = 112,471 × −1,388 0.999 0.011 0.037
SyA 275 0.05–30 y = 73,322 × −3,752 0.999 0.012 0.040
CoA 309 0.05–30 y = 180,726 × −16,730 0.999 0.010 0.033
FeA 322 0.05–30 y = 126,839 × −10,972 0.999 0.011 0.037
SiA 323 0.1–30 y = 90,686 × −25,890 0.999 0.023 0.076

a Analyses were performed in triplicate for each set of analytes standard.

The selectivity of developed method for the simultaneous determination of PAs in powder to prepare lemon flavour drink enriched with black tea extract was evaluated, using a diode array detector (DAD). The DAD can provide absorbance and spectral data that can be used for quantitation and identification of chromatographic peaks [46]. In order to find and choice the most suitable wavelength for the analysis of target analytes, different wavelengths were tried with DAD detector of HPLC system between 211 and 400 nm, when standard solutions were analyzed. The best wavelength provides the higher chromatographic peak area and characteristic UV-spectrum of phenolic acids. The chromatograms of standard solutions, blank samples, and spiked samples of all analytes to different concentrations were compared. The chromatographic peak identity was confirmed by retention times and UV-spectrum obtained to specific wavelength of each phenolic acid. In Table 2, the wavelengths selected as optimal for the identification and quantification of each of the phenolic acids are shown. As can be seen in Fig. 5, the obtained results show that no interferences between analytes and matrix sample were found for all phenolic acids, except for GaA. Since in blank and spiked samples, a signal can be seen that overlaps with the GaA, chromatographic peak, which avoid its identification and subsequent quantification, therefore the precision and accuracy for this analyte could not be estimated. Consequently, the proposed method is selective for the other six phenolic acids, which can be successfully determined in the samples.

Fig. 5.
Fig. 5.

Representative chromatograms obtained for blank sample without added analytes (red line) and blank sample with added analytes at 8 μg mL−1 (green line). Both chromatograms were obtained with mobile phase of solvent A (0.15% formic acid aqueous solution-0.25% DES 1:4) and solvent B (methanol) at 80:20 proportion, flow rate 0.2 mL min−1 with isocratic elution, injection volume of 5 µL, the column was maintained at 35 °C and detection to 295 nm

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01055

The precision was performed in terms of repeatability (intra-day) on same day and reproducibility (inter-day) on three consecutive days, analysing the same three concentrations of the spiked samples for all analytes, under optimal conditions. Table 3 shows the %RSD for each analyte concentrations for all phenolic acids. The highest % RSD values were for inter-day precision, finding values between 1.24 and 2.94%, while for intra-day precision they were lower 0.96–2.59%. In both cases the highest values were for lowest concentration level. However, the values for %RSD were less than 3% for all analytes at evaluated concentrations, expressing a good precision.

Table 3.

Precision assessment of the proposed method for analysis of selected phenolic acids

Analyte Theorical concentration (µg mL−1) Intra-daya Inter-dayb
Calculated concentration mean ± SDc_ % RSDd Calculated concentration mean ± SDc % RSDd
Low level
PrA 0.5 0.52 ± 0.011 2.10 0.54 ± 0.015 2.85
CaA 0.53 ± 0.009 1.64 0.55 ± 0.010 1.83
SyA 0.51 ± 0.013 2.59 0.53 ± 0.016 2.94
CoA 0.53 ± 0.012 2.31 0.54 ± 0.014 2.65
FeA 0.54 ± 0.009 1.56 0.55 ± 0.012 2.16
SiA 0.55 ± 0.006 1.04 0.55 ± 0.010 1.87
Medium level
PrA 8.0 7.84 ± 0.101 1.29 7.90 ± 0.108 1.37
CaA 8.36 ± 0.114 1.37 8.32 ± 0.105 1.26
SyA 8.08 ± 0.093 1.15 8.11 ± 0.154 1.90
CoA 7.97 ± 0.086 1.08 8.01 ± 0.128 1.59
FeA 8.48 ± 0.086 1.02 8.20 ± 0.123 1.50
SiA 8.38 ± 0.101 0.96 8.08 ± 0.100 1.24
High level
PrA 25.0 24.65 ± 0.133 0.54 25.52 ± 0.157 0.62
CaA 25.87 ± 0.282 1.09 25.72 ± 0.300 1.17
SyA 25.39 ± 0.173 0.68 25.43 ± 0.241 0.95
CoA 25.53 ± 0.145 0.57 25.53 ± 0.336 1.32
FeA 24.81 ± 0.059 0.24 24.82 ± 0.173 0.70
SiA 25.36 ± 0.257 1.01 25.84 ± 0.278 1.07

a within the same day.

b three consecutive days.

c SD = standard deviation of three determinations.

d % RSD = relative standard deviation of three determinations.

To evaluate accuracy, a recovery test was carried out. The samples were added to three different concentrations of the target analytes covering the linear interval. Then, the recovery was calculated comparing the concentrations of analytes on the sample (measured value) and the spiked concentration on sample (known or theorical value). At low level, the lowest recoveries were for SyA and the highest for SiA, finding % R values of 101.56 and 110.67%, respectively. While than at medium level, the lowest were for PrA and the highest for FeA, finding values of 97.97 and 106.04%, respectively. On the other hand, for the high level, the lowest %R were for PrA with 98.59% and the highest for FeA with 102.12%. In general, all recoveries were between 99.97 and 109.00%, as can be seen in Table 4. These results indicate an adequate accuracy and therefore a good quantification of phenolic acids.

Table 4.

Accuracy assessment of the proposed method for analysis of selected phenolic acids

Analyte Theorical concentration (µg mL−1) Calculated concentration
Mean ± SDa % R b
Low level
PrA 0.5 0.52 ± 0.011 104.12
CaA 0.53 ± 0.009 106.63
SyA 0.51 ± 0.013 101.56
CoA 0.53 ± 0.012 105.98
FeA 0.54 ± 0.009 109.00
SiA 0.55 ± 0.006 110.67
Medium level
PrA 8.0 7.84 ± 0.101 97.97
CaA 8.36 ± 0.114 104.49
SyA 8.08 ± 0.093 100.96
CoA 7.97 ± 0.086 99.58
FeA 8.48 ± 0.086 106.04
SiA 8.38 ± 0.101 104.92
High level
PrA 25.0 24.65 ± 0.133 98.59
CaA 25.87 ± 0.282 103.49
SyA 25.39 ± 0.173 101.55
CoA 25.53 ± 0.145 102.12
FeA 24.81 ± 0.059 99.23
SiA 25.36 ± 0.257 101.43

a SD = standard deviation of three determinations.

b % R = average recovery of three determinations.

The LOD and LOQ under optimal chromatographic conditions were determined by signal-to-noise (S/N) ratio of 3 and 10, respectively. Table 2, shown the LOD and LOQ for each compound. The found values were in the range of 0.009–0.023 mg mL−1 for the LOD and 0.030–0.076 mg mL−1.

Method applicability

In order to demonstrate the applicability of the developed method for the separation and determination of phenolic acids, three different samples were analyzed by triplicate using the validated methodology. The samples were powder to prepare lemon flavour drink enriched with black tea extract of three different brands marketed at Monterrey, Nuevo León, México. The experimental results are displayed in Table 5. As can be seen on this table, the phenolic acids PrA, CaA, SyA, CoA, and FeA were found in the ZUKO® and TANG® samples, while in the NESTEA® sample only PrA and CoA were found. ZUKO® and TANG®, presented the same acids, for both cases the most abundant was CoA and the least abundant was FeA, finding values of 11.09 mg kg−1 and 5.89 mg kg−1, respectively. On the other hand, the least abundant in both cases was FeA, presenting values of 1.08 mg kg−1 for ZUKO® and 0.85 mg kg−1 for TANG®. While for NESTEA®, PrA and CoA were found at levels of 4.10 and 2.06 mg kg−1, respectively. Figure 6 shows some representative chromatograms obtained from sample analysis.

Table 5.

Results of determination of selected phenolic acids in powder to prepare lemon flavour drink enriched with black tea extract

Sample brand Analyte Mean calculated concentration (mg kg−1) SDa % RSDb
ZUKO® PrA 5.37 0.18 3.36
CaA 2.12 0.06 2.71
SyA 1.32 0.06 4.26
CoA 11.07 0.42 3.76
FeA 1.08 0.05 4.74
NESTEA® PrA 4.10 0.09 2.17
CoA 2.06 0.06 3.01
TANG PrA 3.54 0.08 2.20
CaA 1.21 0.04 3.19
SyA 1.19 0.04 3.30
CoA 5.89 0.25 4.30
FeA 0.85 0.03 3.38

a SD = standard deviation of three determinations.

b % RSD = relative standard deviation of three determinations.

Fig. 6.
Fig. 6.

Representative chromatograms obtained for analyzed samples: ZUKO® (red line), NESETEA® (green line) and TANG® (blue line). Chromatograms were obtained with mobile phase of solvent A (0.15% formic acid aqueous solution-0.25% DES 1:4) and solvent B (methanol) at 80:20 proportion, flow rate 0.2 mL min−1 with isocratic elution, injection volume of 5 µL, and the column was maintained at 35 °C as column temperature and detection to 295 nm. Sinapic acid (SiA) was not found in any of the samples tested

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01055

The results indicate that there are differences in the amount and number of phenolic acids between brands of powdered drinks enriched with tea extracts. A slight shift in analyte retention times was seen, however, the identity of all analytes was confirmed by their UV spectrum with the DAD detector and its comparison with the corresponding standard. The samples are from different manufacturers, making each matrix different. This may be the cause of the shifting in retention times, since each sample probably has different ingredients, such as sweeteners, thickeners, colorants, among other compounds; that can interact with the column and target analytes, contributing to deviations in chromatographic behavior [47, 48]. Nevertheless, according to our results, the new method allowed the determination of the phenolic acids in the powdered flavour drinks without the need for a previous extraction or pre-concentration step.

Conclusions

This work presents the simultaneous separation of seven phenolic acids (caffeic, coumaric, gallic, ferulic, protocatechuic, sinapic, and syringic acid) in a new RP-HPLC isocratic method using an acidic mobile phase with a deep eutectic mixture of choline chloride and glycerol as additive. The series of DESs used as additives, were synthesized through a fast, safe, and simple process. The DESs were prepared to ratio of 1:2, 1:3 and 1:4 of choline chloride and glycerol, respectively. These eutectic mixtures were characterized by infrared spectroscopy, where the existence of hydrogen bonds between its components was evidenced. When DESs, are added to mobile phase, generally, the retention times were shortened with the increase of DES concentration (0.05, 0.15 and 0.25%), especially for analytes coumaric, ferulic, and sinapic acid. The observed results show that the best separation conditions for the seven target compounds were obtained by adding 0.25% of DES 1:4, since under this condition the most adequate resolution for quantification of the target analytes was found. Due to the interest of establishing the identity or quantity of phenolic acids present in various samples, the proposed method was validated, and proved to be linear, precise and accuracy. The method was not selective for gallic acid, therefore, this analyte could not be identified or quantified in the analyzed samples. For method applicability evaluation, the selected samples were powder to prepare flavoured beverages enriched with black tea extract. The application was successful, since in two of the samples, the phenolic acids found were caffeic, coumaric, ferulic, protocatechuic, and syringic acid, while in the other sample only protocatechuic and coumaric acid were found. The retention times reduction without use of gradient elution and employ of DAD detection, contribute to method simplicity, and expand its applicability. Furthermore, aqueous mobile phase with DES as additives can be used to improve the chromatographic behavior of molecules like those studied in this work or inclusive other pharmaceutical compounds. In this work it was shown that adding DES as additives to mobile phase increases of the separation performance by decreasing the retention times of the molecules and increasing efficiency parameters such as resolution. On the other hand, ChCl-Gly based deep eutectic solvents emerge as a cheaper, ecological and easy-to-prepare new alternative to ionic liquids when they are used as additives to the mobile phase. The proposed method is simple and therefore, can be conducted on traditional HPLC systems, which allows its routine use in assay labs.

Acknowledgment

This research has been supported by UANL-PAICYT 2021 funds (project number: SA1911-21). The authors acknowledge the participation of students Paola Cobos Cervantes and Mónica Díaz Hernández in the development of this work.

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Senior editors

Editor(s)-in-Chief: Kowalska, Teresa

Editor(s)-in-Chief: Sajewicz, Mieczyslaw

Editors(s)

  • Danica Agbaba (University of Belgrade, Belgrade, Serbia)
  • Ivana Stanimirova-Daszykowska (University of Silesia, Katowice, Poland)
  • Monika Waksmundzka-Hajnos (Medical University of Lublin, Lublin, Poland)

Editorial Board

  • R. Bhushan (The Indian Institute of Technology, Roorkee, India)
  • J. Bojarski (Jagiellonian University, Kraków, Poland)
  • B. Chankvetadze (State University of Tbilisi, Tbilisi, Georgia)
  • M. Daszykowski (University of Silesia, Katowice, Poland)
  • T.H. Dzido (Medical University of Lublin, Lublin, Poland)
  • A. Felinger (University of Pécs, Pécs, Hungary)
  • K. Glowniak (Medical University of Lublin, Lublin, Poland)
  • B. Glód (Siedlce University of Natural Sciences and Humanities, Siedlce, Poland)
  • A. Gumieniczek (Medical University of Lublin, Lublin, Poland)
  • U. Hubicka (Jagiellonian University, Kraków, Poland)
  • K. Kaczmarski (Rzeszow University of Technology, Rzeszów, Poland)
  • H. Kalász (Semmelweis University, Budapest, Hungary)
  • K. Karljiković Rajić (University of Belgrade, Belgrade, Serbia)
  • I. Klebovich (Semmelweis University, Budapest, Hungary)
  • A. Koch (Private Pharmacy, Hamburg, Germany)
  • Ł. Komsta (Medical University of Lublin, Lublin, Poland)
  • P. Kus (Univerity of Silesia, Katowice, Poland)
  • D. Mangelings (Free University of Brussels, Brussels, Belgium)
  • E. Mincsovics (Corvinus University of Budapest, Budapest, Hungary)
  • Á. M. Móricz (Centre for Agricultural Research, Budapest, Hungary)
  • G. Morlock (Giessen University, Giessen, Germany)
  • A. Petruczynik (Medical University of Lublin, Lublin, Poland)
  • R. Skibiński (Medical University of Lublin, Lublin, Poland)
  • B. Spangenberg (Offenburg University of Applied Sciences, Germany)
  • T. Tuzimski (Medical University of Lublin, Lublin, Poland)
  • Y. Vander Heyden (Free University of Brussels, Brussels, Belgium)
  • A. Voelkel (Poznań University of Technology, Poznań, Poland)
  • B. Walczak (University of Silesia, Katowice, Poland)
  • W. Wasiak (Adam Mickiewicz University, Poznań, Poland)
  • I.G. Zenkevich (St. Petersburg State University, St. Petersburg, Russian Federation)

 

KOWALSKA, TERESA
E-mail: kowalska@us.edu.pl

SAJEWICZ, MIECZYSLAW
E-mail:msajewic@us.edu.pl

Indexing and Abstracting Services:

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2021  
Web of Science  
Total Cites
WoS
652
Journal Impact Factor 2,011
Rank by Impact Factor Chemistry, Analytical 66/87
Impact Factor
without
Journal Self Cites
1,789
5 Year
Impact Factor
1,350
Journal Citation Indicator 0,40
Rank by Journal Citation Indicator Chemistry, Analytical 72/99
Scimago  
Scimago
H-index
29
Scimago
Journal Rank
0,27
Scimago Quartile Score Chemistry (miscellaneous) (Q3)
Scopus  
Scopus
Cite Score
2,8
Scopus
CIte Score Rank
General Chemistry 210/409 (Q3)
Scopus
SNIP
0,586

2020
 
Total Cites
650
WoS
Journal
Impact Factor
1,639
Rank by
Chemistry, Analytical 71/83 (Q4)
Impact Factor
 
Impact Factor
1,412
without
Journal Self Cites
5 Year
1,301
Impact Factor
Journal
0,34
Citation Indicator
 
Rank by Journal
Chemistry, Analytical 75/93 (Q4)
Citation Indicator
 
Citable
45
Items
Total
43
Articles
Total
2
Reviews
Scimago
28
H-index
Scimago
0,316
Journal Rank
Scimago
Chemistry (miscellaneous) Q3
Quartile Score
 
Scopus
393/181=2,2
Scite Score
 
Scopus
General Chemistry 215/398 (Q3)
Scite Score Rank
 
Scopus
0,560
SNIP
 
Days from
58
submission
 
to acceptance
 
Days from
68
acceptance
 
to publication
 
Acceptance
51%
Rate

2019  
Total Cites
WoS
495
Impact Factor 1,418
Impact Factor
without
Journal Self Cites
1,374
5 Year
Impact Factor
0,936
Immediacy
Index
0,460
Citable
Items
50
Total
Articles
50
Total
Reviews
0
Cited
Half-Life
6,2
Citing
Half-Life
8,3
Eigenfactor
Score
0,00048
Article Influence
Score
0,164
% Articles
in
Citable Items
100,00
Normalized
Eigenfactor
0,05895
Average
IF
Percentile
20,349
Scimago
H-index
26
Scimago
Journal Rank
0,255
Scopus
Scite Score
226/167=1,4
Scopus
Scite Score Rank
Chemistry (miscellaneous) 240/398 (Q3)
Scopus
SNIP
0,494
Acceptance
Rate
41%

 

Acta Chromatographica
Publication Model Online only
Gold Open Access
Submission Fee none
Article Processing Charge 400 EUR/article
Regional discounts on country of the funding agency World Bank Lower-middle-income economies: 50%
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Acta Chromatographica
Language English
Size A4
Year of
Foundation
1992
Volumes
per Year
1
Issues
per Year
4
Founder Institute of Chemistry, University of Silesia
Founder's
Address
PL-40-007 Katowice, Poland, Bankowa 12
Publisher Akadémiai Kiadó
Publisher's
Address
H-1117 Budapest, Hungary 1516 Budapest, PO Box 245.
Responsible
Publisher
Chief Executive Officer, Akadémiai Kiadó
ISSN 2083-5736 (Online)

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