A capillary electrophoresis method to determine aliphatic organic acids in bracatinga honeydew honey and floral honey

Journal of Food Composition and Analysis 82 (2019) 103243

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Original Research Article

A capillary electrophoresis method to determine aliphatic organic acids in bracatinga honeydew honey and floral honey

T

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Patricia Brugnerotto , Fabiana Della Betta, Luciano Valdemiro Gonzaga, Roseane Fett, ⁎ Ana Carolina Oliveira Costa Department of Food Science and Technology, Federal University of Santa Catarina, Florianopolis, SC, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Analytical validation Apis mellifera Capillary electrophoresis Gluconic acid Honey composition Lactic acid Organic acids Peakmaster®

Aliphatic organic acids (AOA) make a strong contribution to flavor, taste, acidity, pH and electrical conductivity. In this paper, a new and fast capillary electrophoresis method was developed and validated to determine 14 AOA simultaneously in bracatinga (Mimosa scabrella Bentham) honeydew honey and floral honey. Separation of fourteen compounds occurred in 9 min using glyoxylic acid as an internal standard, and the background electrolyte was comprised of phthalic acid (20 mmol L−1,) Tris (14 mmol L−1), CTAB(1.6 mmol L−1), CaCl2 (1 mmol L−1), pH 3.3. The performance and fitness-for-purpose of this method were assessed based on the results obtained for the parameters: system suitability, linearity, matrix effects, selectivity, precision, accuracy, detection and quantification limits, and robustness. Gluconic acid was the main acid in both kinds of honey. Malonic and glycolic acids were quantified in honeydew honey and floral honey for the first time, while lactic acid was quantified for the first time in honeydew honey.

1. Introduction Apis mellifera can produce honey from the nectar of flowers (floral honey), secretions of living parts of plants or excretions of plant-sucking insects on the living parts (honeydew honey) (European Comission, 2002). One type of honeydew honey that has a large occurrence in the southern Brazilian states of Paraná, Santa Catarina and Rio Grande do Sul is bracatinga (Mimosa scabrella Bentham) honeydew honey. This honey is produced from the excretions of plant-sucking insects (Tachardiella sp. or Stigmacoccus paranaensis Foldi) that infest the bracatinga (Mimosa scabrella Bentham), in months of scarcity of nectar and pollen, usually between January and June in biannual periods (Mazuchowski et al., 2014; Wolff et al., 2015). The distinct origin of honeys (nectar or honeydew) causes dissimilarity in the chemical composition of these products, mainly due to the presence of approximately 200 chemical compounds in honeys (Da Silva et al., 2016; Seraglio et al., 2019). Despite the small number of studies about aliphatic organic acids (AOA) in honey, these compounds, although comprising a small proportion, are very important because they make a strong contribution to the honey stability, conservation, physical, chemical and sensorial properties (Da Silva et al., 2016; Mato et al., 2003; Navarrete et al., 2005; Tezcan et al., 2011). AOA origin in honeys is not fully known, although many of them

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may be natural intermediates in the metabolic pathways of microorganisms, of Krebs cycle (acids: citric, succinic, glutaric, fumaric and oxaloacetic) or enzymatic reactions. They can also be synthesized from glucose, fructose and sucrose from nectar, by the enzymatic action of bees, or may come directly from plant secretion and also from plantsucking insects’ excretion (Mato et al., 2003; Yin et al., 2015). Diverse techniques coupled to distinct detectors can be used to evaluate AOA in fruits and vegetables (Flores et al., 2012), in beverages including juices and wines (Mato et al., 2006a), in cheese (Zeppa et al., 2001), in dairy products (Tormo and Izco, 2004), in coffee (Galli and Barbas, 2004), in soil (Baziramakenga et al., 1995), in urine (Awad et al., 2019; Tůma et al., 2011) and in honeys (Haroun et al., 2012; Sanz et al., 2005). To evaluate AOA composition in honey, high-performance liquid chromatography is most often applied (Haroun et al., 2012; Nozal et al., 2003a; Suárez-Luque et al., 2002). However other reported methods used gas chromatography coupled with mass spectrometry (Sanz et al., 2005) and capillary electrophoresis (Mato et al., 2006b; Tezcan et al., 2011). The use of methods based on capillary electrophoresis (CE) is interesting, because this is a versatile analytical technique, capable of detecting compounds with low molar absorptivity, such as AOA. This kind of compound can be analyzed by indirect mode with a short time of analysis, using an environmentally friendly technique (Kaljurand and

Corresponding authors. E-mail addresses: [email protected] (P. Brugnerotto), [email protected] (A.C. Oliveira Costa).

https://doi.org/10.1016/j.jfca.2019.103243 Received 5 April 2019; Received in revised form 17 June 2019; Accepted 19 June 2019 Available online 20 June 2019 0889-1575/ © 2019 Published by Elsevier Inc.

Journal of Food Composition and Analysis 82 (2019) 103243

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temperature (25 ± 2 °C) and stored in the freezer (−20 ± 2 °C) until analysis. For analysis, each sample was thawed, homogenized and five mixtures of honeydew (n = 5) and floral honey (n = 5) were prepared. An aliquot of 0.3 g of each honey (three independent replicates) was placed in a 2-mL polypropylene microtube and 1.2 g of ultrapure water was added. The samples were stirred using a vortex stirrer (Fisatom, Sao Paulo, Brazil) for 1 min and centrifuged (MiniSpin® plus) at 9861 g for 8 min. Aliquots of the supernatant were transferred to microtubes and dilutions were performed for each sample. Considering the different concentrations of analytes in the samples and linearity of the method, three dilutions were performed for each sample: 1:5; 1:10 and 1:100 (w/w) for floral honey and 1:10; 1:30 and 1:150 (w/w) for bracatinga honeydew honey. The resulting solutions were diluted in the ratio 9:1 (v/v) with I.S. (final concentration 14.8 mg L−1) for subsequent injection into the CE system.

Koel, 2011). Despite these advantages, the determination of AOA with CE methods in honeys is very limited, with few studies exploring these methods (Mato et al., 2006b; Navarrete et al., 2005; Tezcan et al., 2011). The composition of honeys (bracatinga honeydew and/or floral honeys) from Santa Catarina (Brazil) state has been studied in several works, including the physicochemical characteristics (Bergamo et al., 2019), amino acids (Azevedo et al., 2017a), proteins (Azevedo et al., 2017b), phenolic compounds (Seraglio et al., 2016; da Silva et al., 2019), minerals (Bergamo et al., 2018; Rizelio et al., 2012a), and carbohydrates (Rizelio et al., 2012b). However, to this date, the AOA composition was not studied in these matrices. In this context, this study aimed to develop a method for the determination of AOA using CE. The analytical method was developed, validated and applied to the determination of AOA in samples of bracatinga (Mimosa scabrella Bentham) honeydew honey and floral honey from Santa Catarina state.

2.3. CE analysis 2. Material and methods The experiments were conducted using a capillary electrophoresis system (model 7100, Agilent Technologies, Santa Clara, CA), equipped with a diode array detector, set at 230 nm (indirect detection, with a reference at 360 nm for peak inversion), and with the temperature control device maintained at 20 °C. The acquisition and data treatment were done using HP ChemStation® software (rev A.06.01). Separations were performed using an uncoated fused silica capillary (Polymicro Technologies, Phoenix, AZ) with dimensions of 60.5 cm total length, 52 cm effective length, 75 μm inner diameter and 375 μm outer diameter. Before the first run, the capillary was conditioned by flushing 1 mol L−1 NaOH (30 min) and water (30 min). At the beginning of each day the capillary was also conditioned, this time by flushing 1 mol L−1 NaOH (15 min) followed by deionized water (15 min) and BGE (30 min). In between runs, the capillary was reconditioned with the BGE solution (3 min flush). At the end of each working day, the capillary was rinsed using 1 mol L−1 NaOH (15 min) and water (15 min). The standards and samples were introduced into the capillary at the end farthest from the detector (inlet) with a hydrodynamic pressure of 50 mbar for 3 s. The applied separation voltage was 15 kV, with negative polarity on the inlet.

2.1. Chemicals and preparation of standard solution Maleic, malonic, fumaric, tartaric, formic, citric, malic, glycolic, lactic, gluconic, glutaric, succinic, acetic, propionic, oxalic, aspartic, phthalic, glyoxylic, tris(hydroxymethyl)aminomethane (Tris) and cetyltrimethylammonium bromide (CTAB) were purchased from SigmaAldrich (St. Louis, MO); calcium chloride was purchased from Vetec (Rio de Janeiro, RJ, Brazil); sodium hydroxide and methanol were purchased from Neon (São Paulo, SP, Brazil). Standard stock solutions of fumaric and glutaric acids were prepared in methanol, and all others were prepared in ultrapure water (Milli-Q®, Millipore, Bedford, MA) and stored at 4 ± 2 °C until the analysis. The working concentration levels (linear range) are shown in Table 1. The background electrolyte (BGE) was composed of 20 mmol L−1 phthalic acid, 14 mmol L−1 Tris, 1.6 mmol L−1 CTAB, and 1 mmol L−1 CaCl2 at pH 3.3. An internal standard (I.S.) glyoxylic acid was used (final concentration 14.8 mg L−1). 2.2. Samples and sample preparation

2.4. Method validation

Bracatinga (Mimosa scabrella Bentham) honeydew honey (n = 5) and floral (n = 5) samples, produced by Apis mellifera, were obtained directly from beekeepers from five distinct geographic regions (Lages, São Joaquim, Urubici, Urupema, and Bom Retiro) of Santa Catarina state during the harvest of 2016. Samples were transported at room

The system suitability, linearity, matrix effect, selectivity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ) and robustness were evaluated, to confirm the fitness-for-purpose of this

Table 1 Analytical performance of the optimized CE method: linear range (mg L−1), regression equation, linear determination coefficient (R2), detection and quantification limits evaluated in standard and matrix solutions for 14 aliphatic organic acids (AOA). AOA

maleic malonic fumaric tartaric formic citric malic glycolic lactic gluconic succinic glutaric acetic propionic

linear range (mg L−1)

3.2–64 2.9–58 3.2–64 4.2–83 1.3–25 5.3–107 3.7–74 2.1–42 2.5–50 6.1–121 3.3–66 3.7–73 1.7–33 2.1–41

regression equation

y = 1.957x y = 4.700x y = 1.963x y = 6.280x y = 2.704x y = 6.309x y = 5.842x y = 4.471x y = 3.980x y = 4.895x y = 5.085x y = 5.452x y = 3.965x y = 4.112x

– 0.008 – 0.003 + 0.014 – 0.005 – 0.004 – 0.044 + 0.001 + 0.015 + 0.025 + 0.013 + 0.019 + 0.020 + 0.020 + 2x10−5

R2

0.999 0.999 0.998 0.999 0.995 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999

2

standard solution

matrix solution

LOD (mg L−1)

LOQ (mg L−1)

LOD (mg L−1)

LOQ (mg L−1)

0.71 0.12 0.72 0.30 0.21 0.52 0.60 0.09 0.17 0.30 0.34 0.07 0.20 0.19

2.38 0.39 2.42 1.01 0.70 1.72 1.99 0.30 0.56 1.00 1.14 0.25 0.68 0.62

1.12 0.99 1.30 0.77 0.40 1.17 1.17 0.29 0.23 0.74 0.53 0.30 0.48 0.48

3.72 3.30 4.34 2.57 1.32 3.90 3.91 0.96 0.77 2.46 1.78 0.98 1.61 1.60

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2.5. Method applicability

method (Eurachem, 2014; Faria et al., 2008). Before the analytical validation, the suitability of the CE equipment to provide reliable data was assessed, by considering the relative standard deviation (RSD) of the parameters corrected peak area (area(analyte)/area(I.S.)) and the corrected migration time (time(analyte)/ time(I.S.)), which were obtained from the seventh point of the calibration curve mean value for 10 consecutive injections. Linearity was evaluated using standard solution and matrix calibration curves, built at seven equally spaced concentration levels (1.3–121 mg L−1) in three independent replicates. Each replicate was prepared on each day of analysis and samples of each concentration were randomly tested. The matrix calibration curves (floral and bracatinga honeydew honey) were built using standard additions. The linearity of the standard solution and matrix calibration curves was assessed by the ordinary least squares method (OLSM). Residual plots were examined for clear patterns and the presence of discrepant points (outliers). After visual identification, the possible outliers were tested by applying the Grubbs test (Grubbs, 1969), and none of them was confirmed as a outlier. The regressions were also evaluated for possible violation of the assumptions: normality by Shapiro-Wilk test (Shapiro and Cochran, 1965); homoscedasticity by Cochran test (Cochran, 1941); independence by Durbin-Watson test (Durbin and Watson, 1951); and lack of fit by F-test (Snedecor and Cochran, 1989). The matrix effect was evaluated by the comparison of slopes obtained for the aqueous and matrix solutions calibration curves. The ttest was used for each analyte, testing the significant difference, considering a 99% confidence level. Selectivity was evaluated experimentally, by analyzing the possible interference of some anions (oxalic acid and aspartic acid) on the 14 desired AOA separation under separation conditions previously described in Section 2.3. The intra-day precision was tested by injecting on the same day three independent replicates of the analytes at three distinct concentration levels. The inter-day precision was determined by injection results using the same three concentration levels in three independent replicates over three different days. In intra- and inter-day precision, the concentration of I.S. was the same in all replicates. The results were expressed as % RSD for two parameters, the corrected migration time and the corrected peak area. The accuracy was obtained using the recovery of fortified samples. The samples of floral and bracatinga honeydew honey were prepared in three independent replicates, spiked at three concentration levels within the range used for the calibration curves. The LOD and LOQ limits were determined using the standard and matrix solutions and were calculated as described by Faria, Souza, and Oliveira (2008) using the equations

LOD =

3 × Sb × Cs Hmax − Hmin

(1)

LOQ =

10 × Sb × Cs Hmax − Hmin

(2)

After validation, the proposed CE method was successfully applied to determine maleic, malonic, fumaric, tartaric, formic, citric, malic, glycolic, lactic, gluconic, succinic, glutaric, acetic and propionic acids in five samples of bracatinga (Mimosa scabrella Bentham) honeydew honey and in five samples of floral honey from five distinct geographic locations (Lages, São Joaquim, Urubici, Urupema, and Bom Retiro) across the Santa Catarina (Brazil) state. 2.6. Statistical analysis In order to increase the reliability of results, all statistical tests were performed at 99% confidence level. The verification of the assumptions of normality (Shapiro-Wilk test) and homoscedasticity (Cochran test) was performed by using Assistat 7.7 (free software), while the tests of independence (Durbin-Watson test) and lack-of-fit were accomplished using the software Statistic 13.0. All quantification analysis was performed in three independent replicates and the results were reported as mean ± standard deviation. 3. Results and discussion 3.1. CE method The selection of a suitable BGE for simultaneous determination of 14 AOA was achieved using simulations on the Peakmaster® software version 5.3, which allowed reduction of the number of experiments and unnecessary reagent consumption on the development phase. Since AOA have low molar absorptivity in the UV–Vis region, all simulations were carried out using indirect detection mode. Taking into account the curve of effective mobility versus pH and the pKa of the studied AOA, that range from 1.94 to 4.43, it was verified that the best separation condition was at pH 3.3, a condition in which all analytes are partially ionized. Phthalic acid was selected as a BGE component, since it can act as co-ion (having the same analyte charge), has a chromophore in the UV–vis region (230 nm) and presents mobility close to the analytes’ mean mobility, which minimizes the electromigration dispersion (EMD), one of the causes of the zones enlargement. The concentration of the co-ion was defined through simulations using Peakmaster® software as 20 mmol L−1 (data not shown). After the co-ion concentration was set, the counter-ion concentration (Tris) was simulated (Peakmaster®) between 2 and 16 mmol L−1 (2 mmol L−1 intervals), and the best condition was found to be 14 mmol L−1 (data not shown). At this concentration, the requirements of a good BGE to separate the anions are attended to: low conductivity (0.087 S cm−1), low ionic strength (17.7 mmol L−1) and buffer capacity (11.1 mmol L−1). AOA are cyclic or acyclic compounds of low molecular mass (45–220 g mol−1), water-soluble compounds, containing one or more carboxylic acid groups, which can be classified as monocarboxylic, dicarboxylic or tricarboxylic (Galli et al., 2003; Tůma et al., 2011). Most of the AOA are slow analytes, having similar chemical structure and electrophoretic mobility, which can result in co-migration and/or long separation time, especially at acidic pH, where the electroosmotic flow (EOF) is almost negligible. In this regard, a cationic surfactant (CTAB) was added to the BGE at 1.6 mmol L−1, acting as a flow inverter, to ensure that all analytes reach the detector faster. Although the strategies mentioned above were used to ensure adequate resolution and peak efficiency, lactic and gluconic acids still showed co-migration. Therefore, calcium chloride (CaCl2) was added at a 1 mmol L−1 in the BGE, since some inorganic salts can selectively complex with analytes, changing their mobility and improving the separation (Mato et al., 2006b). By using BGE, the resolution was improved, especially between lactic and gluconic acids.

where Sb is the standard deviation of the baseline, Cs the concentration of the analyte, Hmax is the maximum peak height and Hmin the minimum peak height. To assess the robustness of the method, the Youden test (Youden and Steiner, 1975) was performed. For this test, small variations in the parameters voltage separation (−15 and −15.5 kV); pH of the BGE (3.3 and 3.4); cartridge temperature (20 and 21 °C); injection pressure (50 and 49 mbar); wavelength (230 and 227 nm); flush time between runs (120 and 117 s); and injection time (3 and 4 s) were compared. Samples and standards were analyzed under the nominal and altered conditions to determine the influence of these variations on performance parameters. The selected performance parameters were analyte concentration (mmol L−1), corrected peak area, corrected migration time, peak symmetry and resolution. 3

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Fig. 1. Electropherogram of an aliphatic organic acid standard solution: (1) maleic (2) malonic, (3) fumaric (4) tartaric, (I.S.) glyoxylic, (5) formic, (6) citric, (7) malic, (8) glycolic, (9) lactic, (10) gluconic, (11) succinic, (12) glutaric, (13) acetic, (14) propionic.

succinic acids, respectively (floral honey). These results were acceptable (AOAC, 2012; Bruce et al., 1998) and confirmed the accuracy of the method.

The voltage of 15 kV (negative polarity) was used to generate low electrical current during the analyses, in order to reduce undesirable interferences and deformation of peaks caused by the Joule effect, improving the resolution of the analytes. The control of the capillary temperature is an important practice to ensure repeatability of separations. In this regard, the temperature was set as 20 °C. The chosen internal standard (I.S.) was glyoxylic acid, which is an anion not present in the samples with electrophoretic mobility close to the analytes’ mean mobility. Fig. 1 shows an electropherogram of the 14 analytes obtained under optimum conditions.

3.2.5. Detection and quantification limits For the standard solution, the LOD values varied from 0.07 to 0.72 mg L−1 and the LOQ varied between 0.25 and 2.42 mg L−1, for glutaric and fumaric acids, respectively. For the matrix solution, the LOD values ranged between 0.30 and 1.30 mg L−1 for glutaric and fumaric acids and the LOQ varied from 0.77 to 4.34 mg L−1 for lactic and fumaric acids. The LOD and LOQ were also evaluated in the matrix solution, because this case represents accurately the real limits when compared with the ones obtained using the aqueous solution. The results are presented in Table 1.

3.2. Method validation 3.2.1. System suitability For the corrected migration time the RSD ranged from 0.01% for fumaric, tartaric and formic acids to 0.15% for maleic and glycolic acids. The corrected peak area varied from 1.16% for gluconic acid to 2.65% for formic acid. All RSD values were below 2.7%, attesting to the suitability of the CE system for the obtainment of accurate and reproducible data.

3.2.6. Robustness The method robustness was evaluated for all desired AOA, with the results of five parameters being monitored, while small changes were made in the other seven parameters (previously described in Section 2.4). The monitored parameters were corrected area, corrected migration time, concentration, symmetry, and resolution, while the manipulated parameters were injection time, wash time, wavelength, pressure, temperature, pH and voltage. The test showed that the variations in the monitored parameters were not sufficient to affect the performance of the method, demonstrating its robustness.

3.2.2. Linearity Linearity was verified for the standard solution calibration curves of all analytes in the working range (Table 1), with all the curves presenting acceptable determination coefficients (R2 > 0.99), and the residual plots profile showed no pattern, being normally distributed, homoscedastic, independent and not presenting lack-of-fit (p > 0.01).

3.3. Method application

3.2.3. Matrix effect Linearity was also evaluated in the matrix calibration curves of all tested parameters, with an R2 > 0.99, the absence of outliers, homoscedasticity, residuals independence, and no lack-of-fit, verifying that the method is linear. The t-test was used to evaluate the matrix effect, by the comparison of both curves’ slopes. For all AOA, both matrices (honeydew and floral honeys) did not interfere with the analytical signal, since, the calculated t was less than the tabulated t. With no matrix effect, the AOA quantification in the samples was performed using the calibration curve obtained for aqueous solution.

3.3.1. Bracatinga honeydew honeys This is the first report of the determination of AOA in bracatinga (Mimosa scabrella Bentham) honeydew honey. All the investigated AOA were identified and quantified, except for fumaric and tartaric acids. Fig. 2 shows the electropherograms of the AOA separation obtained from bracatinga honeydew honey (Urubici geographical region). The AOA quantification results are summarized in Table 2. The AOA levels ranged between 40.9 mg 100 g−1 for maleic acid (sample from Lages) and 7415 mg 100 g−1 for gluconic acid (sample from Urupema). The gluconic and lactic acids were the major AOA in the bracatinga honeydew honey samples, presenting concentrations between 4478 and 7415 mg 100 g−1 for gluconic acid and between 931 and 1543 mg 100 g−1 for lactic acid. Normally, gluconic acid is the main AOA in honeys. This acid can be obtained by the conversion of D-glucose by the action of D-glucose oxidase. This enzyme comes from the hypopharyngeal gland of Apis mellifera (Cherchi et al., 1994; Seraglio et al., 2019). Part of the gluconic acid found in honeys may have bacterial origin. Gluconobacter spp. bacteria are present in the bee´s gut and also during the maturing of honeys. Therefore, they can produce large amounts of gluconic acid under aerobic conditions and high concentrations of glucose (Mato et al., 2003). There have been no previous reports of the quantification of lactic acid in honeydew honeys. Olofsson and Vásquez (2008) discovered new bacterial flora composed of lactic acid bacteria of the genus Lactobacillus and Bifidobacterium, originating in the stomach of Apis mellifera. Therefore, it is evident that the presence of lactic acid in the bracatinga honeydew honeys may come from a natural fermentation process that occurs in the digestive system of the bees. The concentrations of malonic and glycolic acids in the honeydew

3.2.4. Selectivity, precision, and accuracy For the selected separation conditions, the results indicated that the method was selective, presenting adequate separation of all the compounds, even in the presence of possible interfering compounds, oxalic and aspartic acids. Oxalic acid migrates before maleic acid, and aspartic acid migrates between gluconic and succinic acids (data not shown). The RSD results for intra-day precision were 0.29–4.47 % for corrected peak area and 0.01 to 0.46% for corrected migration time. For inter-day precision, the results for corrected peak area and for corrected migration time were 1.07–7.34% and 0.05 to 0.85%, respectively. The variability of the precision is considered acceptable, with all RSDs being lower than the maximum allowed variability, which is 20% (European Comission, 2002). The recovery obtained at the three levels for all analytes ranged from 80.55 to 106.9% for citric and lactic acids, respectively (bracatinga honeydew honey) and from 81.13 to 106.3% for malonic and 4

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Fig. 2. Electropherograms of aliphatic organic acids in bracatinga honeydew honey from Urubici (a1) 1:100 (w/w) and (a2) 1:10 (w/w); floral honey from Lages (b) 1:10 (w/w): (1) maleic (2) malonic, (3) fumaric (4) tartaric, (I.S.) glyoxylic, (5) formic, (6) citric, (7) malic, (8) glycolic, (9) lactic, (10) gluconic, (11) succinic, (12) glutaric, (13) acetic, (14) propionic.

Table 2 Aliphatic organic acids (AOA, mg 100 g−1) present in bracatinga honeydew honeys from different cities of Santa Catarina state. AOA

Lages

São Joaquim

Urubici

Urupema

Bom Retiro

maleic malonic fumaric tartaric formic citric malic glycolic lactic gluconic succinic glutaric acetic propionic Mean

40.9 ± 1.50 259 ± 10.6 < LOD < LOD 39.8 ± 1.06 259 ± 8.57 302 ± 3.65 90.1 ± 3.12 1543 ± 19.7 6198 ± 152 660 ± 23.4 94.5 ± 1.35 63.1 ± 0.93 196 ± 6.77 9748 ± 164

41.6 ± 1.23 309 ± 7.63 < LOD < LOD 72.9 ± 2.56 244 ± 5.99 595 ± 25.9 97.8 ± 3.25 1046 ± 32.8 4478 ± 131 651 ± 8.68 119 ± 4.28 111 ± 4,00 297 ± 7.90 8062 ± 26.8

53.9 ± 1.45 330 ± 11.9 < LOD < LOD 43.7 ± 1.33 157 ± 5.20 446 ± 4.59 101 ± 3.85 931 ± 39.9 5582 ± 96.5 537 ± 18.6 89.1 ± 2.98 105 ± 3.33 181 ± 6.69 8557 ± 151

72.7 ± 2.15 288 ± 11.5 < LOD < LOD 87.8 ± 2.23 171 ± 3.16 415 ± 16.6 90.1 ± 3.10 1272 ± 34.4 7415 ± 292 484 ± 5.68 111 ± 3.39 80.6 ± 2.74 242 ± 6.03 10730 ± 278

79.1 ± 1.53 330 ± 13.6 < LOD < LOD 54.7 ± 0.758 227 ± 2.27 390 ± 11.3 98.1 ± 0.974 1372 ± 12.6 6590 ± 143 672 ± 7.33 114 ± 0.724 135 ± 4.09 258 ± 4.50 10321 ± 139

LOD: Limit of detection in matrix solution for fumaric acid 1.30 mg L−1 and tartaric acid 0.77 mg L−1. Table 3 Aliphatic organic acids (AOA, mg 100 g−1) present in floral honey from different cities of Santa Catarina state. AOA

Lages

São Joaquim

Urubici

Urupema

Bom Retiro

maleic malonic fumaric tartaric formic citric malic glycolic lactic gluconic succinic glutaric acetic propionic Mean

< LOD 82.2 ± 0.432 < LOD < LOD 37.9 ± 0.478 108 ± 0.310 23.4 ± 0.328 27.8 ± 1.36 72.6 ± 0.256 4523 ± 36.7 12.7 ± 0.174 < LOD 20.5 ± 0.693 10.3 ± 0.364 4917 ± 36.7

10.2 ± 0.240 118 ± 3.79 < LOD < LOD 46.3 ± 1.97 506 ± 16.5 19.9 ± 0.182 42.2 ± 0.662 68.0 ± 1.05 4566 ± 201 13.3 ± 3.50 < LOD 58.4 ± 2.40 16.3 ± 0.343 5464 ± 222

14.3 ± 0.161 109 ± 0.842 < LOD < LOD 61.5 ± 0.613 62.7 ± 1.73 33.0 ± 0.515 29.6 ± 0.791 31.2 ± 0.688 8712 ± 244.06 25.4 ± 0.102 < LOD 26.7 ± 0.704 22.8 ± 0.640 9128 ± 244

12.3 ± 0.360 134 ± 1.74 < LOD < LOD 67.9 ± 1.43 70.9 ± 0.982 132 ± 3.99 43.7 ± 1.18 84.3 ± 1.35 5449 ± 61.87 96.4 ± 0.767 21.2 ± 0.450 27.6 ± 0,662 55.8 ± 1.72 6195 ± 67.9

9.09 ± 0.347 87.7 ± 1.01 < LOD < LOD 18.8 ± 0.768 48.2 ± 0.861 49.6 ± 0.652 27.5 ± 1.24 87.3 ± 0.954 4481 ± 98.47 49.0 ± 1.45 < LOD 13.7 ± 0.315 21.5 ± 0.734 4893 ± 86.7

LOD: Limit of detection in matrix solution for fumaric acid 1.30 mg L−1, tartaric acid 0.77 mg L−1 and glutaric acid 0.30 mg L−1.

5

Floral/ Spain

Floral/ Spain

Floral/ Spain

Suárez-Luque et al. (2002)

Mato et al. (2006)

Navarrete et al. (2005) Nozal et al. (2003a)

6

CE–DAD/ 9 min

NMR/ (n.i.)

HPLC–DAD/ (n.i.)

Dilute in water

Dilute in water, pH adjustment to 10.5 (NaOH 0.1 M) and pH 5.0 (HCl 0.1 M). Solid phase extraction Dilute in water, pH adjustment to 4.5 (NaOH 1 M)

Dilute in water

Liquid–liquid extraction

GC–MS/ 12 min

CE–DAD/ 6 min

Liquid–liquid extraction

Liquid–liquid extraction

Dilute in water, pH adjustment to 10.5 (NaOH 0.1 M) and pH 5.0 (HCl 0.1 M). Solid-phase extraction Dilute in water, pH adjustment to 10.5 (NaOH 0.1 M) and pH 5.0 (HCl 0.1 M) Dilute in water

Sample preparation

HPLC–DAD/ 60 min

CE–UV-Vis/ 18 min HPLC–UV-Vis/ 60 min

CE–UV-Vis/ 4 min

HPLC–UV-Vis/ 14 min

Technique/run time (min)

Maleic, malonic, fumaric, tartaric, formic, citric, malic, glycolic, lactic, gluconic, succinic, glutaric, acetic and propionic

Formic, fumaric, pyruvic, tartaric, malic, citric, succinic

Oxalic, formic, malic, citric, succinic and gluconic Tartaric, malic, maleic, citric, succinic and fumaric

Maleic, malonic, formic, citric, malic, glycolic, lactic, gluconic, succinic, glutaric, acetic and propionic

All

Formic, malic, citric, succinic and gluconic All

Quinic and gluconic

Maleic, malonic, formic, citric, malic, glycolic, lactic, gluconic, succinic, glutaric, acetic and propionic

Formic, fumaric, tartaric, citric, succinic

Formic, malic, citric and gluconic All

Quinic and gluconic

Oxalic

– –

–

– –

–

0.25–2.42

–

–

0.07–0.72

0.18– 72.5

0.18– 72.5

0.33–50.87

12.0 – 78.0

0.025– 10.93

LOQ (mg L−1)

0.05– 21.75

0.10– 15.23 0.05– 21.75

–

Oxalic

0.40– 38.0

–

Oxalic, formic, malic, succinic, pyruvic, acetic, lactic, citric and gluconic oxalic, fumaric, maleic and malic D-glucuronic, oxalic, citric, Dgluconic, quinic, formic, glutaric and propionic

Oxalic, formic, tartaric,malic, succinic, maleic, glutaric, pyruvic, acetic, lactic, citric, butyric, sorbic, and gluconic Tartaric, oxalic, fumaric, pyruvic, maleic, propionic, malic and lactic Oxalic, D-glucuronic, citric, D-gluconic, pyruvic, malic, citramalic, quinic, succinic, lactic, formic, glutaric, propionic and butyric Oxalic, D-glucuronic, citric, propionic, pyruvic, malic, citramalic, quinic, Dgluconic, lactic, formic, glutaric, butyric Citric, quinic and gluconic

Oxalic, citric, D-gluconic, glutaric and propionic

0.006– 7.57

–

Honeydew

LOD (mg L−1)

All

Floral

AOA quantified

Malic, maleic, citric, succinic and fumaric

AOA investigated

CE: capillary zone electrophoresis; HPLC: high performance liquid chromatography; UV–vis: ultraviolet-visible detector; DAD: diode array detector; NMR: nuclear magnetic resonance; GC–MS: gas chromatography–mass spectrometry; LOD: limit of detection; LOQ: limit of quantification; n.i. – no information.

This method

Ohmenhaeuser et al. (2013)

Haroun et al. (2012)

Floral and honeydew/ Germany Floral and honeydew/ Brazil

Floral and honeydew/ Spain and Italy Floral and honeydew/ Turkey Floral and honeydew/ Turkey

Sanz et al. (2005)

Tezcan et al. (2011)

Floral and honeydew/ Spain

Nozal et al. (2003b)

Floral and Honeydew/ Spain

Type/Country

Reference

Table 4 Methods used to determine aliphatic organic acids (AOA) in honeydew honey and floral honey.

P. Brugnerotto, et al.

Journal of Food Composition and Analysis 82 (2019) 103243

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P. Brugnerotto, et al.

honey samples varied between 259 and 330 mg 100 g−1 and between 90.1 and 100 mg 100 g−1, respectively. Besides lactic acid, malonic and glycolic acids are also reported in honeydew honey for the first time. Succinic, citric, malic, acetic and propionic acids were found in intermediate concentrations, while formic and glutaric acids were found in small concentrations in the honeydew honey samples evaluated in this paper. The results presented in this paper can be compared to thise for AOA in pine (Pinus sp.) honeydew honey from Turkey (Haroun et al., 2012), and also with Quercus and Quercus ilex honeydew honey originated from Spain (unknown region) (Nozal et al., 2003a; Sanz et al., 2005; Tezcan et al., 2011). Bracatinga honeydew honey presented higher concentrations for all quantified AOA. In this paper some distinct acids were detected, compounds that until this date were not reported in the literature, i.e., lactic, malonic and glycolic acids.

the CE method proposed by Navarrete et al. (2005). Moreover, the last one presented higher LOD and LOQ and was applied only for floral honey. The study of Tezcan et al. (2011) presented a lower analysis time but was not validated for honey samples, did not present LOD and LOQ and evaluated a smaller analyte quantity (6 AOA), compared with the evaluated analytes in this study (14 AOA). The study of Mato et al. (2006b) evaluated the same analyte quantity with lower analysis time. However, pH adjustment stages were applied, a disadvantage when compared with the proposed method, which requires only water dilution. In addition, the method was only applied to floral honeys and obtained higher LOD and LOQ when compared to the limits of te current work.

3.3.2. Floral honeys Generally, there are more studies evaluating AOA in floral honey (Haroun et al., 2012; Mato et al., 2006b; Navarrete et al., 2005; Nozal et al., 2003a; Ohmenhaeuser et al., 2013; Sanz et al., 2005; SuárezLuque et al., 2002; Tezcan et al., 2011) when compared with honeydew honey studies. However, the evaluation of AOA using CE in Brazilian floral honey, proposed in this study, is reported for the first time. In Table 3 the AOA quantification of the floral honey samples is summarized. AOA levels ranged between 9.09 mg 100 g−1 for maleic acid (sample from Bom Retiro) and 8712 mg 100 g−1 for gluconic acid (sample from Urubici). Gluconic acid was found in higher concentrations among all the organic acids quantified in all honey samples, with concentrations ranging between 4523 and 8712 mg 100 g−1, in samples from Lages and Urubici, respectively. Like in honeydew honeys, gluconic acid is the major AOA in floral honeys. Furthermore, in this case, the botanical source, the pollen and nectar of the flowers used by the bee to produce the honey can influence the variability in the concentrations of this acid. Glutaric acid was quantified only in the Urupema sample, with a concentration of 21.2 mg 100 g−1. Malonic acid was found in concentrations ranging between 82.2 and 134 mg 100 g−1, while glycolic acid was found in concentrations ranging between 27.8 and 43.7 mg 100 g−1. This study is the first to report the quantification of malonic and glycolic acids in floral honey samples. Formic, lactic, acetic and propionic acids were found in intermediate concentrations, while citric and malic acids presented variability in their concentrations, which ranged between 48.2 and 506 mg 100 g−1 for citric, and between 19.9 and 132 mg 100 g−1 for malic. These six acids were already evaluated in other floral honey samples from Spain and Turkey (Haroun et al., 2012; Mato et al., 2006b; Nozal et al., 2003a; Suárez-Luque et al., 2002). With the exception of propionic acid, all other acids were reported in lower concentrations than the ones in this study. In all honeys evaluated in this study, fumaric and tartaric acid were not detected and maleic acid was not detected in the Lages sample, as demonstrated in Fig. 2.

This paper presented a novel CE method to quantify simultaneously 14 AOA in less than 10 min. The application of the proposed method enabled the first evaluation of the AOA composition in bracatinga honeydew honey and floral honey of Santa Catarina state. All AOA quantified in the samples of bracatinga honeydew honeys were at concentrations higher than the ones found in floral honey samples. Gluconic acid was the major acid in the two honey classes (floral and honeydew), presenting higher concentrations in honeydew honey when compared to floral honey. From the current literature data, it was observed that malonic acid and glycolic acids were quantified for the first time in honeydew and floral honeys and that lactic acid was quantified for the first time in honeydew honey.

5. Conclusions

Acknowledgments The authors acknowledge the financial support of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Finance code 001), Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC) and the collaborating beekeepers of the mountain plateau region in the state of Santa Catarina (Brazil). References AOAC, 2012. Appendix F : guidelines for standard method performance requirements. Off. Methods Anal. AOAC Int. 1–17 Appendix. Awad, H., Allen, K.J.H., Adamko, D.J., El-Aneed, A., 2019. Development of a new quantification method for organic acids in urine as potential biomarkers for respiratory illness. J. Chromatogr. B 1122–1123, 29–38. https://doi.org/10.1016/j. jchromb.2019.05.021. Azevedo, M.S., Pirassol, G., Fett, R., Micke, G.A., Vitali, L., Costa, A.C.O., 2014. Screening and determination of aliphatic organic acids in commercial Brazilian sugarcane spirits employing a new method involving capillary electrophoresis and a semi-permanent adsorbed polymer coating. Food Res. Int. 60, 123–130. https://doi.org/10. 1016/j.foodres.2013.11.007. Azevedo, M.S., Seraglio, S.K.T., Rocha, G., Balderas, C.B., Piovezan, M., Gonzaga, L.V., Falkenberg, D., de, B., Fett, R., de Oliveira, M.A.L., Costa, A.C.O., 2017a. Free amino acid determination by GC-MS combined with a chemometric approach for geographical classification of bracatinga honeydew honey (Mimosa scabrella Bentham). Food Control 78, 383–392. https://doi.org/10.1016/j.foodcont.2017.03.008. Azevedo, M.S., Valentim-Neto, P.A., Seraglio, S.K.T., da Luz, C.F.P., Arisi, A.C.M., Costa, A.C.O., 2017b. Proteome comparison for discrimination between honeydew and floral honeys from botanical species Mimosa scabrella Bentham by principal component analysis. J. Sci. Food Agric. 97, 4515–4519. https://doi.org/10.1002/jsfa. 8317. Baziramakenga, R., Simard, R.R., Leroux, G.D., 1995. Determination of organic acids in soil extracts by ion chromatography. Soil Biol. Biochem. 27, 349–356. https://doi. org/10.1016/0038-0717(94)00178-4. Bergamo, G., Seraglio, S.K.T., Gonzaga, L.V., Fett, R., Costa, A.C.O., 2019. Physicochemical characteristics of bracatinga honeydew honey and blossom honey produced in the state of Santa Catarina: an approach to honey differentiation. Food Res. Int. 116, 745–754. https://doi.org/10.1016/j.foodres.2018.09.007. Bergamo, G., Seraglio, S.K.T., Gonzaga, L.V., Fett, R., Costa, A.C.O., 2018. Mineral profile as a potential parameter for verifying the authenticity of bracatinga honeydew honeys. LWT - Food Sci. Technol. 97, 390–395. https://doi.org/10.1016/j.lwt.2018. 07.028. Bruce, P., Minkkinen, P., Riekkola, M.-L., 1998. Practical method validation: validation

4. Comparison with other methods The main studies on the determination of AOA in honeys are summarized in Table 4, considering studies of the last 10 years. These studies applied distinct analytical techniques and sample preparation, and obtained different analysis times and LOD and LOQ. Generally, methods that apply CE have lower analysis time and operational cost, presenting larger resolution and efficiency, and consuming fewer chemicals and samples, when compared with high-performance liquid chromatography (Azevedo et al., 2014; Nozal et al., 2003a). The proposed method (CE–DAD) presented lower analysis times than the methods based on liquid chromatography (Nozal et al., 2003b, 2003a; Suárez-Luque et al., 2002), gas chromatography (Sanz et al., 2005) and 7

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