Screening and Determination of Aliphatic Organic Acids In

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Food Research International 60 (2014) 123–130

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Food Research International

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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

Mônia Stremel Azevedo a,GabrielaPirassola,RoseaneFetta, Gustavo A. Micke b, Luciano Vitali a, Ana Carolina Oliveira Costa a,⁎

a Department of Science and Food Technology, Federal University of Santa Catarina, Florianopolis, SC, Brazil b Department of Chemistry, Federal University of Santa Catarina, Florianopolis, SC, Brazil

article info abstract

Article history: A simple method for the screening and determination of short chain aliphatic carboxylic acids in Brazilian sugar- Received 10 June 2013 cane spirits by indirect capillary zone electrophoresis was developed and validated according to Eurachem guide- Received in revised form 28 October 2013 lines. The separations were performed in a coated capillary (Ltot 73 cm × Ldet 64.5 cm) with 2-hydroxy- Accepted 6 November 2013 propyltrimethyl-ammonium chloride chitosan. The background electrolyte was optimized using Peakmaster® Available online 20 November 2013 software and was composed of 21 mmol L−1 β-alanine and 10 mmol L−1 3,5 dinitrobenzoic acid, at pH 3.6, with an applied voltage of −30 kV. The method was validated for formic, lactic and acetic acids. Linearity was Keywords: ﬁ −1 Aliphatic organic acids demonstrated and signi cant matrix effects were not observed at between 2.8 and 144 mg L . The recovery Brazilian sugarcane spirits (means of 89.6–117.9%) was evaluated, and ANOVA was carried out to determine the RSD values for the repeat- Method validation ability (2.63–8.35%) and reproducibility (1.92–6.62%). The LOD and LOQ ranged from 0.69 to 2.70 and 2.76 to Peakmaster® software 8.11 mg L−1, respectively. The concentrations detected in the samples were 0.19–106.43 mg per 100 g. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction (maximum capacity of 700 L) for a period of not less than 1 year. Premi- um and extra premium sugarcane spirits are spirits that were whole Aliphatic organic acids are found in a wide range of foods and bever- aged for 1 year and 3 years, respectively (Brasil, 2005). In aged alcoholic ages (Klampfl,2007). In the case of alcoholic beverages that are beverages, the concentration of aliphatic organic acids is directly related fermented or distilled, determination of the aliphatic organic acids al- to the aging time and does not have a direct relationship with the aging lows control over the evolution of the acidity during the production process applied (Schawan, Mendonça, Silva, Rodrigues, & Wheals, 2001; steps (alcoholic fermentation, malolactic fermentation and aging pro- Schwarz, Rodríguez, Guillén, & Barroso, 2011). Thus, the profile of these cess). In wine, acetic and lactic acids are associated with contamination compounds can be used for the hygiene control, differentiation, classifi- by microorganisms and may be indicators of poor hygiene conditions cation, origin identification and/or detection of the adulteration of alco- (Sádecká, Májek, & Thóthová, 2008). The presence of aliphatic organic holic beverages (Sádecká et al., 2008). This chemical monitoring acids in the distillate is also related to the control of the processes occur- contributes to the quality improvement and standardization of bever- ring during the production and storage of the beverages. In sugarcane ages such as sugarcane spirit, an alcoholic beverage widely consumed spirit (known in Brazil as cachaça), aliphatic organic acids originate di- in Brazil and also appreciated on the international market (Nóbrega rectly from the distillation and/or fermentation of the sugarcane juice et al., 2009). and they exert a strong influence on the quality of the sensory proper- Major aliphatic organic acids previously detected in sugarcane spirit ties of this alcoholic beverage (Campos, Figueiredo, Hogg, & Couto, are: acetic, citric, formic, glycolic, lactic, maleic, malic, malonic, succinic, 2009). The Brazilian law establishes that aged sugarcane spirit should and tartaric (Chen, Lin, Chen, Misra, & Liu, 2003; Moreno, Jurado, & contain at least 50% of the spirit matured in appropriate wooden casks Barroso, 2003; Park, Kim, & Kim, 1999; Sádecká et al., 2008). Several methods to determine these aliphatic organic acids in food and

Abbreviations: SCACA, short chain aliphatic carboxylic acids; CZE, capillary zone elec- beverages have been described in the literature based on different trophoresis; EOF, electroosmotic flow; BGE, background electrolyte; HACC, 2-hydroxy- separation techniques including gas chromatography–mass spectrometry propyltrimethyl-ammonium chloride chitosan; AOAC, Association of Official Analytical (GC–MS) (Gallagher et al., 2010; Jurado-Sánchez, Ballesteros, & Gallego, Chemists; RS, resolution; I.S., internal standard; tm, migration time; OLSM, ordinary least 2011), gas chromatography–flame ionization detection (GC–FID) squares method; α,significance level; L , total length; L , effective length; TU, Tiselius tot det (Ortega, Lopez, Cacho, & Ferreira, 2001; Park et al., 1999), high perfor- unit; p, significance. – – – ⁎ Corresponding author. Tel.: +55 48 3721 2975; fax: +55 48 3721 9943. mance liquid chromatography ultraviolet visible detection (HPLC UV/ E-mail address: [email protected] (A.C.O. Costa). Vis) (Zhang et al., 2011), HPLC-conductivity detection (HPLC-CD)

(Schwarz et al., 2011), two-dimensional liquid chromatography using ion 2. Materials and methods chromatography × reversed-phase liquid chromatography (LC × LC) (Brudin, Shellie, Haddad, & Schoenmakers, 2010), and capillary 2.1. Reagents and solutions electrophoresis-UV/Vis (CE-UV/Vis) (Chen et al., 2003; Rovio, Kalliola, Siréna, & Tamminen, 2010; Sádecká et al., 2008). The technique of All chemicals used in the experiments were of analytical reagent CE-UV/Vis offers the advantages of ease of application and reduced grade. Short chain aliphatic carboxylic acids (maleic, malonic, tartaric, sample preparation time, allowing the high frequency provided by formic, citric, malic, glycolic, lactic, succinic, acetic), aspartic acid (inter- CE, good resolution and efficient separation of aliphatic organic nal standard — IS), 3,5-dinitrobenzoic acid, and β-alanine were obtained acids, simple instrumentation, low consumption of chemicals, great from Sigma-Aldrich (St. Louis, CO, USA). Deionized water with a resis- potential for appropriate detection and quantification limits in food tivity of 18.2 M Ω cm was obtained from a Milli-Q system (Millipore, and beverage analysis, which are fundamental requirements in the Bedford, MA, USA) and used to prepare all solutions. HACC synthesized development of analytical methods for quality control, and auxiliary by Vitali et al. (2011) was kindly provided for the coating of the software can be used for the rational optimization of buffer parame- capillaries. A stock solution of 1.0% (w/v) HACC was prepared in ters for obtaining suitable separation of the analytes (Bianchi, Careri, 2mmolL–1 hydrochloric acid, filtered through a 45 μm membrane & Corradini, 2005; Gaš,Jaroš,Hrušca,Zuskova,&Štědrý, 2005; Izco, and applied to modify the surface of the capillary. Standard stock solu- Tormo, & Flores, 2002; Johns, Breadmore, Bruno, & Haddad, 2009; tions containing 1000 mg L–1 of the aliphatic organic acids were Mato, Suárez-Luque, & Huidobro, 2007; Santalad, Teerapornchaisit, prepared in deionized water. Calibration solutions were prepared by Burakham, & Srijaranai, 2007). Anion separation by CE normally re- diluting the stock solutions with deionized water. quires the use of an electroosmotic flow (EOF) inverter, which gener- ates an anodic EOF to promote the separation in co-electroosmotic 2.2. Capillary electrophoresis system and capillary coating mode. This is required because in uncoated capillaries the anion separation occurs in counter-electroosmotic mode (cathodic EOF). CE assays were conducted in a capillary electrophoresis system In these capillaries, the EOF mobility varies over a wide range and (model 7100, Agilent Technologies, Palo Alto, CA, USA), equipped with is strongly dependent on the pH of the background electrolyte a diode array detector (set at 254 nm; indirect detection, with a refer- (BGE), which may adversely affect the separation of aliphatic organic ence at 360 nm for peak inversion), a temperature-control device acids with respect to time (long separation times) and simultaneity (maintained at 25 °C), and data acquisition and treatment software (anions with a higher or lower degree of mobility than the EOF) supplied by the manufacturer (HP ChemStation, rev. A.06.01). The cap- due to differences in terms of the mobility of these compounds. illary was employed in separations and was coated by a procedure Capillary modifiers, such as tetradecyltrimethylammonium hydrox- adapted from Vitali et al. (2011) using fused-silica capillaries ide (Mato et al., 2007; Moreno et al., 2003), cetyltrimehylammonium (Polymicro Technologies, Phoenix, AZ, USA) with dimensions of 73 cm bromide (Esteves, Lima, Lima, & Duarte, 2004; Galli & Barbas, 2004; total length, 64.5 cm effective length and 75 μm inner diameter. The ac- Rovio et al., 2010), tetraethylenepentamine (Fung & Lau, 2003)and tivation of the capillary surface by dissociation of the silanol groups was hexadimethrine bromide (Bianchi et al., 2005) have been used. In performed by flushing with 1 mol L–1 NaOH for 30 min and then with most cases, these modifiers have been employed as additives in the deionized water for 15 min. After preconditioning, the capillary BGE. However, hexadimethrine bromide can be used as a semi- was modified by applying a semi-permanent coating of HACC through permanent coating where the modifier is adsorbed on the surface the following procedure: flush 10 min with HACC 0.2% (w/v), maintain of the fused silica capillary and does not need to be added to the in static contact for 10 min and, finally, flush the capillary for 5 min with BGE. The semi-permanent coating offers different advantages to BGE (without HACC). The reconstruction of the coating on different other types of modifications, such as simplicity of coating formation, days was carried out by repeating these steps. The BGE used to possibility of coating regeneration, access to aprioriknowledge of determine the aliphatic organic acids was composed of 21 mmol L–1 the coating polymer properties, and reduced dependence of the β-alanine and 10 mmol L–1 3,5-dinitrobenzoic acid, at pH 3.6. The stan- coating process on surface chemistry (Lucy,MacDonald,&Gulcev, dards and samples were introduced at the end of the capillary farthest 2008). Furthermore, semi-permanent modifications can lead to a re- from the detector and injected using a hydrodynamic pressure of duction in the baseline noise, compatibility with mass detectors, and 50 mbar for 3 s. The separation voltage applied was 30 kV, with nega- no undesirable peaks in the electropherogram for the counter-ions, tive polarity on the injection side. The capillary was flushed between such as bromide, which is present in many modifiers and used as a runsfor1.0minwithBGE. BGE additive. A disadvantage in some cases is the low stability of the coating formed, for instance, hexadimethrine bromide resists 2.3. Samples for only ten runs. Thus, new polymeric modifiers need to be tested as polymers containing quaternized cationic groups for formation Samples of sugarcane spirit (with and without aging) were kindly of the anodic EOF; for example, quaternary ammonium chitosan provided by a local Brazilian sugarcane spirit industry production (2-hydroxy-propyltrimethyl-ammonium chloride chitosan; HACC) plant and the Brazilian Ministry of Agriculture and Livestock (MAPA): has been previously employed for the determination of anions and two non-aged samples (named 1-NA and 2-NA) and ten samples aged good results were obtained (Ma et al., 2007; Vitali, Horst, Heller, for 2 to 16 years (3-A2Y, 4-A2Y, 5-A3Y, 6-A3Y, 7-A3Y, 8-A4Y, 9-A5Y, 10- Fávere, & Micke, 2011). A6Y, 11-A12Y, 12-A16Y). Before injection into the CE equipment, the The aim of this study was to develop a simple method for the screen- samples were diluted to give 9:1 (v/v) sample:I.S. solution (I.S. injected ing and determination of 10 aliphatic organic acids in commercial at 200 mg L–1). Brazilian sugarcane spirits employing capillary zone electrophoresis (CZE) and using a semi-permanent capillary coating comprised of HACC. 2.4. Validation procedure and statistical analysis The development of the separation method, selection of the BGE compounds and instrumental parameters and evaluation of the dispersion The method was validated according to Eurachem (1998) and effects of the analytes were performed using the Peakmaster® software Inmetro (2003) guidelines employing assays with standard solutions, (Gaš et al., 2005). The method was validated based on the evaluation blank samples and spiked samples. System suitability, linearity, matrix parameters recommended in the Eurachem (1998) and Inmetro effects, selectivity, precision, recovery, detection and quantification (2003) guidelines and then applied in the determination of acids previ- limits, and robustness were studied. The fitness-for-purpose of this ously identified in Brazilian sugarcane spirits: formic, lactic and acetic. method was assessed based on the results obtained for pre- M.S. Azevedo et al. / Food Research International 60 (2014) 123–130 125 established performance characteristics (Eurachem, 1998; Inmetro, 2.8. Selectivity 2003; ISO—International Standards Organization, 1994; Thompson, Ellison, & Wood, 2002). The Peakmaster® software was used to evaluate the selectivity of the proposed method. With this important analytical tool it was possi- 2.5. System suitability ble to simulate the presence of compounds in sugarcane spirits which can interfere in the analysis. For the optimized BGE (3,5-dinitrobenzoic In this study, the system suitability was tested by means of ten con- acid and 21 mmol L−1 β-alanine), the following parameters were secutive injections of the standard mixture using the I.S. and consider- inserted in the software: all the compounds of interest (oxalic, maleic, ing the relative standard deviation (RSD). Repeatability was evaluated malonic, tartaric, phosphoric, formic, citric, malic, glycolic, lactic, using the corrected peak area calculated as area(analyte)/area(I.S.), gluconic, succinic, and acetic acids); voltage (30000 V); the total length corrected migration time (tm(analyte)/tm(I.S.)), and resolution (RS). of the capillary (Ltot =73cm;Ldet = 64.5 cm); injection polarity (negative); marker flow determined experimentally (11 min); selec- 2.6. Linearity tion of the correction of ionic strength; and signal input (direct). Other organic acids that can be found in sugarcane spirit were also added to The screening method was optimized for the separation of 10 short the simulation (glucolic, phosphoric and oxalic acids). The method chain aliphatic carboxylic acids (SCACA) commonly found in distilled selectivity was established from the near baseline separation of the spirits. After performing a preliminary assessment and screening with organic acids in the simulated electropherogram, evaluation of the the proposed method and then verifying the presence of formic, lactic sample blank and addition of standard solutions to the samples. and acetic acids in the samples, the method was validated for these compounds in order to determine their presence in samples of com- 2.9. Precision mercial Brazilian sugarcane spirit. Linearity was assessed by obtaining three calibration curves, prepared on different days at the levels of 2.8, The precision (repeatability) was determined through the analysis 4.1, 5.5, 6.9, 8.3, 9.6 and 11 mg L−1of formic acid, 8.11, 12.2, 16.2, of 10 independent replicates of a mixture of SCACA standards at the 20.2, 24.3, 28.4 and 32.4 mg L−1 of lactic acid and 36, 54, 72, 90, 108, seven levels used to obtain the calibration curves. All analyses were car- 126 and 144 mg L−1 of acetic acid with three independent replicates. ried out on the same day, by the same analyst, using the same method- The range for the calibration curves comprised seven equidistant levels ology and equipment. To evaluate the partial reproducibility, firstly 10 and the standard solutions were prepared from different stock solutions independent replicates were analyzed at the seven levels used to obtain on each day of analysis and run in a random order. Blanks were also the calibration curve for the SCACA, using the same methodology and prepared, in triplicate, for each curve to set zero on the equipment. equipment, with samples prepared by the same analyst with an interval The linear model that relates the analyte concentration (X) to the signal of 7 days. The same procedure was then performed by a different ana- provided by the measurement instrument (Y)is, lyst with an interval of 14 days. Precision, repeatability and partial reproducibility conditions, were Y ¼ a þ bX þ e; estimated by ANOVA (ISO—International Standards Organization, 1994; Kuttatharmmakul, Massart, & Smeyers-Verbeke, 1999)and where α is the intercept, β the slope, and ε the error or residual. The or- expressed in terms of relative standard deviation for the replicates of dinary least squares method (OLSM), which minimizes the sum of the the standard mixture solution at each concentration level calculated as squared residuals, was used to calculate α and β. This method assumes tm(analyte)/tm(I.S.) and area(analyte)/area(I.S.),respectively. that the residuals are normally distributed, with constant variance and that they are independent of each other (Draper & Smith, 1981). After 2.10. Accuracy an exploratory fitting using the OLSM, the residual plots were examined for obvious patterns, with outliers indicated by points out of the range The apparent recovery was investigated through the mean obtained with the square root of the residual variance. Outliers were determined for the six independent replicates of a spiked sample at seven levels in by Grubbs standardized residuals test which was applied successively the same range used for the calibration curves. The concentrations for until no further outliers were detected or until a drop of 22.2% in the the spiked sample were calculated using the calibration curves prepared original number of results was observed (Horwitz, 1982). Violations of in deionized water, since no matrix effects were detected. The minimum assumptions underlying OLSM residuals were evaluated through the trueness criteria ranged from ±20% (Brasil, 2003). following tests: residual normality (Ryan & Joiner, 2000), independence (Durbin & Watson, 1951) and homoscedasticity (Bartlett, 1937). F-tests 2.11. Detection and quantification limits were carried out to check the fitting with the model through the evaluation of the regression and lack of fitsignificances (Draper & Smith, The limit of quantification (LOQ) was defined as the lowest concen- 1981). tration at which the method could operate with acceptable precision (signal/noise ≥ 10). The limit of detection (LOD) was considered as 2.7. Matrix effects the lowest concentrations of formic, lactic, and acetic acids that were detectable in all replicates but not necessarily quantified and distin- Matrix effects were verified by applying the method of standard ad- guished from zero (signal/noise ≥ 3). These limits were established ditions. Matrix-matched curves were prepared according to the proce- based on the mean and relative standard results obtained for 10 inde- dure used to assess the linearity for the matrix free of the SCACA. pendent replicates of these concentrations. Three independent replicates were run in a random order on the same day. Blanks were also prepared to set zero on the equipment. The values 2.12. Robustness for the slope and intercept, and the respective variances, of both curves were calculated by OLSM. All assumptions tested in assessing the linear- The robustness of the electrophoretic method for the SCACA quanti- ity were also verified for these curves. The slopes and intercepts obtain- tation was evaluated using the method proposed by Youden & Steiner ed for the solvent and matrix-matched calibration functions were (1975). Seven analytical parameters were selected and small variations compared to verify significant differences applying the t-test were induced in the nominal values of the method. Eight runs were then (Armitage & Berry, 1994). The hypothesis tests were performed at the performed to determine the influence of each parameter on the final re- α = 0.05 level. sult. The seven analytical parameters were employed at the nominal 126 M.S. Azevedo et al. / Food Research International 60 (2014) 123–130 values, and the conditions, with small variations, included: voltage sep- 3.2. Method validation aration (30 and 25 kV); pH of BGE (3.63 and 3.73); cartridge temperature (25 and 20 °C); injection pressure (50 and 55 mbar); wavelength 3.2.1. System suitability for indirect UV detection (254 and 258 nm); flush between runs (60 Before obtaining the figures of merit in validation experiments, the and 30 s); and injection time (3 and 4 s). For each combination, three system used for the analysis must be evaluated to verify that it provides injections of a sample (sample 12-A16Y) and standard solution were data of acceptable quality. Therefore, suitability tests need to be carried out, at the working concentration (middle of the calibration performed. In this study, the system suitability was determined by curve). The results obtained for each combination were: formic, lactic means of ten consecutive injections of the standard mixture using the −1 and acetic acid contents (mg L ); migration time (tm); peak symme- internal standard and considering the relative standard deviation (RSD). try; corrected peak area (area(analyte)/area(I.S.)); and resolution (RS). To Repeatability was evaluated using the corrected peak area calculated as determine the influence of variations in each parameter on the final area(analyte)/area(I.S.), corrected migration time (tm(analyte)/tm(I.S.)) and result, the mean of the five values corresponding to the nominal condi- resolution (RS). Table 1 shows the data obtained. tions was compared to the mean of the five values corresponding to the As shown in Table 1, the results indicate that the RSD% ranged from altered conditions. By means of Youden's test, it is possible to precisely 0.02 to 0.19% for the corrected migration time and 0.47 to 1.74% for establish the parameters which have the greatest influence on the final corrected peak area. The values obtained show that the instrumental result of the analysis and gain more rigorous control of the variations in system is suitable for use in the validation procedure. these parameters that may occur during routine analysis. 3.2.2. Linearity and matrix effects 3. Results and discussion In the residual plots outliers were not detected for the solvent and matrix-matched curves on applying the Grubbs standardized residual 3.1. Background electrolyte optimization test. The assumption that the residuals are normally distributed was confirmed. The Ryan–Joiner correlation coefficients were 0.980, 0.984 The optimization of the BGE for the proposed screening and quanti- and 0.980 for formic, lactic and acetic acids, respectively for the 3 sol- fication method employing CZE was performed considering the separa- vent curves and 0.988, 0.976 and 0.980 for formic, lactic and acetic tion of aliphatic organic acids normally found in samples of sugarcane acids, respectively, for the 3 matrix-matched curves, indicating no sig- spirits listed in the introduction of this paper. To select the optimum nificant deviation from normality (p N 0.10). No autocorrelation was separation conditions, data related to the pH, co-ion and counter-ion observed and the Durbin–Watson statistics were 1.15, 1.74, and 2.75 of the BGE, internal standard and other system parameters of separation for formic, lactic and acetic acids, respectively for the 3 solvent curves were used to create curves of effective mobility versus pH using and 0.832, 1.831, and 1.734 for formic, lactic and acetic acids, respective- Peakmaster® software. The pH was selected considering the ionized ly, for the 3 matrix-matched curves. This shows that the residuals were analytes, and the difference in mobility required for separation was statistically independent (p N 0.05). Homoscedasticity was confirmed. 3.64. The components of the BGE selected to provide this pH condition The residual variability across all concentration levels was constant, were 3,5-dinitrobenzoic acid as the co-ion and β-alanine as the since the Bartlett t-statistics were not significant (p N 0.05) (Table 2). counter-ion, at concentrations of 10 and 21 mmol L−1, respectively. The high degree of significance (p b 0.001) of the regression can be This co-ion was selected because it has chromophore groups in its struc- seen in Table 3, while the lack-of-fit was not significant (p N 0.05) for ture, enabling the indirect detection of analytes that do not absorb in the the solvent and matrix-matched curves, indicating linearity in the UV–Vis range, and also due to its effective mobility (−23 TU) being range of 2.8–11.0 mg L−1 for formic acid, 8.1–32.4 mg L−1 for lactic close to the average effective mobility of the analytes (~−21 TU), acid and 36.0–144.0 mg L−1 for acetic acid. ensuring a low dispersion through electromigration of the analyte All of the OLMS assumptions were confirmed for the solvent and peaks (−2.8 to 6.5 × 10−3 mol m2 S−1). β-alanine was chosen as the matrix-matched curves. No matrix effects were detected in the range counter-ion since it has a pKa of 3.43, a value very close to the separa- studied. Lack of significance (p N 0.05) was observed when the slope tion pH, thereby conferring an adequate buffering capacity to this meth- (t = 0.842) and the intercept (t = 0.419) from the solvent curve od (~15 mmol L−1). The ionic strength and the conductivity obtained were compared with those from the matrix-matched curve. Based on from the proposed composition of the BGE were 8.8 mmol L−1 and these results, it is possible to conclude that the formic, lactic and acetic 6.0 × 10−2 mS−1, respectively. The internal standard selected was acid solvent solutions, prepared as described herein, gave the same sig- aspartic acid, which has characteristics similar to those of the analytes nal as a matrix sample containing the same concentration of these and was not present in the sample matrix. To promote the separation SCACA. Solvent curves were used in the subsequent steps to calculate of aliphatic organic acids in the shortest possible time, a capillary coated the organic acid concentrations in samples of Brazilian sugarcane spirit. with HACC was used, generating an anodic EOF of around of −24 TU. In relation to the applicability of the proposed screening method to The effective separation of the analytes in co-electroosmotic mode in other distilled beverages, it is important to evaluate each specificcase, less than 10 min was achieved with the application of 30 kV in a capil- which will require new tests to determine the matrix effects. lary of 64.5 cm (length to the detector) coated with HACC. The performance of the semi-permanent HACC coating was found to be superior 3.2.3. Selectivity, precision and accuracy to that of other coatings used in the separation of aliphatic organic A simulated electropherogram obtained by the software Peakmaster® acids described previously, resisting for up to 40 runs. The electrophero- indicates the selectivity of the proposed method in relation to the grams were obtained through the simulation of the separation of 10 organic acids evaluated, with baseline separation of the simulated SCACA using Peakmaster® software. The experimental electrophero- peaks. The selectivity was also verified through the evaluation of the gram for the acid standards and the electropherogram of a sample of sample blanks and SCACA were not detected (signal/noise b 3). Finally, commercial Brazilian sugarcane spirit employing the optimized condi- the selectivity of the method was investigated through the addition of tions are shown in Fig. 1. A good similarity was observed between the the standards in the samples analyzed, making it possible to verify simulated and experimental electropherograms for the standards, and identify all of the SCACA added to obtain the experimental electro- verifying that the separation conditions chosen were appropriate. The pherogram (Fig. 1). The coated capillaries provided a higher precision in application of the screening method developed to the sugarcane spirit terms of the migration times than the uncoated capillaries, which samples revealed the presence of formic, lactic and acetic acids. Thus, permits the use of this observation as a selectivity parameter. the validation procedure for quantification purposes, described below, ANOVA was carried out to estimate the RSD and the values for the was applied only to these analytes. repeatability were 4.20–7.94%, 2.89–8.35%, and 2.63–5.38% for formic, M.S. Azevedo et al. / Food Research International 60 (2014) 123–130 127 eof 5

6 3 10 2 7 8 1 PI 9

10 40 mAU PI 4 C 8

3 4 5 6 7 8 9 min

Fig. 1. Electropherogram of SCACA studied under (A) simulated conditions; (B) experimental conditions; and for (C) commercial Brazilian sugarcane spirit, respectively. For the analytical conditions see Materials and methods section. Legend peaks: 1, maleic; 2, malonic; 3, tartaric; 4, formic; 5, citric; 6, malic; 7, glycolic; 8, lactic; 9, succinic; 10, acetic acids. I.S., aspartic acid. lactic and acetic acids, respectively. The results for the partial reproduc- Acceptable mean recoveries were obtained in the range of 103.3– ibility of 10 independent replicates using the same equipment and pre- 117.9%, 105.5–123.8%, and 89.6–104.6% for formic, lactic and acetic pared by the same analyst with a 7-day interval ranged from 4.04 to acids. 7.49%, 5.16 to 7.79%, and 1.70 to 5.32% for formic, lactic and acetic acids, respectively. When the same procedure was executed by a differ- 3.2.4. Detection and quantiﬁcation limits ent analyst with an interval of 14 days the corresponding results obtain- The limits of detection and quantiﬁcation for the SCACA studied ed were 3.16–6.60%, 3.81–10.5%, and 1.92–6.62%, respectively. were determined for standard solutions prepared in the solvent, since there was no matrix effect observed. The LOD and LOQ values were determined for 10 independent preparations of the standard with signal/ noise ratios of ≥3 and 10, respectively. The LOD values for formic, lactic Table 1 and acetic acid were 0.69 ± 0.04, 2.70 ± 0.2, 1.8 ± 0.12 mg L−1 and System suitability.

Acid Corrected peak area Corrected migration time Resolution⁎

Maleic 1.67 0.19 8.72 Rs1,2 Table 2 Malonic 1.20 0.16 10.27 Rs2,3 Residual homoscedasticity evaluation by Bartlett test for solvent and matrix-matched cal- Tartaric 0.47 0.15 4.09 Rs3,4 ibration curves. Formic 1.30 0.12 13.37 Rs4,5

Citric 1.14 0.09 4.22 Rs5,6 Acid Solvent Matrix-matched Malic 1.74 0.07 8.54 Rs6,7 ⁎ ⁎ nt pntp Glicolic 0.89 0.05 8.47 Rs7,8

Lactic 0.68 0.02 21.01 Rs8,9 Formic 21 11.8 0.067 21 8.3 0.219

Succinic 0.87 0.04 13.17 Rs9,10 Lactic 21 7.11 0.310 21 8.91 0.178 Acetic 0.94 0.12 Acetic 21 9.00 0.173 21 6.35 0.385 ⁎ ⁎ RS =tm2 − tm1/w0.5,1 + w0.5,2. n:numberofobservations,t :Bartlettt-statistic, p: signiﬁcance. 128 M.S. Azevedo et al. / Food Research International 60 (2014) 123–130

Table 3 3.4. Comparison with other methods ANOVA statistics for regression including lack of fit test of solvent and matrix-matched calibration curves. The comparative analysis (summarized in Table 5)ofsomecharac- Acids in Regression Lack of fit teristics of the method presented in this paper and those of other migration Equation Coefficient Fp Fp methods employed to determine SCACA in beverages using indirect order (y = ax + b) (R2) UV detection showed that the proposed method using HACC as the semi-permanent EOF modifier required less conditioning time between Formics* y =0.311x − 0.001 0.9907 455 p b 0.001 1.12 0.395

Formicm** y =0.307x + 0.003 0.9959 230 p b 0.001 0.15 0.977 runs and provided a shorter separation time. b Lactics* y =0.194x + 0.007 0.9905 293 p 0.001 0.49 0.781 Bianchi et al. (2005) did not report LOD and LOQ values, resulting in b Lacticm** y =0.258x + 0.007 0.9835 95.5 p 0.001 1.36 0.297 incomplete validation of the method for the analysis of SCACA. Further- Acetic y =0.580x + 0.006 0.9994 2824 p b 0.001 0.25 0.931 s* more, although their method is the fastest of those shown in Table 5,it Aceticm** y =0.536x + 0.122 0.9983 581 p b 0.001 2.46 0.084 was used in the separation of only five SCACA. fi F: variance ratio, p:signi cance, s*: solvent calibration curve, m**: matrix matched Chen et al. (2003) used a chemically-modified capillary method calibration curve. involving toxic organic solvents such as toluene, a long coating preparation time (approximately 23 h) and several steps. Also, the high temperatures required during the coating procedure (110 °C and − the LOQ values were 2.76 ± 0.22, 8.11 ± 0.7, and 5.40 ± 0.41 mg L 1, 120 °C) makes the modification of the capillary complex and respectively. The values for the relative standard deviations were better laborious. than 11.5%, which is acceptable for indirect capillary electrophoresis Esteves et al. (2004) used CTAB as an EOF inverter and, probably methods. For the applicability of the method, these values are adequate because of the presence of this additive, the LOD was adversely since the concentrations of organic acids exceeded these amounts in affected, being higher than the values obtained for the method de- the samples analyzed. scribed herein. Their method was also used to separate only five SCACA with the same separation time as the method proposed in this article, which can separate ten SCACA. The time required for 3.2.5. Robustness the conditioning with BGE between runs was 4 min, which is consid- Youden's test is a reliable method to evaluate the robustness of ana- ered to be long and is longer than the time required in the method lytical methods by means of an experiment design, which involves proposed herein. seven analytical parameters combined in eight tests. Using the criteria Fung and Lau (2003) reported the use of an amine (TEPA) as an ad- of Youden's test, the electrophoretic method was shown to be robust re- ditive for the BGE to promote a reduction in the EOF at pH 8.4 and ob- garding the formic, lactic and acetic acid contents when variations in tain an effective separation of the analytes in the shortest possible seven analytical parameters were introduced, showing very low values time. However, in this case the additive used in the BGE was a BGE re- which would not be not significant in routine analysis. ducer, which is not recommended for detection with mass spectrometry. Moreover, the LODs obtained were of the order of μmol L−1 which is very low, but with a longer separation time compared to the method 3.3. Sample analysis proposed in this article, and 2 min was used as the conditioning time between runs with BGE. The results for the determination of SCACA in aged and non-aged Rovio et al. (2010) used CTAB as the EOF inverter and encoun- Brazilian sugarcane spirit samples are reported in Table 4. The samples tered the disadvantages mentioned above, principally the incom- were prepared with three independent replicates. Brazilian regulations patibility with the use of a mass detector. Also, in this method the recommend acid–base titration to determine the total acidity, which is BGE consisted of 2 mmol L−1 urea, which is a somewhat extreme related to the presence of organic acids in distilled beverages, especially condition and the conditioning between runs was 2 min with acetic acid, this being a quality parameter for this product (Instrução 0.1 mmol L−1 NaOHand5minwithBGE,whichrepresentsalong normativa, 2005). The results ranged from 0.19 to 2.99 mg per waiting time between runs. In the method proposed herein only 100 mL of sample (2-NA and 3-A2Y) for formic acid, 1.02 to 16.48 mg 1 min of conditioning with BGE is employed between runs, and per 100 mL (2-NA and 3-A2Y) for lactic acid, and 5.39 to 106.43 mg the coating resistance is around 40 runs (6.7 h of analysis with the per 100 mL (2-NA and 4-A2Y) for acetic acid. same coating).

4. Conclusions Table 4 Total acidity and concentration of formic, lactic and acetic acids in the Brazilian sugarcane A simple CZE method with indirect UV detection for the analysis spirits, not aged and aged, by CE-UV. of SCACA in commercial Brazilian sugarcane spirits, which can be ⁎ Sample Acid used as an alternative method, was described. This procedure en- b ⁎ ables the rapid ( 10 min separation time) and simultaneous analysis Formic Lactic Acetic Total acidity of the SCACA with no sample pre-treatment other than a simple dilu- 1-NA 0.45 ± 0.001 2.10 ± 0.04 11.5 ± 0.12 14.0 ± 0.10 tion. The capillary coating with HACC used for the generation of an 2-NA 0.19 ± 0.02 1.02 ± 0.25 5.39 ± 0.26 6.60 ± 0.07 anodic EOF contributed to rapid separations and provides good 3-A2Y 2.99 ± 0.15 16.5 ± 1.04 67.4 ± 2.98 86.8 ± 0.56 4-A2Y 1.90 ± 0.07 10.3 ± 0.45 106.4 ± 4.55 118.6 ± 1.81 precision in the migration time, permitting good reliability in the 5-A3Y 1.07 ± 0.06 3.65 ± 1.34 30.3 ± 1.09 35.4 ± 2.10 peak identiﬁcation for the samples. The results obtained demon- 6-A3Y 2.87 ± 0.13 7.88 ± 0.48 50.0 ± 1.12 60.8 ± 0.65 strate the applicability of capillary electrophoresis in the screening 7-A3Y 0.68 ± 0.02 2.53 ± 0.03 9.89 ± 0.55 13.1 ± 0.54 and quantiﬁcation of the SCACA in sugarcane spirits. The validation 8-A4Y 0.59 ± 0.04 2.83 ± 0.55 9.38 ± 0.16 12.8 ± 0.53 9-A5Y 1.49 ± 0.05 2.82 ± 0.28 30.5 ± 1.69 34.8 ± 1.44 procedure applied to the method to determine formic, acetic and 10-A6Y 0.74 ± 0.01 4.41 ± 0.49 11.0 ± 0.38 16.1 ± 0.40 lactic acids in real samples was based on the Eurachem guidelines 11-A12Y 0.88 ± 0.07 6.41 ± 1.01 12.3 ± 0.62 19.6 ± 1.07 and good results were obtained for the parameters evaluated. This 12-A16Y 1.16 ± 0.01 8.51 ± 1.09 14.1 ± 0.48 23.8 ± 1.45 method can be employed in the monitoring of these SCACA in sugar- ⁎ Concentration in mg 100 mL−1. cane spirit samples. M.S. Azevedo et al. / Food Research International 60 (2014) 123–130 129

Table 5 Methodologies developed by CE for determination of SCACA in beverages.

Matrix SCACA Capillary Electrolyte Separation LOD Run Ref mode (mg L−1) time (min)

−1 −1 Red wine Tartaric; malic; succinic; acetic; lactic Ltot = 61.2 cm 22 mmol L benzoic acid, 1.0 mol L TRIS, CZE NA 3.5 Bianchi

Ldet =50cm 35% MeOH (v/v), pH 6.1 Indirect UV et al. F =75μm detection (2005) Hexadimethrine bromide (HDB) as coating agent. −1 Brandy and juices Oxalic; formic; succinic; malonic; Ltot =70cm 6 mmol L benzoate buffer, pH 4.6. CZE 0.6 to 24 25 Chen −1 lactic; citric; malic; tartaric; acetic Ldet =50cm Indirect UV (μgL ) et al. F =75μm detection (2003) Wall-coated macrocyclic polyamine −1 −1 Port wine Tartaric; malic; lactic; succinic; acetic Ltot =78cm 5 mmol L PDC, 0.5 mmol L CTAB pH 5 CZE 1.78 to 36. 10 Esteves

Ldet =67cm Indirect UV et al. F =75μm detection (2004) −1 −1 Wines and juices Oxalic; tartaric; malic; succinic; Ltot =65cm 3 mmol L BTA, 1.5 mmol L TEPA, CZE 1to4 14 Fung phosphate, maleic; acetic; lactic; F =50μm 15 mmol L−1TRIS, pH 8.4 Indirect UV (μmol L−1) and Lau citric; glucuronic among other detection (2003) inorganic cations −1 Red wine, white wine, Oxalic; formic; tartaric; malic; Ltot =60cm 7.5 mmol L phosphate buffer (2.5 mmol L CZE 0.01 to 0.94 3.5 Mato −1 −1 grape juice, apple juice, succinic; maleic; glutaric; pyruvic; F =75μm NaH2PO4 and Na2HPO4), 2.5 mmol L UV direct at et al. −1 orange juice and acetic; lactic; citric; butyric; benzoic; TTAOH (EOF modiﬁer), 0.24 mmol L CaCl2 185 nm (2007) pineapple juice sorbic; gluconic pH 6.4 −1 −1 Must, wine, brandy, and Formic; fumaric; succinic; oxalic; Ltot =60cm 10 mmol L TBS, 0.5 mmol L TTAOH CZE 0.662 to 20 Moreno

vinegar malic; tartaric; acetic; lactic; citric Ldet =53cm (EOF modiﬁer), UV direct at 5.550 et al. F =75μm using complexing agents: Ca+2 and Mg+2 185 nm (2003) (10 mg L−1) −1 −1 Commercial softwood, Sulfuric; oxalic; formic; malonic; Ltot =60cm 20 mmol L 2,3-PyDC, 65 mmol L CZE 110Rovio −1 −1 hardwood kraft lignin fumaric; maleic; succinic; malic; Ldet =50cm tricine, 2 mmol L BaCl2,0.5mmolL Indirect UV et al. and red wine tartaric; phosphoric; acetic; glycolic; F =50μm CTAB, 2 mol L−1 urea, pH 8 detection (2010) citric; lactic −1 Brandy and wine Oxalic; maleic; phosphoric; malonic; Ltot =80mm 10 mmol L HCl, 0.1% methylhydroxyethyl CITP 3to5 20 Sádecká distillate tartaric; formic; malic; glycolic; lactic; F =0,8mm cellulose adjusted with β-alanine to pH 2.9; Conductivity (μmol L−1) et al. gluconic; succinic the terminating electrolyte: 5 mmol L−1 detector (2008) acetic acid −1 Wine, beer and juices Acetic; lactic; suberic; glutaric; Ltot =40cm 10 mmol L borate buffer, pH 10, CZE 2to10 12 Santalad −1 succinic; malic; malonic; tartaric; Ldet =30cm containing 100 mL L ACN UV direct at et al. citric (with derivatization) F =75μm 230 nm (2007) −1 −1 Sugarcane spirit Maleic; malonic; tartaric; formic; Ltot =73cm 21 mmol L β-alanine, 10 mmol L 3,5 CZE 0.69 to 2.70 10 This

citric; malic; glycolic; lactic; succinic; Ldet =64.5cm dinitrobenzoic acid, pH 3.6 for formic, work acetic F =75μm lactic and acetic

Legend: SCACA, short chain aliphatic carboxylic acids; CZE, capillary zone electrophoresis; Ltot, total length; Ldet, effective length; F, inner diameter; TBS, sodium tetraborate; TTAOH, tetradecyltrimethylammonium hydroxide; PDC, 2,6-pyridinedicarboxylic acid; CTAB, cetyltrimethylammonium bromide; TRIS, tris(hydroxymethyl)aminomethane; 2,3-PyDC, 2,3- pyrazinedicarboxylic acid; BTA, 1,3,5-benzenetricarboxylic acid; TEPA, tetraethylenepentaamine; CITP: capillary isotachophoresis; NA, not available.

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