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Optimization of the determination of organic acids and sugars in fruit juices by ion-exclusion liquid chromatography
ARTICLE IN PRESS
JOURNAL OF FOOD COMPOSITION AND ANALYSIS
Journal of Food Composition and Analysis 18 (2005) 121–130 www.elsevier.com/locate/jfca
Original Article
Optimization of the determination of organic acids and sugars in fruit juices by ion-exclusion liquid chromatography Fabio Chinnici*, Umberto Spinabelli, Claudio Riponi, Aureliano Amati Dipartimento di Scienze degli Alimenti, Universita" di Bologna, Via Fanin 40, Bologna 40127, Italy Received 11 October 2002; received in revised form 21 January 2004; accepted 26 January 2004
Abstract An HPLC method for the determination of the main organic acids and sugars in fruit juices is proposed. Nine acids (including oxalic, citric, malic, quinic, galacturonic, ascorbic, succinic, and fumaric acid) and three sugars (sucrose, glucose and fructose) were separated by ion-exclusion chromatography using a resinbased Aminex HPX 87H column after a sample clean-up with Sax cartridges. In spite of the suitable chromatographic conditions, quantification of ascorbic and dehydroascorbic acids was affected by oncolumn degradation of the former into the latter. For all the other analytes the method showed a good precision and linearity and, as an application, eighteen commercial juices from 4 different fruits were tested. Independently from the fruit, citric and malic acids were the main acids in juices, with amounts varying from 0.05 to 3.23 g/L and from 0.52 to 5.61 g/L, respectively. Compared to other fruits, pears demonstrated the highest content of succinic acid, possibly due to the coelution of relatively high quantities of shikimic acid. In some juices, small amounts of quinic, fumaric and galacturonic acids were also found. Finally, peach juices were demonstrated to be the richest in sugars, with apple juices being the poorest. r 2004 Elsevier Inc. All rights reserved. Keywords: Organic acids; Sugars; Fruit juices; HPLC; Ion-exclusion chromatography
1. Introduction The determination of organic acids and sugars in foods is very important. Their presence and relative ratio, in fact, can affect the chemical and sensorial characteristics of the matrix (e.g., pH, total acidity, microbial stability, sweetness, global acceptability) and can provide precious information on food wholesomeness or on how to optimize some selected technological processes.
*Corresponding author. Fax: +39-051-209-6017. E-mail address: [email protected] (F. Chinnici). 0889-1575/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2004.01.005
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Several methods have been published for sugars and/or organic acids determination in foods such as cheese (Mullin and Emmons, 1997; Zeppa et al., 2001), tomatoes (Velterop and Vos, 2001), green beans (Vazquez Oderiz et al., 1994), ground coffee (Rogers et al., 1999), fruits such as apricots (Bartolozzi et al., 1997), apples and pears (Drake and Eisele, 1999), kiwi (Walton and De Jong, 1990; Castaldo et al., 1982), prickly pears (El Kossori et al., 1998), blackberry, blackcurrant, babaco, passion fruit (Romero Rodriguez et al., 1992), fruit juices (Saccani et al., 1995; Hong and Wrolstad, 1986; Lee and Wrolstad, 1998), musts and wines (Auguste and Bertrand, 1980; Callul et al., 1992; Castellari et al., 2000). A number of these methods are based on GC or HPLC separation (Molna! r-Perl, 1999), sometimes with simultaneous quantification of both acids and sugars. For fruit and fruit juices, methods based on GC analysis are available (Molna! r-Perl and Morvai, 1992; Timothy et al., 1997) which, in spite of excellent separation and sensitivity, need tedious and time-consuming derivatization steps, often using toxic derivatization agents. Moreover, the high temperatures required for these analyses may lead to artifacts and sample decomposition. On the other hand, drawbacks of HPLC methods reside in the low resolution and high limits of detection. Nevertheless, due to its simplicity and to the more suitable chromatographic conditions, HPLC separation is thought to be attractive for the fast and quantitative separation of the main organic acids and sugars in fruit juices. Various HPLC methods have been proposed for fruit juices (Saccani et al., 1995; Hong and Wrolstad, 1986; Hiroshi, 2000; Badoud and Pratz, 1986; Trifiro" et al., 1997). All of these methods, however, produce the separation of a limited number of compounds or need a derivatization step. Our focus was to develop a simple and reproducible HPLC method for the rapid separation and quantification of the major organic acids and sugars in fruit juices, which could be useful for technological and screening purposes.
2. Materials and methods 2.1. Standard solutions and samples Standards of sugars and organic acids (Sigma Chemicals) commonly found in fruit juices were used within concentration ranges typical of these matrices (Table 1). Commercial apple, peach, pear and apricot juices were purchased from local stores. 2.2. Sample preparation In order to achieve the best resolution and precision, two procedures were compared: direct injection and sample clean-up with Bond Elutes Sax cartridge (Varian, Harbor City, CA). Samples, diluted 1:10 (v/v) with the mobile phase, were filtered through a 0.22 mm celluloseacetate membrane before direct injection. In comparison with this simplified procedure, pre-treatment with Sax cartridge was tested with the aim of separating the neutral compounds (e.g., sugars and alcohols) from the acidic ones. The cartridge (3 mL/500 mg) was preliminarily conditioned with methanol (3 mL) and redistilled water
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Table 1 Chromatographic parameters, concentration ranges and detection system of standard organic acids and sugars No. Compound
Detection Capacity Range tR (min) system factor (k) (mg/L)
Intercept
Slope
R2 LOD (n ¼ 5) (mg/L)
1 2 3 4 5 6 7 8 9 10 11 12 13
8.55 10.50 10.95 11.70 12.55 12.75 13.40 13.85 13.85 14.35 17.65 17.65 19.20
49,459 105,005 8791 3291 1,030,576 — 1417 92,639 10,665 — 8018 — 1,370,333
28,070 1,235,281 3349 1447 1,390,399 — 2589 1,277,484 1513 — 1608 — 293,859
0.983 0.999 0.999 0.999 0.999 — 0.999 0.998 0.999 — 0.999 — 0.998
Oxalic acid Sucrose Citric acid Galacturonic acid Glucose Dehydroascorbic acid Malic acid Fructose Quinic acid Ascorbic acid Succinic acid Shikimic acid Fumaric acid
UV RI UV UV RI UV UV RI UV UV UV UV UV
0.005 0.24 0.29 0.38 0.48 0.50 0.57 0.63 0.63 0.68 1.08 1.08 1.26
10–200 1000–25,000 50–1000 10–200 1000–25,000 — 50–1000 1000–25,000 50–1000 — 25–500 — 5–100
2.0 80 3.3 2.3 70 — 1.8 70 7.3 — 5.1 — 0.5
(3 mL). Then the sample (0.5 mL) adjusted to pH9–pH10 with 1 n NaOH, was loaded and slowly eluted (0.5 mL/min) through the cartridge. The recovery of neutral compounds was performed washing twice with 1 mL of redistilled water adjusted to pH 7 with 0.1 n NaOH, while acids were eluted with 0.5 mL of 1 n HCl (5 times). The two fractions (neutral and acidic compounds) were diluted with the mobile phase to a final volume of 5 mL, filtered through 0.22 mm membrane and finally injected in the HPLC apparatus. 2.3. Equipment and operating conditions The HPLC system (Jasco Inc., Tokyo, Japan), was equipped with an isocratic pump (PU 980), a variable wavelength detector (UV 970) set at 210 nm connected in series with a refractive index detector (RI 830) and an injection valve (Rheodyne Inc, Cotati, CA) fitted with a 20 mL loop. The samples were isocratically separated at 0.4 mL/min, using a Bio-Rad Aminex HPX 87H Hydrogen form cation exchange resin-based column (300 7.8 mm i.d.) at 25 C. For method optimization, both sulfuric and phosphoric acids were tested at concentrations ranging from 0.1 to 0.001 n. Acetonitrile and methanol were also tested as organic modifiers. Chromatographic peaks were identified by comparing retention times (tR ) with those of standards and by spiking samples with pure compounds while quantification was carried out using the external standard method. Peaks data were collected with Borwins 5.0 software (JMBS developments, Grenoble, France). 2.4. Calibration and calculation Repeatability of the method was determined by calculating the CV% of the analytes concentrations after five repeated HPLC runs of a standard solution containing each compound at the level commonly found in juices. Linearity was obtained with five solutions derived by
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sequentially diluting a concentrated standard solution. The concentration ranges of these solutions are shown in Table 1. The same calibration runs were used to determine the detection limits (LODs), which were calculated considering a signal-to-noise ratio (S/N) of 3. Recoveries were calculated by spiking fruit juices with known amounts of each compound.
3. Results and discussion Ion-exclusion chromatography is recognized as a useful technique for the separation of organic and inorganic weak acids (Tanaka et al., 1999; Ohta et al., 1996; Klampf et al., 1997). Typically, resin-based polystyrene-divinylbenzene (PS-DVB) columns in the H+ form are used, combined with aqueous solutions of sulfuric acid as eluent. In a preliminary phase, we tested both sulfuric and phosphoric acid in order to obtain the ideal chromatographic conditions. Due to better baseline stability and lower background noise, phosphoric acid was chosen and used for all the successive experiments. For organic acids, the best separation was obtained with 0.005 n phosphoric acid as eluent (Fig. 1a). Using these conditions the nine compounds were separated in less than 20 min. Oxalic acid, due to its low pKa ; was almost totally ionized and eluted near the column exclusion region which was verified for unretained compounds (sulfuric acid) at 8.50tR : For this analyte, the capacity factor was very low (Table 1) and the presence of large amounts of excluded ions or polymers was expected to represent an obstacle for its determination in fruit juices. Many efforts were made for the separation of malic, quinic and ascorbic acids, the coelution of which was resolved only by exploiting their differences in acidic strength by varying the eluent molarity. Unfortunately this was not the case for succinic and shikimic acids. Their separation, in fact, proved to be unaffected by any variation of the chromatographic conditions we tested (including use of acetonitrile and methanol as organic modifiers). This drawback was already reported by other authors (Castellari et al., 2000), and seems to be a characteristic behavior of this kind of column. So, for these acids, no separation was obtained. Runs of freshly made ascorbic acid standard solutions showed two peaks, identified as ascorbic and dehydroascorbic acid. A progressive transformation of ascorbic acid into dehydroascorbic acid was verified by repeated injections of these solutions. The high instability of ascorbate in aqueous solutions was reported earlier (Iwase and Ono, 1998; Iwase, 2000) and metaphosphoric acid is generally used as a stabilizing agent. However, even after the addition of metaphosphoric acid, a residual and progressive on-column decomposition was observed, probably related to an interaction between the analyte and the sulfonated stationary phase. Due to this problem, the calibration curves for ascorbic and dehydroascorbic acid could not be defined (Table 1) and their quantification in fruit juices was not performed (even though their presence was observed in several samples). In a separate run, mixtures of standard sugars were injected (Fig. 1b) and analyzed under the same chromatographic conditions used for organic acids. Moreover, in order to obtain a satisfactory response factor, RI detection was adopted.
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Fig. 1. Chromatograms of standard solutions. (a) UV detection and (b) RI detection. Peaks: (1) oxalic acid; (2) sucrose; (3) citric acid; (4) galacturonic acid; (5) glucose; (6) dehydroascorbic acid; (7) malic acid; (8) fructose; (9) quinic acid; (10) ascorbic acid; (11) succinic acid; (12) shikimic acid; (13) fumaric acid. Chromatographic conditions: eluent: 0.005 n phosphoric acid; flow: 0.4 mL/min; temperature: 25 C.
Thanks to the favorable elution conditions (e.g., weak acidic strength and low elution temperature), sugars showed a good linearity in the range of 1–25 g/L and suitable limits of detection (Table 1). Sucrose and glucose, which do not absorb UV wavelengths, did not interfere with the detection of organic acids. Conversely, fructose (which shows an appreciable UV absorption) eluted with the same tR of quinic acid (Table 1), obstructing its quantification. Owing to the broad diffusion of quinic acid in fruit juices, the separation of the organic acids from the sugars appeared to be necessary and was carried out by using strong anion exchange (SAX) cartridges. Table 2 reports the method precision and the recovery of spiked actual samples after the cleanup with SAX cartridges. As expected for oxalic acid, the precision was low owing to the closeness of the exclusion region, which obstructed the recovery calculation. For all the other analytes, the method showed good precision and satisfactory recoveries.
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Table 2 Method precision and recoveries of standards added to commercial fruit juices (n ¼ 5) after sample clean-up with SAXs cartridges Compound
Precision n ¼ 5 (CV%)
Recovery n ¼ 5 (%)
Oxalic acid Citric acid Galacturonic acid Malic acid Quinic acid Succinic acid Shikimic acid Fumaric acid Glucose Fructose Sucrose
3.42 1.12 0.71 0.23 0.39 0.48 — 0.82 0.94 1.05 1.23
— 97.1 94.2 97.8 101.2 98.0 — 98.8 102.3 97.1 99.1
Fig. 2. Chromatograms of fruit juice after clean-up with SAX cartridges. (a) UV detection and (b) RI detection. Peak identification and chromatographic conditions as for Fig. 1.
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Table 3 Mean content (n ¼ 3) of organic acids and sugars in commercial fruit juices (AP=apricots; AL=apples; PR=pears; PC=peaches) Sample Citric acid Galacturonic Malic acid Quinic acid Succinic Fumaric Sucrose Glucose Fructose (g/L) acid (g/L) (g/L) (g/L) acida acid (mg/L) (g/L) (g/L) (g/L) (g/L) AP 1 AP 2 AP 3 AP 4 AP 5 AL 1 AL 2 AL 3 AL 4 AL 5 PR 1 PR 2 PR 3 PR 4 PC 1 PC 2 PC 3 PC 4
2.11 2.69 3.23 2.85 3.15 oLOQc oLOD 1.09 0.84 0.05 1.45 1.81 1.94 1.98 1.04 1.49 1.77 1.91
oLODb oLOD oLOD oLOD oLOD 0.09 oLOD oLOD oLOQ oLOD oLOQ 0.02 oLOD oLOD oLOD oLOD oLOD oLOD
5.11 2.25 4.41 3.44 3.71 3.32 5.18 1.43 1.37 5.61 0.59 0.52 0.82 1.18 5.61 1.99 1.49 2.35
oLOD 0.36 0.15 oLOD 0.17 oLOQ oLOQ oLOD oLOQ 0.18 oLOQ oLOQ 0.07 0.03 0.66 0.18 0.64 0.43
0.10 0.76 0.16 0.22 0.13 0.25 0.84 0.10 0.19 0.39 1.04 0.36 3.26 0.95 1.49 0.23 0.55 0.14
2.68 2.24 3.24 2.86 2.93 2.35 1.03 0.69 0.64 1.04 0.94 0.72 1.19 1.12 3.93 1.28 1.18 1.52
54.8 34.9 55.3 69.4 39.4 7.6 15.6 38.5 30.7 17.5 42.8 54.1 53.8 43.6 19.9 65.4 53.5 77.2
45.9 57.1 52.2 45.3 62.7 32.2 31.7 45.0 34.6 24.6 34.7 37.2 32.9 33.8 40.7 42.6 36.5 38.4
35.4 40.7 33.4 41.6 44.1 63.6 53.2 45.8 44.0 62.1 29.5 51.0 58.4 45.2 75.4 38.9 22.2 34.3
a
Sum of succinic and shikimic acids. LOD=limit of detection. c LOQ=limit of quantification. b
An initial low recovery for galacturonic acid (o90%) was improved by adjusting to pH 7 the redistilled water used for the elution of neutral compounds during the sample clean-up. This expedient improved the recovery of all the acidic compounds. As an application, 18 commercial juices from 4 different fruits were evaluated for their acidic and sugar compositions through two separate runs (Figs. 2a and b). For each sample, the mean content of the identified analytes is reported in Table 3. Citric and malic acids were the main acids. Fumaric acid is believed to be an important parameter to reveal microbial spoilage or the processing of decayed fruits (Trifiro" et al., 1997). Its natural concentration in apple juices usually does not exceed 3 mg/L and higher levels can arise from microbic growth or from the addition of synthetic malic acid (Kvasnicka and Voldrich, 2000). In our samples, the content of fumaric acid was compatible with well-processed juices, with apricots and peaches showing a higher content than apples and pears. In pears, succinic acid seemed to be the principal acid, probably because of the concomitant presence of shikimic acid (which possesses a high UV response). Several fruit juices showed the presence of quinic acid, inversely to galacturonic acid whose presence in clarified juices is presumably due to the use of pectolytic formulations during the technological process.
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Fig. 3. UV detection of sugars fraction separated by SAX cartridge, showing the presence of two neutral compounds with tR of 17.70 and 19.74, potentially coeluting with succinic and/or fumaric acid.
The concentration of sugars was between about 100 g/L (in apples) and 150 g/L (in peaches and apricots). Sorbitol, which lacks UV absorption, was also detected at 14.55tR : The RI detector revealed this polyalcohol, not included in the standard mixture, in the sugars fraction of actual samples. Its identification was confirmed by the spiking technique and by comparing the retention time of a standard compound. Probably due to the sample dilutions related to their pre-treatment (1:10 v/v), only pear juices revealed a detectable sorbitol content ranging from 500 to 750 mg/L (quantified through an external standard curve). During the analysis of sugars fractions, two broad peaks absorbing at UV wavelengths and with tR of 17.70 and 19.70 were recorded (Fig. 3). Attempts to identify these peaks were unsuccessful. Nevertheless, runs carried out on a HPLC apparatus with both diode array and RI detectors led us to exclude (poly)alcohols (owing to the UV spectrum and the lack of response to RI detection) and to hypothesize some furanic compounds resulting from Maillard reactions. However, because of their likely coelution with succinic and/or fumaric acid, the usefulness of the preventive separation of acids from neutral compounds was definitely demonstrated for actual samples.
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4. Conclusions In this work we propose the use of a very common column (Aminex HPX 87H) for the quantification of 7 organic acids and 3 sugars, after a sample clean-up with SAX cartridges. After separation of the acidic and neutral fractions, they were independently analyzed by using the same chromatographic conditions within 35 min (total analysis time). A dual detection system (UV and IR) was used. The specificity of RI detector in revealing sugars and alcohols was exploited to obtain an accurate quantification of these compounds. In spite of the mild chromatographic conditions, ascorbic acid was affected by on-column degradation and its quantification could not be performed. Eighteen juices from four different fruits were analyzed and their acidic and sugars content was reported. Additionally, sorbitol was found in pear juices. Due to the extreme variability, among commercialized juices, in fruit composition (e.g., CV employed, degree of maturation, soundness) and technological parameters (e.g., percentage of water, utilization of enzymes, sugars addition), collected data could not be compared with other published data. This method can be easily applied for routine analysis in the fruit juices industry and for the determination of the main parameters related to sensory evaluation. Moreover, information on wholesomeness of juices and exogenous additions can be provided.
References # et les vins par Auguste, M.H., Bertrand, A., 1980. Dosage des acides tartrique, amilique et citrique dans les mouts chromatographie en phase liquide a" haute pression. Bulletin de l’OIV 53, 1–4. Badoud, R., Pratz, G., 1986. Improved high-performance liquid chromatographic analysis of some carboxylic acids in food and beverages as their p-nitrobenzyl esters. Journal of Chromatography A 360, 119–136. Bartolozzi, F., Bertazza, G., Bassi, D., Cristoferi, G., 1997. Simultaneous determination of soluble sugars and organic acids as their trimethylsilyl derivatives in apricot fruits by gas–liquid chromatography. Journal of Chromatography A 758, 99–107. Callul, M., Merc!e, R.M., Borrul, F., 1992. Determination of carboxylic acids, sugars, glycerol and ethanol in wine and grape must by ion-exchange chromatography with refractive index detection. Journal of Chromatography A 590, 215–222. " A., Gherardi, S., 1982. Composition of Italian kiwi (Actinidia chinensis) puree. Journal of Castaldo, D., Lo, A., Trifiro, Agricultural and Food Chemistry 40, 594–598. Castellari, M., Versari, A., Spinabelli, U., Galassi, S., Amati, A., 2000. An improved HPLC method for the analysis of organic acids, carbohydrates and alcohols in grape musts and wines. Journal of Liquid Chromatography and Related Technologies 23, 2047–2056. Drake, S.R., Eisele, T.A., 1999. Carbohydrate and acid content of Gala apples and Bartlett pears from regular and controlled atmosphere storage. Journal of Agricultural and Food Chemistry 47, 3181–3184. El Kossori, R.L., Villaume, C., El Boustami, E., Sauvaire, Y., M!ejean, L., 1998. Composition of pulp, skin and seeds of prickly pears (Opuntia ficus indica sp.). Plant Foods for Human Nutrition 52, 263–270. Hiroshi, M., 2000. High-performance liquid chromatographic determination of mono-, poly- and hydroxycarboxylic acids in foods and beverages as their 2-nitrophenylhydrazides. Journal of Chromatography A 881 (1–2), 365–385. Hong, V., Wrolstad, R.E., 1986. Cranberry juice composition. Journal of Association of Official Analytical Chemists 69, 594–597. Iwase, H., Ono, I., 1998. Determination of ascorbic acid in food by column liquid chromatography with electrochemical detection using eluent for pre-run sample stabilization. Journal of Chromatography A 806, 361–364.
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Iwase, I., 2000. Use of an amino acid in the mobile phase for the determination of ascorbic acid in food by highperformance liquid chromatography with electrochemical detection. Journal of Chromatography A 881, 317–326. Klampf, C.W., Buchberger, W., Riede, G., Bonn, G.K., 1997. Retention behavior of carboxylic acids on highly crosslinked poly(styrene-divinylbenzene)-based and silica based cation exchangers. Journal of Chromatography A 770, 23–28. Kvasnicka, F., Voldrich, M., 2000. Determination of fumaric acid in apple juice by on-line coupled capillary isotachophoresis-capillary zone electrophoresis with UV detection. Journal of Chromatography A 891, 175–181. Lee, J.S., Wrolstad, R.E., 1998. Apple juice composition: sugar, nonvolatile acid and phenolics profiles. Journal of AOAC International 71, 789–794. Molnar-Perl, I., 1999. Simultaneous quantitation of acids and sugars by chromatography: gas or high-performance liquid chromatography? Journal of Chromatography A 845, 181–195. Moln!ar-Perl, I., Morvai, M., 1992. Rapid method for the simultaneous GC quantitation of acids and sugars in fruits and vegetables. Food Additives and Contaminants 9, 505–514. Mullin, W.J., Emmons, D.B., 1997. Determination of organic acids and sugars in cheese, milk and whey by high performance liquid chromatography. Food Research International 30, 147–151. Ohta, K., Tanaka, K., Hadda, P.R., 1996. Ion-exclusion chromatography of aliphatic carboxylic acids on an unmodified silica gel column. Journal of Chromatography A 739, 359–365. Rogers, W.J., Michaux, S., Bastin, M., Buchelli, P., 1999. Changes to the content of sugars, sugar alcohols, myoinositol, carboxylic acids and inorganic anions in developing grains from different varieties of Robusta (Coffea canephora) and arabica (C. arabica) coffees. Plant Science 149 (2), 115–123. Romero Rodriguez, M.A., Vazquez Oderiz, M.L., Lopez Hernandez, J., Simal Lozano, J., 1992. Determination of vitamin C and organic acids in various fruits by HPLC. Journal of Chromatographic Science 30 (11), 433–437. " A., Soresi Bordini, C., Calza, M., Freddi, C., 1995. Use of ion chromatography for Saccani, G., Gherardi, S., Trifiro, the measurement of organic acids in fruit juices. Journal of Chromatography A 706 (1–2), 395–403. Tanaka, K., Chikara, H., Hu, W., Hasebe, K., 1999. Separation of carboxylic acids on a weakly acidic cation-exchange resin by ion-exclusion chromatography. Journal of Chromatography A 850, 187–196. Timothy, J., Barden, M.Y., Croft, E., Murby, J., Wells, J.R., 1997. Gas chromatographic determination of organic acids from fruit juices by combined resin mediated methylation and extraction in supercritical carbon dioxide. Journal of Chromatography A 785 (1–2), 251–261. " A., Saccani, G., Gherardi, S., Vicini, E., Spotti, E., Previdi, M.P., Ndagijimana, M., Cavalli, S., Reschiotto, C., Trifiro, 1997. Use of ion chromatography for monitoring spoilage in the fruit juice industry. Journal of Chromatography A 770, 243–252. Vazquez Oderiz, M.L., Vazquez Blanco, M.E., Lopez Hernandez, J., Simal Lozano, J., Romero Rodriguez, M.A., 1994. Simultaneous determination of organic acids and vitamin C in green beans by liquid chromatography. Journal of AOAC International 77 (4), 1056–1059. Velterop, J.S., Vos, F., 2001. A rapid and inexpensive microplate assay for the enzymatic determination of glucose, fructose, sucrose, l-malate and citrate in tomato (Lycopersicon esculentum) extracts and in orange juice. Phytochemical Analysis 12 (5), 299–304. Walton, E.F., De Jong, T.M., 1990. Growth and compositional changes in kiwifruit berries from three Californian locations. Annals of Botany 66, 285–298. Zeppa, G., Conterno, L., Gerbi, V., 2001. Determination of organic acids, sugars, diacetyl, and acetoin in cheese by high-performance liquid chromatography. Journal of Agricultural and Food Chemistry 49, 2722–2726.
JOURNAL OF FOOD COMPOSITION AND ANALYSIS
Journal of Food Composition and Analysis 18 (2005) 121–130 www.elsevier.com/locate/jfca
Original Article
Optimization of the determination of organic acids and sugars in fruit juices by ion-exclusion liquid chromatography Fabio Chinnici*, Umberto Spinabelli, Claudio Riponi, Aureliano Amati Dipartimento di Scienze degli Alimenti, Universita" di Bologna, Via Fanin 40, Bologna 40127, Italy Received 11 October 2002; received in revised form 21 January 2004; accepted 26 January 2004
Abstract An HPLC method for the determination of the main organic acids and sugars in fruit juices is proposed. Nine acids (including oxalic, citric, malic, quinic, galacturonic, ascorbic, succinic, and fumaric acid) and three sugars (sucrose, glucose and fructose) were separated by ion-exclusion chromatography using a resinbased Aminex HPX 87H column after a sample clean-up with Sax cartridges. In spite of the suitable chromatographic conditions, quantification of ascorbic and dehydroascorbic acids was affected by oncolumn degradation of the former into the latter. For all the other analytes the method showed a good precision and linearity and, as an application, eighteen commercial juices from 4 different fruits were tested. Independently from the fruit, citric and malic acids were the main acids in juices, with amounts varying from 0.05 to 3.23 g/L and from 0.52 to 5.61 g/L, respectively. Compared to other fruits, pears demonstrated the highest content of succinic acid, possibly due to the coelution of relatively high quantities of shikimic acid. In some juices, small amounts of quinic, fumaric and galacturonic acids were also found. Finally, peach juices were demonstrated to be the richest in sugars, with apple juices being the poorest. r 2004 Elsevier Inc. All rights reserved. Keywords: Organic acids; Sugars; Fruit juices; HPLC; Ion-exclusion chromatography
1. Introduction The determination of organic acids and sugars in foods is very important. Their presence and relative ratio, in fact, can affect the chemical and sensorial characteristics of the matrix (e.g., pH, total acidity, microbial stability, sweetness, global acceptability) and can provide precious information on food wholesomeness or on how to optimize some selected technological processes.
*Corresponding author. Fax: +39-051-209-6017. E-mail address: [email protected] (F. Chinnici). 0889-1575/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2004.01.005
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Several methods have been published for sugars and/or organic acids determination in foods such as cheese (Mullin and Emmons, 1997; Zeppa et al., 2001), tomatoes (Velterop and Vos, 2001), green beans (Vazquez Oderiz et al., 1994), ground coffee (Rogers et al., 1999), fruits such as apricots (Bartolozzi et al., 1997), apples and pears (Drake and Eisele, 1999), kiwi (Walton and De Jong, 1990; Castaldo et al., 1982), prickly pears (El Kossori et al., 1998), blackberry, blackcurrant, babaco, passion fruit (Romero Rodriguez et al., 1992), fruit juices (Saccani et al., 1995; Hong and Wrolstad, 1986; Lee and Wrolstad, 1998), musts and wines (Auguste and Bertrand, 1980; Callul et al., 1992; Castellari et al., 2000). A number of these methods are based on GC or HPLC separation (Molna! r-Perl, 1999), sometimes with simultaneous quantification of both acids and sugars. For fruit and fruit juices, methods based on GC analysis are available (Molna! r-Perl and Morvai, 1992; Timothy et al., 1997) which, in spite of excellent separation and sensitivity, need tedious and time-consuming derivatization steps, often using toxic derivatization agents. Moreover, the high temperatures required for these analyses may lead to artifacts and sample decomposition. On the other hand, drawbacks of HPLC methods reside in the low resolution and high limits of detection. Nevertheless, due to its simplicity and to the more suitable chromatographic conditions, HPLC separation is thought to be attractive for the fast and quantitative separation of the main organic acids and sugars in fruit juices. Various HPLC methods have been proposed for fruit juices (Saccani et al., 1995; Hong and Wrolstad, 1986; Hiroshi, 2000; Badoud and Pratz, 1986; Trifiro" et al., 1997). All of these methods, however, produce the separation of a limited number of compounds or need a derivatization step. Our focus was to develop a simple and reproducible HPLC method for the rapid separation and quantification of the major organic acids and sugars in fruit juices, which could be useful for technological and screening purposes.
2. Materials and methods 2.1. Standard solutions and samples Standards of sugars and organic acids (Sigma Chemicals) commonly found in fruit juices were used within concentration ranges typical of these matrices (Table 1). Commercial apple, peach, pear and apricot juices were purchased from local stores. 2.2. Sample preparation In order to achieve the best resolution and precision, two procedures were compared: direct injection and sample clean-up with Bond Elutes Sax cartridge (Varian, Harbor City, CA). Samples, diluted 1:10 (v/v) with the mobile phase, were filtered through a 0.22 mm celluloseacetate membrane before direct injection. In comparison with this simplified procedure, pre-treatment with Sax cartridge was tested with the aim of separating the neutral compounds (e.g., sugars and alcohols) from the acidic ones. The cartridge (3 mL/500 mg) was preliminarily conditioned with methanol (3 mL) and redistilled water
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Table 1 Chromatographic parameters, concentration ranges and detection system of standard organic acids and sugars No. Compound
Detection Capacity Range tR (min) system factor (k) (mg/L)
Intercept
Slope
R2 LOD (n ¼ 5) (mg/L)
1 2 3 4 5 6 7 8 9 10 11 12 13
8.55 10.50 10.95 11.70 12.55 12.75 13.40 13.85 13.85 14.35 17.65 17.65 19.20
49,459 105,005 8791 3291 1,030,576 — 1417 92,639 10,665 — 8018 — 1,370,333
28,070 1,235,281 3349 1447 1,390,399 — 2589 1,277,484 1513 — 1608 — 293,859
0.983 0.999 0.999 0.999 0.999 — 0.999 0.998 0.999 — 0.999 — 0.998
Oxalic acid Sucrose Citric acid Galacturonic acid Glucose Dehydroascorbic acid Malic acid Fructose Quinic acid Ascorbic acid Succinic acid Shikimic acid Fumaric acid
UV RI UV UV RI UV UV RI UV UV UV UV UV
0.005 0.24 0.29 0.38 0.48 0.50 0.57 0.63 0.63 0.68 1.08 1.08 1.26
10–200 1000–25,000 50–1000 10–200 1000–25,000 — 50–1000 1000–25,000 50–1000 — 25–500 — 5–100
2.0 80 3.3 2.3 70 — 1.8 70 7.3 — 5.1 — 0.5
(3 mL). Then the sample (0.5 mL) adjusted to pH9–pH10 with 1 n NaOH, was loaded and slowly eluted (0.5 mL/min) through the cartridge. The recovery of neutral compounds was performed washing twice with 1 mL of redistilled water adjusted to pH 7 with 0.1 n NaOH, while acids were eluted with 0.5 mL of 1 n HCl (5 times). The two fractions (neutral and acidic compounds) were diluted with the mobile phase to a final volume of 5 mL, filtered through 0.22 mm membrane and finally injected in the HPLC apparatus. 2.3. Equipment and operating conditions The HPLC system (Jasco Inc., Tokyo, Japan), was equipped with an isocratic pump (PU 980), a variable wavelength detector (UV 970) set at 210 nm connected in series with a refractive index detector (RI 830) and an injection valve (Rheodyne Inc, Cotati, CA) fitted with a 20 mL loop. The samples were isocratically separated at 0.4 mL/min, using a Bio-Rad Aminex HPX 87H Hydrogen form cation exchange resin-based column (300 7.8 mm i.d.) at 25 C. For method optimization, both sulfuric and phosphoric acids were tested at concentrations ranging from 0.1 to 0.001 n. Acetonitrile and methanol were also tested as organic modifiers. Chromatographic peaks were identified by comparing retention times (tR ) with those of standards and by spiking samples with pure compounds while quantification was carried out using the external standard method. Peaks data were collected with Borwins 5.0 software (JMBS developments, Grenoble, France). 2.4. Calibration and calculation Repeatability of the method was determined by calculating the CV% of the analytes concentrations after five repeated HPLC runs of a standard solution containing each compound at the level commonly found in juices. Linearity was obtained with five solutions derived by
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sequentially diluting a concentrated standard solution. The concentration ranges of these solutions are shown in Table 1. The same calibration runs were used to determine the detection limits (LODs), which were calculated considering a signal-to-noise ratio (S/N) of 3. Recoveries were calculated by spiking fruit juices with known amounts of each compound.
3. Results and discussion Ion-exclusion chromatography is recognized as a useful technique for the separation of organic and inorganic weak acids (Tanaka et al., 1999; Ohta et al., 1996; Klampf et al., 1997). Typically, resin-based polystyrene-divinylbenzene (PS-DVB) columns in the H+ form are used, combined with aqueous solutions of sulfuric acid as eluent. In a preliminary phase, we tested both sulfuric and phosphoric acid in order to obtain the ideal chromatographic conditions. Due to better baseline stability and lower background noise, phosphoric acid was chosen and used for all the successive experiments. For organic acids, the best separation was obtained with 0.005 n phosphoric acid as eluent (Fig. 1a). Using these conditions the nine compounds were separated in less than 20 min. Oxalic acid, due to its low pKa ; was almost totally ionized and eluted near the column exclusion region which was verified for unretained compounds (sulfuric acid) at 8.50tR : For this analyte, the capacity factor was very low (Table 1) and the presence of large amounts of excluded ions or polymers was expected to represent an obstacle for its determination in fruit juices. Many efforts were made for the separation of malic, quinic and ascorbic acids, the coelution of which was resolved only by exploiting their differences in acidic strength by varying the eluent molarity. Unfortunately this was not the case for succinic and shikimic acids. Their separation, in fact, proved to be unaffected by any variation of the chromatographic conditions we tested (including use of acetonitrile and methanol as organic modifiers). This drawback was already reported by other authors (Castellari et al., 2000), and seems to be a characteristic behavior of this kind of column. So, for these acids, no separation was obtained. Runs of freshly made ascorbic acid standard solutions showed two peaks, identified as ascorbic and dehydroascorbic acid. A progressive transformation of ascorbic acid into dehydroascorbic acid was verified by repeated injections of these solutions. The high instability of ascorbate in aqueous solutions was reported earlier (Iwase and Ono, 1998; Iwase, 2000) and metaphosphoric acid is generally used as a stabilizing agent. However, even after the addition of metaphosphoric acid, a residual and progressive on-column decomposition was observed, probably related to an interaction between the analyte and the sulfonated stationary phase. Due to this problem, the calibration curves for ascorbic and dehydroascorbic acid could not be defined (Table 1) and their quantification in fruit juices was not performed (even though their presence was observed in several samples). In a separate run, mixtures of standard sugars were injected (Fig. 1b) and analyzed under the same chromatographic conditions used for organic acids. Moreover, in order to obtain a satisfactory response factor, RI detection was adopted.
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Fig. 1. Chromatograms of standard solutions. (a) UV detection and (b) RI detection. Peaks: (1) oxalic acid; (2) sucrose; (3) citric acid; (4) galacturonic acid; (5) glucose; (6) dehydroascorbic acid; (7) malic acid; (8) fructose; (9) quinic acid; (10) ascorbic acid; (11) succinic acid; (12) shikimic acid; (13) fumaric acid. Chromatographic conditions: eluent: 0.005 n phosphoric acid; flow: 0.4 mL/min; temperature: 25 C.
Thanks to the favorable elution conditions (e.g., weak acidic strength and low elution temperature), sugars showed a good linearity in the range of 1–25 g/L and suitable limits of detection (Table 1). Sucrose and glucose, which do not absorb UV wavelengths, did not interfere with the detection of organic acids. Conversely, fructose (which shows an appreciable UV absorption) eluted with the same tR of quinic acid (Table 1), obstructing its quantification. Owing to the broad diffusion of quinic acid in fruit juices, the separation of the organic acids from the sugars appeared to be necessary and was carried out by using strong anion exchange (SAX) cartridges. Table 2 reports the method precision and the recovery of spiked actual samples after the cleanup with SAX cartridges. As expected for oxalic acid, the precision was low owing to the closeness of the exclusion region, which obstructed the recovery calculation. For all the other analytes, the method showed good precision and satisfactory recoveries.
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Table 2 Method precision and recoveries of standards added to commercial fruit juices (n ¼ 5) after sample clean-up with SAXs cartridges Compound
Precision n ¼ 5 (CV%)
Recovery n ¼ 5 (%)
Oxalic acid Citric acid Galacturonic acid Malic acid Quinic acid Succinic acid Shikimic acid Fumaric acid Glucose Fructose Sucrose
3.42 1.12 0.71 0.23 0.39 0.48 — 0.82 0.94 1.05 1.23
— 97.1 94.2 97.8 101.2 98.0 — 98.8 102.3 97.1 99.1
Fig. 2. Chromatograms of fruit juice after clean-up with SAX cartridges. (a) UV detection and (b) RI detection. Peak identification and chromatographic conditions as for Fig. 1.
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Table 3 Mean content (n ¼ 3) of organic acids and sugars in commercial fruit juices (AP=apricots; AL=apples; PR=pears; PC=peaches) Sample Citric acid Galacturonic Malic acid Quinic acid Succinic Fumaric Sucrose Glucose Fructose (g/L) acid (g/L) (g/L) (g/L) acida acid (mg/L) (g/L) (g/L) (g/L) (g/L) AP 1 AP 2 AP 3 AP 4 AP 5 AL 1 AL 2 AL 3 AL 4 AL 5 PR 1 PR 2 PR 3 PR 4 PC 1 PC 2 PC 3 PC 4
2.11 2.69 3.23 2.85 3.15 oLOQc oLOD 1.09 0.84 0.05 1.45 1.81 1.94 1.98 1.04 1.49 1.77 1.91
oLODb oLOD oLOD oLOD oLOD 0.09 oLOD oLOD oLOQ oLOD oLOQ 0.02 oLOD oLOD oLOD oLOD oLOD oLOD
5.11 2.25 4.41 3.44 3.71 3.32 5.18 1.43 1.37 5.61 0.59 0.52 0.82 1.18 5.61 1.99 1.49 2.35
oLOD 0.36 0.15 oLOD 0.17 oLOQ oLOQ oLOD oLOQ 0.18 oLOQ oLOQ 0.07 0.03 0.66 0.18 0.64 0.43
0.10 0.76 0.16 0.22 0.13 0.25 0.84 0.10 0.19 0.39 1.04 0.36 3.26 0.95 1.49 0.23 0.55 0.14
2.68 2.24 3.24 2.86 2.93 2.35 1.03 0.69 0.64 1.04 0.94 0.72 1.19 1.12 3.93 1.28 1.18 1.52
54.8 34.9 55.3 69.4 39.4 7.6 15.6 38.5 30.7 17.5 42.8 54.1 53.8 43.6 19.9 65.4 53.5 77.2
45.9 57.1 52.2 45.3 62.7 32.2 31.7 45.0 34.6 24.6 34.7 37.2 32.9 33.8 40.7 42.6 36.5 38.4
35.4 40.7 33.4 41.6 44.1 63.6 53.2 45.8 44.0 62.1 29.5 51.0 58.4 45.2 75.4 38.9 22.2 34.3
a
Sum of succinic and shikimic acids. LOD=limit of detection. c LOQ=limit of quantification. b
An initial low recovery for galacturonic acid (o90%) was improved by adjusting to pH 7 the redistilled water used for the elution of neutral compounds during the sample clean-up. This expedient improved the recovery of all the acidic compounds. As an application, 18 commercial juices from 4 different fruits were evaluated for their acidic and sugar compositions through two separate runs (Figs. 2a and b). For each sample, the mean content of the identified analytes is reported in Table 3. Citric and malic acids were the main acids. Fumaric acid is believed to be an important parameter to reveal microbial spoilage or the processing of decayed fruits (Trifiro" et al., 1997). Its natural concentration in apple juices usually does not exceed 3 mg/L and higher levels can arise from microbic growth or from the addition of synthetic malic acid (Kvasnicka and Voldrich, 2000). In our samples, the content of fumaric acid was compatible with well-processed juices, with apricots and peaches showing a higher content than apples and pears. In pears, succinic acid seemed to be the principal acid, probably because of the concomitant presence of shikimic acid (which possesses a high UV response). Several fruit juices showed the presence of quinic acid, inversely to galacturonic acid whose presence in clarified juices is presumably due to the use of pectolytic formulations during the technological process.
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Fig. 3. UV detection of sugars fraction separated by SAX cartridge, showing the presence of two neutral compounds with tR of 17.70 and 19.74, potentially coeluting with succinic and/or fumaric acid.
The concentration of sugars was between about 100 g/L (in apples) and 150 g/L (in peaches and apricots). Sorbitol, which lacks UV absorption, was also detected at 14.55tR : The RI detector revealed this polyalcohol, not included in the standard mixture, in the sugars fraction of actual samples. Its identification was confirmed by the spiking technique and by comparing the retention time of a standard compound. Probably due to the sample dilutions related to their pre-treatment (1:10 v/v), only pear juices revealed a detectable sorbitol content ranging from 500 to 750 mg/L (quantified through an external standard curve). During the analysis of sugars fractions, two broad peaks absorbing at UV wavelengths and with tR of 17.70 and 19.70 were recorded (Fig. 3). Attempts to identify these peaks were unsuccessful. Nevertheless, runs carried out on a HPLC apparatus with both diode array and RI detectors led us to exclude (poly)alcohols (owing to the UV spectrum and the lack of response to RI detection) and to hypothesize some furanic compounds resulting from Maillard reactions. However, because of their likely coelution with succinic and/or fumaric acid, the usefulness of the preventive separation of acids from neutral compounds was definitely demonstrated for actual samples.
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4. Conclusions In this work we propose the use of a very common column (Aminex HPX 87H) for the quantification of 7 organic acids and 3 sugars, after a sample clean-up with SAX cartridges. After separation of the acidic and neutral fractions, they were independently analyzed by using the same chromatographic conditions within 35 min (total analysis time). A dual detection system (UV and IR) was used. The specificity of RI detector in revealing sugars and alcohols was exploited to obtain an accurate quantification of these compounds. In spite of the mild chromatographic conditions, ascorbic acid was affected by on-column degradation and its quantification could not be performed. Eighteen juices from four different fruits were analyzed and their acidic and sugars content was reported. Additionally, sorbitol was found in pear juices. Due to the extreme variability, among commercialized juices, in fruit composition (e.g., CV employed, degree of maturation, soundness) and technological parameters (e.g., percentage of water, utilization of enzymes, sugars addition), collected data could not be compared with other published data. This method can be easily applied for routine analysis in the fruit juices industry and for the determination of the main parameters related to sensory evaluation. Moreover, information on wholesomeness of juices and exogenous additions can be provided.
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