Quantitation determination of chlorogenic acid in cider apple juices by ...

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Keywords: Chlorogenic acid; Apple juice; Nuclear magnetic resonance. 1. Introduction .... was achieved with MestRe-C software package [16]. 2.4. Calibration ...
Analytica Chimica Acta 486 (2003) 269–274

Quantitation determination of chlorogenic acid in cider apple juices by 1H NMR spectrometry Iñaki Berregi a,∗ , José Ignacio Santos a , Gloria del Campo a , José Ignacio Miranda b , Jesus Maria Aizpurua b a b

Applied Chemistry Department, Faculty of Chemistry, University of the Basque Country, Manuel Lardizabal 3, 20018 Donostia-San Sebastián, Spain Organic Chemistry Department, Faculty of Chemistry, University of the Basque Country, Manuel Lardizabal 3, 20018 Donostia-San Sebastián, Spain Received 20 January 2003; accepted 17 April 2003

Abstract This article introduces a method for the determination of chlorogenic acid in apple juices by measuring its signal at 7.20 ppm in the 1 H NMR spectrum. The method is direct and does not need any previous derivatization. The addition to the juice of 1,3,5-benzenetricarboxylic acid as internal standard allows the determination of the absolute concentration of chlorogenic acid. Ascorbic acid is also added to prevent enzymatic oxidation of the phenolic compounds and to adjust the pH to 2.74, since the chemical shifts of some compounds vary with the pH. A standard addition method performed with the juices of three different varieties of apples gave recoveries between 91 and 107%. The precision of the method was tested for repeatability (n = 5) and reproducibility (n = 15), obtaining coefficient of variation of 5.7 and 7.5%, respectively, for a sample of Gezamina apple juice (586 mg l−1 chlorogenic acid). © 2003 Elsevier Science B.V. All rights reserved. Keywords: Chlorogenic acid; Apple juice; Nuclear magnetic resonance

1. Introduction Besides water, sugars (glucose, fructose and sucrose) and malic acid are the main components of apple juice and they give, respectively, the dominant taste sensations of sweetness and acidity to all apple products. Phenolic compounds are present in lower proportion but they give bitterness and astringency to the juices, and contribute to the flavour of processed apple products like cider [1]. Moreover, browning of apples is due to enzymatic oxidation of phenolic compounds ∗ Corresponding author. Tel.: +34-943015419; fax: +34-943212236. E-mail address: [email protected] (I. Berregi).

to quinones, which subsequently polymerize to brown products. From the point of view of health, some of these phenolic compounds, such as hydroxycinnamic acid derivatives, are an important source of antioxidants and free radical scavengers [2]. Among the hydroxycinnamic acids presents in apple juice, chlorogenic acid is the most abundant. Moreover, it is one of the main phenolic compounds in cider apples, together with epicatechin and procyanidin B2 [3]. This acid is considered as the preferential natural substrate for polyphenol oxidase and therefore it may have an effect on the oxidation process and colour development during cider making [4]. Analysis for phenolic compounds is generally accomplished by means of liquid chromatography (LC)

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with UV-visible spectrometric detection. Due to the complexity of the analysis, some steps before LC, such as extraction, purification, and concentration, must be carried out [5]. Proton nuclear magnetic resonance spectroscopy (1 H NMR) is becoming of great importance in the analysis of food products because it is non-destructive, selective and capable of simultaneous detection of a number of chemicals (sugars, organic and amino acids, phenolics, etc.) in a single spectrum. This method also offers advantages in terms of rapidity and simplicity of sample preparation. 1 H NMR, together with principal component analysis, has been used to differentiate the origin of wines [6], types of beers [7], and to detect adulteration in olive oils [8] and orange juices [9]. However, to our knowledge, quantification has not been carried out using 1 H NMR, with the exception of the work of Košir et al. [10] that uses peak heights to quantify organic acids in wines. We have found a determination using 31 P NMR that needs previous derivatization of the analyte [11,12] and also a determination of carbohydrates by 13 C NMR, using peak heights and the usual 1,4-dioxan as internal standard [13]. In this article, we report a direct determination method for chlorogenic acid in apple juices by means of its 1 H NMR signals, without any derivatization, using a new specific internal standard and peak areas measurement. 2. Experimental 2.1. Obtaining and storing apple juices Basque cider apples of the varieties Gezamina, Goikoetxea and Txalaka were harvested at maturity during the 2001 season from different plantations of Gipuzkoa (Basque Country, Spain). Each variety was crushed and pressed separately. Ascorbic acid (5 g/100 ml) was added immediately to each juice to prevent enzymatic oxidation of the phenolic compounds and also to adjust the pH to the same value (2.74 ± 0.07) in all the juices because, for some compounds, the chemical shifts in 1 H NMR spectra are dependent on pH [14]. The juices were then clarified by centrifugation (10,000 rpm, 15 min), microfiltered through a 0.45 ␮m pore size filter and stored at −20 ◦ C until recording the 1 H NMR spectra.

2.2. Preparing the D2 O–TSS–BTC solution 0.1250 g of [1,3,5-benzenetricarboxylic acid] (BTC) was dissolved in 10–15 ml of 1 M NaOH. After dissolution, the solution was neutralized with HCl and diluted to 25 ml with water. 0.1 g of the sodium salt of 3-(trimethylsilyl)-1-propanesulfonic acid (TSS) was dissolved in 7 ml of D2 O, 2 ml of the BTC solution and 1 ml of D2 O were added, and the solution was made up to 10 ml with water. The final concentrations were 10 g l−1 of TSS, 1.000 g l−1 of BTC and 70% D2 O. 2.3. Recording of 1 H NMR spectra After thawing a juice, 600 ␮l was placed in a 5 mm outer diameter NMR tube and 100 ␮l of the D2 O–TSS–BTC solution added. The final concentrations of TSS and BTC were 1.4 and 0.1430 g l−1 , respectively. D2 O served as the field frequency lock and all spectra were referenced to the signal from TSS at δ = 0.00 ppm. BTC was added in exactly known concentration as an internal standard which supplied a reference peak for the phenolic region (5.8–8.5 ppm). Using a Bruker DRX-500 spectrometer, 500 MHz 1 H NMR spectra were recorded. One hundred and twenty-eight scans of 64 K data points were acquired with a spectral width of 8012 Hz (16 ppm), acquisition time of 4.09 s, recycle delay of 1 s and flip angle of 90◦ . Solvent suppression was achieved using the Watergate pulse sequence [15]. The data were acquired under an automation procedure, requiring about 11 min per sample. Preliminary data processing was carried out with Bruker software, version 2.5. The FIDs were Fourier transformed (0.4 Hz line broadening) and the spectra were phased and baseline corrected. The resulting spectra were then aligned by right or left shifting as necessary (using the TSS signal as reference), saved as ASCII files and transferred to a PC. Data analysis was achieved with MestRe-C software package [16]. 2.4. Calibration graphs Six hundred microliters mix of adequate volumes of 1000 mg l−1 chlorogenic acid and water were placed in the NMR tubes. After adding 100 ␮l of the D2 O–TSS–BTC solution, 1 H NMR spectra were

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obtained in the same way as before. Final chlorogenic acid concentrations were obtained in the range 0–900 mg l−1 . 2.5. Standard additions After thawing a juice, 200 ␮l was placed in an NMR tube and a further 400 ␮l was made up with appropriate volumes of 1000 mg l−1 chlorogenic acid solution and water. The remaining 100 ␮l was occupied with D2 O–TSS–BTC solution, and 1 H NMR spectra were recorded as before. The final chlorogenic acid concentrations added were from 0 to 500 mg l−1 . 3. Results and discussion Fig. 1 shows the whole NMR spectrum from the apple variety called Gezamina. The other varieties present qualitatively similar spectra and they can be divided into three regions [17]. The high field region

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(0.0–2.5 ppm) contains mainly signals from the alkyl residues of free amino acids, some organic acids and alcohols. The central region (2.5–5.8 ppm) is dominated by signals from the components of highest concentration; glucose, fructose and sucrose. The low field region (5.8–8.5 ppm) contains a number of signals from aromatic protons of phenolic constituents. This is the working region for the determination of chlorogenic acid. It has been enlarged in the figure insert because its signals are the least intense of the spectrum and, therefore, the vertical gain has to be considerably expanded. Fig. 2 is an expansion of the working region of the spectrum. The signals of chlorogenic acid are indicated on the spectrum and correspond to chemical shifts of 7.64, 7.20, 7.13, 6.96 and 6.38 ppm [18]. The 7.20 ppm signal was selected for measurement because it is strong enough and appears alone, hence, its intensity was used for calibration. The 6.96 ppm signal is inadequate since it is overlapped by many phenolic

Fig. 1. 1 H NMR spectrum from apple juice of the variety Gezamina.

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Fig. 2. Low field 1 H NMR spectrum from apple juice of the variety Gezamina. Chlorogenic acid and BTC signals are indicated.

Fig. 3. Low field 1 H NMR spectrum of chlorogenic acid and its formula. Chemical shifts and protons corresponding to the signals are indicated.

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Table 1 Sweetness, acidity and bitterness of the apple varieties studied: total sugar, malic acid and phenolic contents Variety

Number of samples

Total sugar contenta (g l−1 )

Malic acid contentb (g l−1 )

Phenolic compoundsc (g l−1 )

Gezamina Goikoetxea Txalaka

5 8 8

128.7 111.2 96.7

2.98 6.75 9.13

4.89 1.92 2.51

a

Determined enzymatically. Determined by HPLC. c Determined by Folin–Ciocalteu method and expressed as tannic acid. b

signals, as for example, the 6.93 ppm signal of epicatechin, also present in apple juices. In fact, the 6.92 ppm signal is assigned to “polyphenols”. Fig. 3 shows the NMR spectrum of chlorogenic acid with BTC added. Protons corresponding to the signals are also indicated. The distortion observed in the 7.64 and 6.38 ppm peaks is caused by the Watergate pulse sequence used to suppress the solvent signal. This does not give any problem, as these peaks were not used for quantification. To choose the internal standard for phenolic compounds, three factors were taken into account: the standard had to be absent from the sample, it had to be soluble in water and it had to give a clear signal, preferably a singlet, in the low field (phenolic) region of the spectrum. BTC is not present in apple juices, is soluble in water, although a basic medium is necessary to dissolve it, and it gives a clear and strong singlet signal, also indicated in Fig. 3. This signal does not appear at a fixed chemical shift but it is always situated in the 8.5–8.8 ppm interval and, what is most important, it is never overlapped by any other signal from the apple juices. Therefore, BTC was considered to be a very suitable compound to be used as an internal standard for phenolics. A calibration graph was obtained by plotting the ratio between the peak areas of chlorogenic acid (A) and the internal standard BTC (AST ) against chlorogenic acid concentration (C, mg l−1 ), at the selected chemical shifts. Processing of the data with the aid of the program SPSS 11.0 for Windows generated the following equation: A/AST = (1.922 ± 0.034) × 10−3 C −(2.843 ± 1.802) × 10 (n = 6,

r = 0.9994,

−2

Sy/x = 2.6780 × 10−2 )

The limit of detection, calculated from “3Sy/x + intercept”, was 42 mg l−1 . To evaluate potential matrix effects, a standard addition method was applied to the juices of the apple varieties Gezamina, Goikoetxea and Txalaka. These varieties were selected by considering that, owing to their differences in sweetness, acidity and bitterness (see Table 1), they are representative of a large apple ensemble and, consequently, the method can also be applied to other apple juice matrices. The juices were spiked with chlorogenic acid (see Section 2) and its concentration was calculated from the 1 H NMR spectra with the calibration equation. The results, listed in Table 2, show that satisfactory recoveries were obtained. This demonstrates that no matrix effects are observed, so the method proposed is valid for the direct determination of chlorogenic acid in apple juices. The fidelity of the method was verified by a repeatability and reproducibility test. It was performed by Table 2 Determination of chlorogenic acid in apple juices from three different varieties using the standard addition method Variety

Chlorogenic acid Added (mg l−1 )

Found (mg l−1 )

Recovery (%)

Gezamina

0 102 205

586 679 805

– 91 107

Goikoetxea

0 151 301 422

153 311 438 584

– 105 95 102

Txalaka

0 102 305 509

45 153 342 557

– 106 97 101

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obtaining the 1 H NMR spectra of many samples corresponding to Gezamina variety apple juice and calculating the concentration of chlorogenic acid for each sample with the calibration equation. A coefficient of variation of 5.7% was obtained for 5 measurements made in the same day (repeatability), and 7.5% for 15 measurements achieved over 3 days (reproducibility), which were considered satisfactory. Acknowledgements We thank the “Gipuzkoako Foru Aldundia” for its financial support and the NMR service of the faculty of Chemistry of Donostia, University of the Basqune Country, Donostia, for its professional work. References [1] D.L. Downing, Processed Apple Products, Van Nostrand Reinhold, New York, 1989 (Chapter 15). [2] J.J. Mangas, R. Rodr´ıguez, B. Suárez, A. Picinelli, E. Dapena, J. Agric. Food Chem. 47 (1999) 4046. [3] R.M. Alonso-Salces, E. Korta, A. Barranco, L.A. Berrueta, B. Gallo, F. Vicente, J. Agric. Food Chem. 49 (2001) 3761.

[4] P. Sanoner, S. Guyot, N. Marnet, D. Molle, J.-F. Drilleau, J. Agric. Food Chem. 47 (1999) 4847. [5] J. J Mangas, B. Suárez, A. Picinelli, J. Moreno, D. Blanco, J. Agric. Food Chem. 45 (1997) 4777. [6] I.J. Košir, J. Kidriˇc, Anal. Chim. Acta 458 (2002) 77. [7] I. Duarte, A. Barros, P.S. Belton, R. Righelato, M. Spraul, E. Humpfer, A.M. Gil, J. Agric. Food Chem. 50 (2002) 2475. [8] C. Fauhl, F. Reniero, C. Guillou, Magn. Reson. Chem. 38 (2000) 436. [9] J.T.W.E. Vogels, L. Terwel, A.C. Tas, F. van den Berg, F. Dukel, J. van der Greef, J. Agric. Food Chem. 44 (1996) 175. [10] I. Košir, M. Kocjanˇciˇc, J. Kidriˇc, Analusis 26 (1998) 97. [11] A. Spyros, P. Dais, J. Agric. Food. Chem. 48 (2000) 802. [12] P. Fronimaki, A. Spyros, S. Christophoridou, P. Dais, J. Agric. Food. Chem. 50 (2002) 2207. [13] V. Mazzoni, P. Bradesi, F. Tomi, J. Casanova, Magn. Reson. Chem. 35 (1997) S81. [14] G. Le Gall, M. Puaud, I.J. Colquhoun, J. Agric. Food. Chem. 49 (2001) 580. [15] M. Liu, X. Mao, C. He, H. Huang, J.K. Nicholson, J.C. Lindon, J. Magn. Reson. 132 (1998) 125. [16] J.C. Cobas, F.J. Sardina, MestRe-C, Beta 3.0.0.2 version, University of Santiago de Compostela, Spain, 2002. [17] I.J. Colquhoun, Spectrosc. Europe 10 (1) (1998) 8. [18] P.S. Belton, I. Delgadillo, A.M. Gil, P. Roma, F. Casuscelli, I.J. Colquhoun, M.J. Dennis, M. Spraul, Magn. Reson. Chem. 35 (1997) S52.