View Article Online / Journal Homepage / Table of Contents for this issue ANALYST, MARCH 1984, VOL 109 263 Chlorogenic Acid Composition of Instant Coffees Published on 01 January 1984 Downloaded by University of Connecticut on 28/10/2014 16:16:40 Luiz C Trugo and Robert Macrae Department of Food Science, University of Reading, London Road, Reading, RG7 5AQ, UK A method, based on high-performance liquid chromatography, is described for the determination of chlorogenic acid isomers in instant coffee Identification of the individual components was assisted by the preparation of isomeric mixtures by the isomerisation of available compounds and quantification based on published ultraviolet molar absorptivities A number of extraction and clearing techniques were studied to determine the optimum conditions for the recovery of all the isomers present The developed method was applied to a wide range of commercially available instant coffees Keywords: Chlorogenic acids; high-performance liquid chromatography; coffee The chlorogenic acid composition of coffee products is extremely complex with at least five major groups of compounds present: caffeoylquinic acids (CQA), dicaffeoylquinic acids (diCQA), feruloylquinic acids (FQA) ,p-coumaroylquinic acids (CoQA) and caffeoylferuloylquinic acids (CFQA).1 The general structure of chlorogenic acids is shown in Fig There are also a wide range of more minor chlorogenic acid components that have received little attention Several attempts have been made to correlate (inversely) the levels of chlorogenic acids with beverage quality and to correlate specific sensory attributes such as astringency, to the presence of specific chlorogenic isomers There is little evidence in the literature to support these ideas but it is generally accepted that Arabica coffee, with a lower chlorogenic acid content, is superior to Robusta coffee.' A significant loss of chlorogenic acids, in some instances up to 90Y0,~takes place on roasting Additionally, the levels found in instant coffees will depend on the extraction conditions These factors, coupled with a large difference between varieties, means that commercial instant coffees would be expected to vary widely in terms of chlorogenic acid composition A number of methods have been described in the literature for the determination of chlorogenic acids in green, roasted and instant coffees The simplest methods for analysis are based on ultraviolet absorption of alcoholic extracts3 or colorimetry.4 The former lacks specificity and the latter even using differential colorimetric techniques is subject to interferences and individual isomers cannot be determined Chromatographic techniques have greatly improved the precision of analytical data and, even though gas chromatography provides excellent resolution ,5 highperformance liquid chromatography (HPLC) is preferred, as it avoids the derivation step in the analysis.6.7 This paper presents an HPLC method for the determination of the nine main chlorogenic acids in a range of commercial instant coffees Particular attention has been paid to extraction efficiency and the optimisation of chromatographic resolution, factors that have received only brief consideration in some published methods Extraction solvents, aqueous methanol (40 and 70% VIV), aqueous propan-2-01 (70% VIV) and aqueous ethanol (70% ViV Chromatography solvents, tripotassium citrate (K3C6H H ) ,0.01 M Adjusted to pH 2.5 with dilute hydrochloric acid and HPLC-grade methanol Chlorogenic acid solutions The IUPAC nomenclature8 is used to name chlorogenic acid isomers 5-Caffeoylquinic acid (commerically termed chlorogenic acid) was obtained from Sigma, UK 5-Feruloylquinic acid solution, mg ml- I Obtained from Dr M Clifford (University of Surrey) Dicaffeoylquinic acid mixture (commercially termed isochlorogenic acid) Obtained from Roth, F.R.G Apparatus and Chromatographic Conditions An Applied Chromatography Systems liquid chromatograph (two pumps, Model 750103, and a programmer, Model 750136) was used Injection was achieved by means of a fixed-loop Rheodyne injection valve, Model 7120 (20 pl) A pre-column (50 x mm i.d.) dry packed with coarse silica (70-150 pm) was placed in-line before the injection valve in order to saturate the mobile phase A Spherisorb 5-ODS column (Phase Separations Ltd., UK, 2.50 x mm i.d.) was used for the chromatographic separations The final conditions used were a gradient of methanol (see Fig 3) in 0.01 M tripotassium citrate solution (pH 5), increasing from 20 to 70% with 5-min linear segments of , , , , , , , 2and 0% min-1 and a flow-rate of ml min-1 Peak integration was achieved by means of a Hewlett-Packard 3390-A integrator im:H H HO I HO-C OH I HO Experimental Reagents All reagents and solvents were of analytical-reagent grade unless otherwise specified Carrez solutions (clearing agent) Solution (I) was prepared by dissolving 21.9 g of crystallised zinc acetate, Zn(C2H302)2.2H20, and ml of glacial acetic acid in distilled water and diluting to 100 ml Solution (11) consisted of 10.6 g of potassium hexacyanoferrate(I1) in 100 ml of distilled water Ammonia solution, approximately M Hydrochloric acid, approximately M ' OH (b) Fig Structure of chlorogenic acids ( a ) D-(-)-Quinic acid ( b ) R = H, 5-p-coumaroylquinic acid; R = OH, 5-caffeoylquinic acid; and R = OCH,, 5-feruloylquinic acid Esterification is also possible at carbon atoms and of the quinic acid View Article Online 264 ANALYST, MARCH 1984, VOL 109 Procedure Published on 01 January 1984 Downloaded by University of Connecticut on 28/10/2014 16:16:40 Extraction Finely ground instant coffee (0.5 g) was dissolved in 80 ml of an aqueous methanol solution (40%) and transferred into a 100-ml calibrated flask Carrez solution (2 ml of each component) was added and the mixture was allowed to stand for 10 after making up to volume The precipitate was removed by filtration under gravity (using Whatman No filter-paper) and the filtrate was used directly for chromatography For testing extraction efficiencies 70% V/V aqueous solutions of propan-2-01, ethanol and methanol were also used Isomerisation of chlorogenic acids 5-Caffeoylquinic acid (200 mg) was dissolved in distilled water (20 ml) and the pH was adjusted to with dilute ammonia solution This solution was heated for 30 in a boiling water-bath The pH was then adjusted to 2.5-3.0 with dilute hydrochloric acid, after cooling to room temperature For the preparation of feruloylquinic acid isomers, 20 1-11 of the standard solution of 5-feruloylquinic acid were added to ml of water, adjusted to pH with dilute ammonia solution and heated for 15 in a boiling water-bath before being evaporated to dryness under reduced pressure and the residue dissolved in 0.1 ml of dilute hydrochloric acid These solutions were then used directly for chromatography Quantification Quantification was achieved by peak-area measurement and by comparison with a 5-caffeoylquinic acid standard It was possible to quantify the individual isomers using their molar absorptivities, according to the equation C= RFX X E2 M,, X before and after precipitation of the chlorogenic acids, is adequate for quality control purposes However, where the determination of individual components is required, HPLC provides the simplest method With reference to the published HPLC methods6.7 there appear to be three areas where improvements could be made (unambiguous peak assignment, greater chromatographic resolution and substantiation of the methods in terms of recovery and precision) and these aspects are addressed in this paper The problem of peak assignment is mainly due to the non-availability of chromatographic standards 5-Caffeoylquinic acid and a mixture containing the three dicaffeoylquinic acids are available This range of standards can be extended by isomerising the 5-caffeoylquinic acid to yield an equilibrium mixture of the 3-, 4- and 5-isomers The normal isomerisation conditions consist of heating the chlorogenic acid, either in a phosphate buffer of pH 7-7.211 or in saturated sodium hydrogen carbonate solution.12 However, a much cleaner reaction can be realised simply by heating the acid in dilute ammonia solution at pH The rate of isomerisation can be readily followed by HPLC, the reaction being stopped by acidification after various intervals (Fig 2) The graphs show clearly that one isomer is initially formed at a greater rate than the other and that the final concentrations are approximately equal This is consistent with the isomerisation taking place via an intramolecular mechanism with the 4-isomer being formed first and subsequently isomerising to yield the 3-isomer On this basis the isomer initially formed at the greater rate was assumed to be 4-CQA This assignment is also supported by a consideration of the polarity of the structures (see Fig 1) The 3-isomer contains two equatorial and one axial hydroxyl groups and the 4-isomer contains one equatorial and two axial hydroxyl groups, conferring greater polarity to the former 13 A x M,, where C is the concentration of the isomer in milligrams per millitre; RF is the response factor of 5-caffeoylquinic acid standard (concentration in milligrams per millilitre per unit area); c1 is the molar absorptivity of 5-caffeoylquinic acid; e2 is the molar absorptivity of the isomer in question; Mr, is the relative molecular mass of 5-caffeoylquinic acid; Mr, is the relative molecular mass of the isomer in question; and A is the area of the peak corresponding to the isomer in question Molar absorptivities ( x lo4) are as follows: at A,,,.330 nm, 5-CQA = 1.95,4-CQA = 1.80,3-CQA = 1.84,3,4-diCQA = 3.18, 3,5-diCQA = 3.16 and 4,5-diCQA = 3.32; and at A,, 325 nm, 5-FQA = 1.93, 4-FQA = 1.95 and 3-FQA = 1.90 mol-1 cm-1.9 Calibration graphs were plottcd using the CQA and diCQA isomer mixtures diluted at five different concentrations These concentrations varied from 10 to 100 pg ml-1 in the CQA group and from to 15 1-18 ml-1 in the diCQA group Recoveries were checked by the method of standard additions The levels of addition to the samples (in grams of isomer per 100 g of sample) were 3-CQA, 0.3-1.6; 4-CQA, 0.3-1.5; 5-CQA, 0.3-1.7; 3,4-diCQA, 0.1-0.8; 3,5-diCQA, 0.2-1.9; and 4,5-diCQA, 0.2-1.7 The samples (0.2 g) were dissolved in aqueous methanol (40%), spiked with the isomer solutions, cleared with Carrez reagent, made up to volume (100 ml) and filtered The filtrates were used directly for chromatography Calibration and recovery data were not obtained for 5-feruloylquinic acid as only trace amounts of the material were available Results and Discussion When only a total chlorogenic acid value is required the AOAC method,l0 based on differential ultraviolet absorption K -+-0 F c Q, C C 10 20 10 30 Ti rnelrn in Fig Isomerisation of (A) chlorogenic acid (5-CQA) yielding (B) 3-CQA and (C) 4-CQA Conditions as described in the text Table Recovery of chlorogenic acid isomers using HPLC Average of five levels of addition for each isomer (values are in grams of isomer per 100 g of sample for wet matter) Isomer 3-CQA 4-CQA 5-CQA 3,4-diCQA 3,5-diCQA 4,5-diCQA Range of addition, g per 100 g 0.3-1.6 0.3-1.5 0.3-1.7 0.1-0.8 0.2-1.9 0.2-1.7 Average recovery, YO 101.7 99.3 104.2 99.9 97.3 97.8 Standard deviation 06 4.9 4.4 4.9 3.6 4.4 5.6 View Article Online 265 ANALYST, MARCH 1984, VOL 109 Published on 01 January 1984 Downloaded by University of Connecticut on 28/10/2014 16:16:40 , I (b' A 70% / I\ / / / / / / 30 Timeimin Fig Chromatograms of ( a ) FQA isomers mixture and ( b )+CQA isomers mixture Peaks as described in Fig and cord'itions as described in the text 30 Timeimin Fig Chromatograms of ( a ) chlorogenic acid standard, ( b ) CQA isomers mixture and (c) instant coffee using Spherisorb 5-ODS and gradient elution (1) 3-CQA; (2) 3-FQA; (3) 4-CQA; (4) 5-CQA; (5) 4-FQA; (6) 5-FQA; (7) 3,4-diCQA; (8) 3,5-diCQA; and (9) 4,5diCQA Detection at 325 nm; flow-rate ml min-1; and solvent and gradient conditions as described in the text The same procedure was adopted with a sample of 5-feruloylquinic acid to allow the assignment of the peaks due to the 4and 5-isomers In each instance the coffee extracts were spiked with standards, or isomerised mixtures, to aid peak assignment Quantification of the isomers in the coffee samples was achieved by comparison of peak areas with a standard of 5-caffeoylquinic acid, allowing for differences in molar absorptivities between the isomer in question and the standard Pure reference compounds were not available and thus the molar absorptivities could not be determined experimentally Consequently literature values were used,9 which were values for each component However, recorded at the ,A the ,A values only varied from 325 to 330 nm and so by using a common detection wavelength (325 nm) only a very small error was introduced From a study of the absorption spectrum of one isomer (5-CQA) this error was calculated to be about 1-1.5%, which is acceptable in terms of the over-all experimental accuracy The major source of error in HPLC methods such as these is not in the chromatographic stages but in the preceding extraction and clean-up procedures A number of extraction systems were evaluated and of these, the following three produced good recoveries: (a) 70% propan-2-01; (b) hot water (80 "C) followed by clearing with Carrez solution; and (c) 40% methanol followed by clearing with Carrez solution System (a) produced a clean extract but the presence of propan-2-01 in the sample produced severe distortion of the chromatogram With this system it is essential to evaporate the solvent and redissolve the residue in water or buffer solution prior to injection, a process that complicates the analytical scheme and may cause losses by oxidation System (b) produced chromatograms with interfering peaks and showed losses of dicaffeoylquinic isomers, compared with systems (a) and (c) Consequently, system (c) was chosen for this study The efficiency of this solvent system based on the recoveries of added amounts of CQA and diCQA isomers from one coffee sample is shown in Table Table Coefficient of variation of chlorogenic acid isomers in an instant coffee by HPLC Results obtained from six different extractions from the same sample (values are in grams of isomer per 100 g of sample for wet matter) Isomer 3-CQA 4-CQA 5-CQA 3-FQA 4-FQA 5-FQA 3,4-diCQA 3,5-diCQA 4,5-diCQA Mean, g per 100 g 1.49 1.67 2.12 0.27 0.37 0.52 0.17 0.12 0.24 Coefficient of variation, '/o 0.8 1.2 o 2.5 3.0 3.0 5.4 4.0 2.2 Linearity of the detection system was established over the concentration range expected from the samples, showing good correlation for all isomers studied (correlation coefficients: 5-CQA, 0.999 92; 4-CQA, 0.999 94; 3-CQA, 0.999 8; 3,4diCQA, 0.999 5; 3,5-diCQA, 0.999 995; and 4,5-diCQA, 0.999 90) The major problem encountered with the chromatographic resolution was between the peaks due to 4-caffeoylquinic acid and 3-feruloylquinic acid, although with careful selection of the gradient and solvent conditions adequate resolution can be achieved Chromatograms of standards, isomer mixtures and of instant coffee samples are shown in Figs and All determinations were carried out in duplicate and one sample was analysed six times to obtain an idea of the precision for each isomer and the results are shown in Table The established method was then applied to the determination of chlorogenic acid isomers in 13 commercial instant coffees, with the results shown in Table The major isomer in all the samples was 5-caffeoylquinic acid accounting for about 30% of the total, whereas the sum of the caffeoylquinic isomers accounts for approximately 70% Similarly, the feruloylquinic acid group and the dicaffeoylquinic acid group represent about 20 and lo%, respectively However, it has been suggested that these low levels of dicaffeoylquinic acids are nonetheless very important for sensory qualities of coffee.14 The highest total level found was 10.7% from a "mild" coffee and the lowest (3.6%) was from a decaffeinated coffee, suggesting that there are considerable losses of chlorogenic acid during processing View Article Online 266 ANALYST, MARCH 1984, VOL 109 Table Chlorogenic acid isomer contents in instant coffees Average of duplicate determinations for dry matter Published on 01 January 1984 Downloaded by University of Connecticut on 28/10/2014 16:16:40 Samples, grams of isomer per 100 g of sample C-3 0.90 1.04 1.38 3.32 0.17 0.44 0.32 0.93 0.10 0.07 0.14 0.31 C-4 0.92 1.03 1.36 3.31 0.18 0.37 0.32 0.87 0.10 0.07 0.14 0.31 C-5 1.89 2.25 3.50 7.64 0.35 0.76 0.82 1.93 0.39 0.29 0.48 0.16 C-6 1.44 1.62 1.89 4.95 0.25 0.29 0.36 0.90 (1.16 0.11 0.17 0.43 C-7 0.75 0.87 1.02 2.64 0.15 0.32 0.27 0.74 0.09 0.07 0.07 0.23 C-8 1.40 1.70 2.28 5.38 0.23 0.54 0.52 1.29 0.22 0.17 0.19 0.58 C-9 1.00 1.16 1.69 3.85 0.21 0.49 0.41 1.11 0.12 0.08 0.10 0.30 C-10 1.43 1.56 1.93 4.92 0.22 0.26 0.28 0.76 0.14 0.10 1.42 0.21 0.16 0.03 0.40 C-2 1.35 1.55 2.15 5.05 0.25 0.34 0.45 1.04 0.20 0.16 0.02 0.38 Totalchlorogenicacids 7.10 6.47 4.56 4.49 10.73 6.29 3.61 7.25 5.26 Isomer 3-CQA 4-CQA 5-CQA TotalCQA 3-FQA 4-FQA , , 5-FQA TotalFQA 3,4-diCQA 3S-diCQA , 4,5-diCQA TotaldiCQA C-1 1.38 1.65 2.25 5.28 0.25 0.59 0.58 The authors thank Conselho Nacional de Desenvolvimento Cientifico Tecnologico, Universidade Federal Rio de Janeiro, Brazil, the Committee of Vice-Chancellors and Principals of the Universities of the UK for sponsorship and Dr M Clifford (University of Surrey) for providing the FQA fraction References Clifford, M N and Wight, J , J Sci Food Agric., 1976,27,73 Clifford, M N , Process Biochem., 1975 May, 13 Weiss, L C., J Assoc Off Agric Chem., 1953, 36, 663 Hansermann, M., and Bradenbergen, H Lehensm Unters Forsch., 1961, 115, 516 Konig, W A and Sturm, R., “Tenth International Scientific Colloquium on Coffee,” Association Scientifique Internationale du Cafe, (ASIC), Paris, in the press Rees, D I., and Theaker, P D., “Eighth International Scientific Colloquium on Coffee,” ASI(:, Paris, 1979, p 79 van der Stegen, G H D , and van Duijn J , “Ninth International Scientific Colloquium on Coffee ,” ASIC, Paris, 1980, p 107 0.38 C-11 1.08 1.25 1.74 4.07 0.20 0.37 0.41 0.98 0.13 0.08 0.13 0.35 C-I2 0.72 0.84 1.07 2.63 0.17 0.43 0.29 0.89 0.08 0.06 0.10 0.24 C-13 0.70 0.81 1.04 2.55 0.15 0.40 0.30 0.85 0.07 0.06 0.09 0.22 6.06 5.45 3.76 3.62 0.14 IUPAC Commission on the Nomenclature of Organic Chemistry and IUPAC - IUB Commission on Biochemical Nomenclature, Biochern J., 1976,153,23 Rubach, K., Dusertation, Technical University of Berlin, 1969 10 “Official Methods of Analysis of the Association of Official Analytical Chemists,” Thirteenth Edition, Association of Official Analytical Chemists Washington, DC, 1980, p 233 11 Scarpati, M L., and Guiso, M., Tetrahedron Lett., 1964 34 285 12 Nagels, L., van Dongen, W , Brucker, J , and Pooter, H., J Chromatogr., 1980,187, 181 13 Corse, J , Lundin, R E Sondheimer, E., and Waiss, A C., Jr., Phytochemistry, 1966, , 767 14 Ohiokpehai, Brumen, G , and Clifford, M N , “Tenth International Scientific Colloquium on Coffee,” ASIC, Paris in the press Paper A311 15 Received April 25th, 1983 Accepted June 21st, 1983