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Determination of Total Monomeric Anthocyanin Pigment Content Of LEE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 5, 2005 1269 DIETARY SUPPLEMENTS Determination of Total Monomeric Anthocyanin Pigment Content of Fruit Juices, Beverages, Natural Colorants, and Wines by the pH Differential Method: Collaborative Study JUNGMIN LEE U.S. Department of Agriculture, Agricultural Research Service, Pacific West Area (PWA), Horticultural Crops Research Laboratory Worksite, 29603 University of Idaho Ln, Parma, ID 83660 ROBERT W. DURST and RONALD E. WROLSTAD Oregon State University, Department of Food Science and Technology, Corvallis, OR 97331 Collaborators: K.W. Barnes; T. Eisele; M.M. Giusti; J. Haché; H. Hofsommer; S. Koswig; D.A. Krueger; S. Kupina; S.K. Martin; B.K. Martinsen; T.C. Miller; F. Paquette; A. Ryabkova; G. Skrede; U. Trenn; J.D. Wightman This collaborative study was conducted to organize, and conduct a collaborative study to validate the pH determine the total monomeric anthocyanin differential method as an AOAC method. concentration by the pH differential method, which Anthocyanins are responsible for the red, purple, and blue is a rapid and simple spectrophotometric method hues present in fruits, vegetables, and grains. There are based on the anthocyanin structural 6 common anthocyanidins (pelargonidin, cyanidin, peonidin, transformation that occurs with a change in pH delphinidin, petunidin, and malvidin), whose structures can (colored at pH 1.0 and colorless at pH 4.5). Eleven vary by glycosidic substitution at the 3 and 5 positions. collaborators representing commercial Additional variations occur by acylation of the sugar groups laboratories, academic institutions, and with organic acids. Figure 1 shows the basic structure of an government laboratories participated. Seven anthocyanin pigment. Youden pair materials representing fruit juices, The pH differential method has been used extensively by beverages, natural colorants, and wines were food technologists and horticulturists to assess the quality of tested. The repeatability relative standard deviation fresh and processed fruits and vegetables. The method can be (RSDr) varied from 1.06 to 4.16%. The used for the determination of total monomeric anthocyanin reproducibility relative standard deviation (RSDR) content, based on the structural change of the anthocyanin ranged from 2.69 to 10.12%. The HorRat values chromophore between pH 1.0 and 4.5 (Figure 2). The were £1.33 for all materials. The Study Director anticipated use of the method is in research and for quality recommends that the method be adopted Official control of anthocyanin-containing fruit juices, wines, natural First Action. colorants, and other beverages. The concept of determining the amount of anthocyanin present in a material by measuring the change in absorbance at nthocyanin pigments are important to food quality 2 different pH values (3.4 and 2.0) was first introduced by because of their contribution to color and appearance. Sondheimer and Kertesz (1) in 1948. Since then, researchers AThere is increasing interest in the anthocyanin content have proposed using the pH values of 1.0 and 4.5 (2–5). of foods and nutraceuticals because of possible health Monomeric anthocyanins undergo a reversible structural benefits. Anthocyanin pigment content can also be a useful transformation as a function of pH (colored oxonium form at criterion in quality control and purchase specifications of fruit pH 1.0 and colorless hemiketal form at pH 4.5; Figure 2). Thus, juices, nutraceuticals, and natural colorants. An AOAC the difference in absorbance at the lvis-max (ca 520 nm) of the method is needed to rapidly and precisely determine total pigment is proportional to the concentration of pigment. monomeric anthocyanin content. Our goal was to design, Figure 3 shows the spectra of huckleberry anthocyanins in buffers at pH 1.0 and 4.5. Degraded anthocyanins in the polymeric form are resistant to color change with change in pH. Submitted for publication May 2005. Therefore, polymerized anthocyanin pigments are not The recommendation was approved by the Methods Committee on Commodity Foods and Commodity Products as First Action. See “Official measured by this method because they absorb both at pH 4.5 Methods Program Actions,” (2005) Inside Laboratory Management, and 1.0. July/August issue. Absorbance should be measured at the l of the Corresponding authors' e-mail: [email protected], vis-max [email protected], and [email protected]. pigment solution, and the pigment content should be calculated by using the molecular weight (MW) and molar 1270 LEE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 5, 2005 extinction coefficient of the major anthocyanin in the matrix. For example, the anthocyanin content of wines is customarily calculated as the content of malvidin-3-glucoside (MW = 493.2) by using a molar extinction coefficient of 28 000 (6). However, natural matrixes normally contain a mixture of anthocyanins, and the proportions of these anthocyanins will vary naturally. This variation makes it unrealistic to assign specific extinction coefficients or molecular weights to routine test results. As a result, the method calls for several measurements to be obtained. For purposes of this collaborative study, analysts were instructed to measure all solutions at 520 nm, which is midrange for the different materials in the study. Results were expressed as equivalents of cyanidin-3-glucoside, which is the most common anthocyanin pigment found in nature (7). Identities of the test materials were unknown to the analysts. Figure 1. Basic structure of an anthocyanin pigment. Collaborative Study A collaborative protocol was prepared by following the guidelines of AOAC INTERNATIONAL (8) and approved by the Methods Committee on Commodity Foods and Commodity Products. Collaborating Laboratories Eleven laboratories representing commercial laboratories, academic institutions, and government laboratories participated. Six of the laboratories were outside the United States. One analyst from each laboratory performed the analysis. Collaborators were not compensated for their participation. Test Materials Seven Youden pair materials were prepared; 1 member of the pair was neat (undiluted, X), and the other was diluted Figure 2. Predominant anthocyanin structural forms with distilled water (Y) to obtain a difference of £5%. The present at different pH levels. formula used was (xc – yc/xc) £ 0.05 where xc is the concentration of analyte in X, and yc is the concentration of analyte in Y (8). Descriptions of the materials used in this study are given in Table 1. Concentrates (strawberry, raspberry, and elderberry) were initially diluted to their reported single strength °Brix (8.0, 11.0, and 9.2, respectively) as listed in 21CFR (Code of Federal Regulations), Part 101, and then diluted appropriately as Youden pairs in a 1 L volumetric flask. The standard solution was prepared by weighing 82.2 mg cyanidin-3-glucoside chloride (76.2 mg cyanidin-3-glucoside; Polyphenols Laboratories As, Sandnes, Norway), dissolving in distilled water, and diluting to a final volume of 1 L in a volumetric flask. Two familiarization materials were included with the test sets. The materials were cranberry juice cocktail and an Figure 3. Spectral characteristics of huckleberry intensely pigmented grape juice concentrate diluted to anthocyanin in buffers at pH 1.0 and 4.5. appropriate strength. After reviewing their values, LEE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 5, 2005 1271 Table 1. Materials and their sources used in the guidelines of AOAC INTERNATIONAL (8). The outliers collaborative study were determined at a = 0.025, and the t-statistics for Materiala Source equivalence of variance were tested at a = 0.05. To assess the homogeneity of the splitting of each material, a 1-way analysis of variance (ANOVA), performed with the PROC Cranberry juice cocktail Local grocery store (Corvallis, OR) ANOVA software of the Statistical Analysis System (SAS; 9), Red wine Local grocery store (Corvallis, OR) was used to determine if the mean °Brix values for the 2 Natural colorantb Canandaigua Concentrate (Madera, CA) randomly selected vials for each material varied significantly Strawberry concentratec Kerr Concentrate Inc. (Salem, OR) between themselves. Homogeneity was evaluated by using a Raspberry concentratec Kerr Concentrate Inc. (Salem, OR) significance level of a = 0.05. Elderberry concentratec Kerr Concentrate Inc. (Salem, OR) Methods of Standard Analyses Cyanidin-3-glucoside Polyphenols Laboratories As d chloride (Sandnes, Norway) To investigate recovery issues for the standard used in the collaborative study, additional cyanidin-3-glucoside chloride a Matrixes were prepared as Youden pairs. was purchased from 2 vendors, Polyphenols Laboratories As b A high colored grape juice concentrate. and Extrasynthese (Lyon, France). Percent purity by liquid c Obtained as a concentrate and diluted to the appropriate °Brix. chromatography (LC), percent purity by molar absorptivity, d Standard used in this study. percent moisture content, and hygroscopicity at 1 relative humidity were determined. Only moisture determination and hygroscopicity were duplicated because of the high cost of the collaborators were notified to continue with the actual standards. laboratory test samples. Standards were prepared by weighing 17.00 ± 0.01 mg into The appropriate anthocyanin content of the materials a glass weighing bottle (with lid) that had been cleaned, dried ranged from 20 to 3000 mg/L (expressed as in an
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