Determination of Total Phenolics - Wiley Online Library

Determination of Total Phenolics

UNIT I1.1

The phenols or phenolics in wine are important to both red and white wines. In red wines, this class of substances contributes to the astringency, bitterness, and other tactile sensations defined as structure or body, as well as to the wine’s red color. In white wines, higher levels of phenolics are generally undesirable, as they contribute to excessive bitterness and to the tendency of the wine to brown when it is exposed to air. Phenolics in grapes and wines include many different substances: phenolic acids (e.g., hydroxybenzoic acids such as gallic acid, hydroxycinnamic acids found in grape juice), three classes of flavonoids found in the skins and seeds (the red anthocyanins, the flavonols, and the abundant flavan-3-ols, which comprise the monomeric catechins), oligomeric proanthocyanidins, and polymeric condensed tannins. (For details on phenolics classes and compound structures, refer to UNITS I1.2 & I1.3.) White wine is made by immediately pressing off the skins and seeds after harvesting, and thus contains only small quantities of flavonoids. In contrast, red wine is a whole-fruit extract made by fermenting with the skins and seeds, and the alcohol thus produced is an excellent solvent for these substances. Measuring these different substances and reporting meaningful values in a single number is an analytical challenge. There are many different procedures for analyzing different classes of phenolic substances, but few are used in wine analysis except for anthocyanin or color measures. HPLC methods (UNIT I1.3) that give specific information on individual substances are not widely used in wineries, but are becoming more common as the significance of particular phenolic substances becomes better understood. There are two widely used methods for the analysis of total phenolics in wine. The Folin-Ciocalteau method (Basic Protocol 1 and the Alternate Protocol) has the advantage of a fairly equivalent response to different phenols, with the disadvantage of responding to sulfur dioxide and sugar. The direct spectral absorbance analysis (Basic Protocol 2) is quick and simple, making it suitable for process monitoring. This method, however, responds differently to the various phenolic classes, making comparisons between different wine types problematic, and also gives significant interference for sorbate. Wine, of course, is not the only food that contains phenolics. Phenolics are found in all foods, though at low levels in most. Notable foods that are high in phenolics include coffee and tea, chocolate, fruits and derived products, some oils, spices, and some whole grains. Although the following methods were developed for—and first applied to—analysis of wines and grapes, they can be adapted for other foodstuffs (also see Commentary). DETERMINATION OF TOTAL PHENOLICS BY FOLIN-CIOCALTEAU COLORIMETRY Folin-Ciocalteau (FC) colorimetry is based on a chemical reduction of the reagent, a mixture of tungsten and molybdenum oxides. Singleton adapted this method to wine analysis (Singleton and Rossi, 1965) and has written two major reviews on its use (Singleton, 1974; Singleton et al., 1999). The products of the metal oxide reduction have a blue color that exhibits a broad light absorption with a maximum at 765 nm. The intensity of light absorption at that wavelength is proportional to the concentration of phenols. The FC method has been adopted as the official procedure for total phenolic levels in wine; the Office International de la Vigne et du Vin (OIV), the one international body that certifies specific procedures for wine analysis, accepts the FC method as the standard procedure for total phenolic analysis (OIV, 1990). An earlier variation was the Folin-Denis procedure, but the FC method has displaced it except in a few historical cases of official procedures that have not been updated (AOAC International, 1995). Contributed by Andrew L. Waterhouse Current Protocols in Food Analytical Chemistry (2002) I1.1.1-I1.1.8 Copyright © 2002 by John Wiley & Sons, Inc.

BASIC PROTOCOL 1

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Color development is slow but can be accelerated by warming the sample. With excessive heating, however, subsequent color loss is quite rapid, and timing the colorimetric measurement becomes difficult to reproduce. The reagent is commercially available, but can be prepared (Singleton and Rossi, 1965). The resulting solutions are treated as hazardous waste, and the scale of the original procedure creates a lot of waste. Fortunately, modern liquid-measuring equipment now allows for microscaling the reaction to the volume of a UV-Vis cuvette, reducing the cost of the reagent and waste disposal (see Alternate Protocol). Materials Sample, e.g., white wine or 10% (v/v) red wine in water Gallic acid calibration standards (see recipe) Folin-Ciocalteau (FC) reagent (Sigma; also Singleton and Rossi, 1965), stored in the dark and discarded if reagent becomes visibly green Sodium carbonate solution (see recipe) 100-ml volumetric flask Spectrophotometer set to 765 nm, with 1-cm, 2-ml plastic or glass cuvettes 1. Place 1 ml sample, a gallic acid calibration standard, or blank (deionized or distilled water) in a 100-ml volumetric flask. Samples and standards should be analyzed in triplicate. If any sample has an absorbance reading above that of the 500 mg/liter standard, it must be diluted adequately and remeasured. White wine can typically be analyzed without dilution. Red wine must be diluted with water (usually ten-fold) to fall into the range of the standards.

2. Add ∼70 ml water, followed by 5 ml FC reagent. Swirl to mix and incubate 1 to 8 min at room temperature. The incubation must not be >8 min (see Critical Parameters, discussion of reaction time and temperature).

3. Add 15 ml sodium carbonate solution. 4. Add water to the 100-ml line, mix, and incubate 2 hr at room temperature. 5. Transfer 2 ml to a 1-cm, 2-ml plastic or glass cuvette and measure its absorbance at 765 nm in a spectrophotometer. 6. Subtract the absorbance of the blank from all readings and create a calibration curve from the standards. 7. Use this curve to determine the corresponding gallic acid concentration of the samples. Be sure to multiply by any dilution factor for the correct concentration (i.e., by ten for red wines). Report values in gallic acid equivalents (GAE) using units of mg/liter (see Critical Parameters, discussion of standardization). ALTERNATE PROTOCOL

MICROSCALE PROTOCOL FOR FOLIN-CIOCALTEAU COLORIMETRY This protocol is adapted for small sample volumes. The reaction is performed directly in a 2-ml cuvette. For a list of materials needed, see Basic Protocol 1. 1. Put 20 µl sample, a gallic acid calibration standard, or blank (deionized or distilled water) into a 1-cm, 2-ml plastic or glass cuvette.


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2. Add 1.58 ml water, followed by 100 µl FC reagent. Mix thoroughly by pipetting or inverting and incubate 1 to 8 min. The incubation must not be >8 min (see Critical Parameters, discussion of reaction time and temperature).

3. Add 300 µl sodium carbonate solution, mix, and incubate 2 hr at room temperature. A final volume of 2 ml must fill the cell adequately for a reading.

4. Measure sample absorbance at 765 nm and analyze as described (see Basic Protocol 1, steps 6 to 7). DETERMINATION OF TOTAL PHENOLICS BY SPECTRAL ANALYSIS Phenolic substances all absorb UV light, and all of them have some absorbance at 280 nm. This property can be used to determine phenolics by spectral analysis. One problem with this method is that each class of phenolic substances has a different absorptivity (extinction coefficient, e) at 280 nm. Thus, the results cannot be related to any specific standard and are reported directly in absorbance units (AU). This also means that disparate wines (or other disparate samples) are difficult to compare with this method, as they are likely to have very different compositions.

BASIC PROTOCOL 2

The value of this method is that it is extremely simple and rapid, requiring only filtration and, in some cases, dilution. It is very suitable for monitoring wines during various stages of processing (e.g., fermentation) and for comparing similar wines (e.g., a single grape variety from different vineyards, or wines from a particular vineyard over different vintages). Materials Sample, e.g., red or white wine Filter membrane, e.g., polytetrafluoroethylene (PTFE) Cuvettes, transparent at 280 nm (e.g., quartz or methacrylate) Spectrophotometer, set to 280 nm 1. Filter a sample or blank (deionized or distilled water) with a PTFE filter membrane or other material to achieve clarity. Nylon or other membranes that absorb phenolics should not be used. Membranes can be tested for phenolic absorption by comparing absorbance after single and double filtration.

2. Transfer an appropriate volume of sample to a quartz or methacrylate cuvette and measure absorbance at 280 nm in a spectrophotometer. If absorbance is not within the acceptable precision of the spectrophotometer (usually A < 2 AU), dilute sample as necessary and repeat. 3. Subtract absorbance of blank, and correct absorbance to original concentration and a 1-cm cuvette path length. Subtract 4 AU to report final value. For instance, if a sample is diluted ten-fold with water and a reading of 0.85 AU is observed with a 2-mm cell, the correction would be as follows: total phenol = [A280 × DF × (1 cm/b)] - 4 = [0.85 × 10 × (1 cm/0.2 cm)] - 4 = 38.5 AU where DF is the dilution factor, b is the cell path length, and 4 is an arbitrary correction for nonphenolic absorbance (see Critical Parameters, discussion of spectral analysis). Polyphenolics

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REAGENTS AND SOLUTIONS Use deionized or distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Gallic acid calibration standards Dissolve 0.5 g gallic acid in 10 ml ethanol and then dilute to 100 ml with water (5 g/liter final). Dilute 1, 2, 5, and 10 ml to 100 ml with water to create standards with 50, 100, 250, and 500 mg/liter concentrations, respectively. Store up to 2 weeks at 4°C. Standards will retain 98% of their potency for 2 weeks if kept closed under refrigeration (4°C), but this potency is retained for only 5 days at room temperature. Commercial gallic acid is usually adequately pure, but can be recrystallized from water if desired.

Sodium carbonate solution Dissolve 200 g anhydrous sodium carbonate in 800 ml water and bring to a boil. After cooling, add a few crystals of sodium carbonate and let sit 24 hr at room temperature. Filter through Whatman no. 1 filter paper and add water to 1 liter. Store indefinitely at room temperature. COMMENTARY Background Information There are many phenolic substances in plants and thus in foods. Rich dietary sources of phenolics include fruits, tea, coffee, cocoa, and processed foods derived from these, such as wine. At high levels, and in particular when sugar levels are low, phenols impart an astringency, bitterness, and color to foods. In red wine, unsweetened tea, and chocolate products, the taste is heavily influenced by the presence of phenolics. Therefore, an assessment of phenolic content in food is of great importance.


Folin-Ciocalteau method The Folin-Ciocalteau (FC) procedure is one of the standard procedures in wine analysis, as well as in tea analysis (Wiseman et al., 2001). One drawback in interpretation is that different classes of phenolics have varying taste attributes, and tests for chemical astringency based on precipitation of proteins have been recently developed (Adams et al., 1999). In addition, if the food product contains sugar, it can mask the bitterness and astringency, as observed in ripe fresh fruit, sweetened chocolates, and tea. The differential sensory effect of phenolics aside, a major advantage of the FC procedure is that it has a fairly equivalent response to different phenolic substances in wine, making it suitable for measuring accurate mass levels of total phenolic substances. Among the abundant phenolics in wine, the mass response factor relative to gallic acid ranges from 0.87 for caffeic acid to 1.10 for epicatechin based on values from Singleton (1974). The glucosides

give lower values, but their mass is increased by the nonphenolic glycosidic substituent. Their response appears to be similar on a phenolic fraction basis. Monohydroxyphenolics such as coumaric acid also give low values, but these constitute a small fraction of the phenolics in wine. In general, the response of a phenolic is due to the number of phenolic groups, and Singleton (1974) describes the controlling factors in detail. The FC method has also been applied to other foods. One example of particular use is for analysis of tea (Wiseman et al., 2001). The method has also been applied to vegetables (Kaur et al., 2002) and fruit (Pearson et al., 1999; Vinson et al., 2001), although in one instance the only corrected interference was ascorbate. For analysis of foodstuffs other than wines and grapes, the analyst must be aware of potential interferences. In other fields, the method has been used for analysis of medicines (Sadler and Jacobs, 1995), trees in wood chemistry (Yu and Dahlgren, 2000), and fresh waters (Thoss et al., 2002). The FC procedure was automated some time ago (Slinkard and Singleton, 1977). Although there have been no more published reports on contemporary automation, there are many laboratories that have adapted the procedure to clinical analyzers (G. Burns and T. Collins, pers. comm.). It seems likely that the procedure could be adapted to other analyzers as well. There are few direct comparisons of total phenol values and antioxidant measurements, although some do exist (e.g., Baderschneider

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et al., 1999). In general, the response of total phenol tests is comparable to antioxidant tests, with better correlations for antioxidant tests based on aqueous systems as opposed to those based on lipid media. Spectral analysis Phenolic substances can also be quantified by measuring absorbance at 280 nm. The applicability of this method is far more limited, however, because absorbance properties of different phenolics vary and cannot be related to a specific standard. Because of this, and because of the extreme ease of the method, spectral analysis is well suited for using total phenolic content for process monitoring. Sample preparation The preparation of extracts from solid foods is not trivial. In general, 70% (v/v) acetone is used to extract proanthocyanins and condensed tannins, and aqueous methanol is typically used for other classes of phenolics (UNIT F1.1). However, the acid levels used in some reported extraction procedures can have a devastating effect on the recovery of particular flavonoids, and this has been carefully studied for HPLC analysis of flavonoids (Merken et al., 2001). Although some transformations simply hydrolyze glycosides, which would not significantly change the total phenolic content, other degradations may significantly decrease the amount of phenolics present. In the absence of a welldeveloped procedure for the extraction of a particular compound from a similar matrix, re-extractions and recovery must be validated for any extraction protocol. For solid foodstuffs, the final results would be expressed in mg/100 g or mg/kg.

Critical Parameters Folin-Ciocalteau method Interferences. Because the color formation of the Folin-Ciocalteau reaction is based on chemical reduction of the reagent, this reaction is general enough to allow for interference from a number of sources. In wine, the principal interfering compounds are sulfur dioxide and ascorbate, though high levels of sugar indirectly enhance the readings of other analytes. Because there can be many other interferences in non-wine samples, however, it is necessary to thoroughly investigate the use of this method for different samples. The most problematic interference may well be sugar, as it is not mentioned in the original report (Singleton and

Rossi, 1965), but can be found at very high levels in, for instance, fruits. Another non-wine issue comes from the fact that the Lowry method for protein analysis is based on the reaction of the FC reagent with tyrosine phenolic groups, so samples with high protein levels will not be suitable for phenolic analysis by FC. In addition, it should be noted that phenolics will interfere with protein analysis by the Lowry method. Some investigators have applied the use of the FC procedure to analyze phenolic levels in human blood fractions, such as plasma (Nigdikar et al., 1998). It is likely, however, that this method will respond to changes in the large amount of constitutive redox-active substances, such as ascorbate, urate, and tocopherol, as well as any proteins, making it difficult to attribute changes in response to the appearance of phenolics in the blood. The FC method does not respond to sulfur dioxide alone, but does respond to sulfur dioxide in the presence of phenolic compounds. Presumably, the phenols are oxidized by the FC reagent and then reduced by the sulfur dioxide, creating an interfering response by a type of catalytic cycle. Unfortunately, the magnitude of the interference is not constant (Saucier et al., 1999), so it is not possible to suggest an accurate correction factor, though approximate mass correction factors of 0.1 to 0.2 have been suggested (thus, 10 mg/liter sulfur dioxide would yield a response of 1 to 2 mg/liter in the FC assay; Singleton, 1988). Generally, the case where the sulfur dioxide interference is significant is in white wines, which have a lower range of total phenolic levels and a high level of sulfur dioxide. It has been suggested that this interference renders the method unusable (Somers and Ziemelis, 1980), but that conclusion has not been accepted by others. Ascorbate is present at very low levels in wine unless it has been added, which is legal but rarely done in the United States, although it is common in some other countries. In other food samples, especially fresh fruits, ascorbate levels can be very high. For kiwifruit, a correction of 1 mg/liter per 1 mg/liter ascorbate must be applied. The interference of sugar is easily corrected, and is only necessary with sweet or semisweet wine (>2% [w/v] sugar). With fruit samples, the sugar levels can be very high, and it is not clear if adequate corrections can be applied. Generally, it is not advisable to analyze must for phenolic levels by the FC method because of the complexity of the sugar correction. Sin-

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Table I1.1.1 Approximate Correction to Folin-Ciocalteau Results for Wines Containing Invert Sugara

Apparent phenol content (mg GAE/liter) 100 200 500 1000 2000

Invert sugar content 2.5% 5 10 20 30 60

5.0% 10 15 30 60 120

10% 20 40 50 100 200

aThe correction value should be subtracted from the apparent phenol content for an accurate value. This

correction applies to analyses conducted at room temperature. Abbreviation: GAE, gallic acid equivalents. Reproduced from Slinkard and Singleton (1977), with permission from the American Society for Enology and Viticulture.


gleton suggests conducting the analysis of the standards with the same level of sugar as the sample, but this is a complex issue because different sugars yield different intereferences (i.e., fructose has a higher response than glucose). Singleton suggests the corrections in Table I1.1.1 when sugar is not added to standards (Slinkard and Singleton, 1977) and describes additional correction factors for warmer temperatures. These correction factors have not been directly applied to other foods, but it would be prudent to test for the presence of interfering substances, in particular sugars and ascorbate in fresh fruits, and to assess their level of interference in the assay. Correction factors for other interferences may need to be specifically developed. Limits of detection and quantitation. Because wines that have total phenol levels lower than 50 mg/liter are quite rare, this is not a significant issue for wine. For other sample types, however, a limit of quantitation of ∼0.027 AU or 20 mg/liter would be expected, based on a sample-to-sample variance of 0.003 AU (Singleton and Rossi, 1965). Standardization. Because the analysis reveals the presence of many different substances in one result (in the case of wine, this could mean thousands of substances if one takes into account the diversity of proanthocyanidins), the only practical standard is a single substance. In the cases of wine and tea, the accepted standard is gallic acid. It is a particularly good standard because it is relatively inexpensive in pure form and is stable in its dry form. Other substances have been used, and in principle any phenol could be used, but gallic acid is strongly recommended for the above reasons and the fact that the use of a single standard makes it easier to compare data.

Gallic acid is quite stable in dry form, but will oxidize once it is in solution. This reaction is enhanced at higher temperatures. The author has found that the standard solutions should be stored in full or nearly full bottles that are kept tightly sealed between uses and are stored under refrigeration. The author has observed that >5% potency is lost after ∼1 week at room temperature, but this same loss takes ∼2 weeks with refrigeration. The authors would expect that greater air exposure will also accelerate decomposition. Because a single substance is used, the result must be reported as a response equivalent to the amount or concentration of that substance. As wine is analyzed on a concentration basis, the result is reported in gallic acid equivalents (GAE) using units of mg/liter. For any standard, the results must always be reported on an equivalent basis to avoid the perception that one is measuring the amount of the standard substance. Reaction time and temperature. The oxidative reaction caused by the FC reagent is slow and is not complete when the reading is taken. In addition, the colored product is unstable. The measurement is based on the kinetics of the process. Thus, the time of the colorimetric reading is most critical, and the temperature will affect the extent of the reaction and degradation. This is one of the reasons that standards are run each time, to accommodate for changes in room temperature, timing, and reagent condition. At higher temperatures, the colored product has a very limited lifetime. For a full discussion, see Singleton et al. (1999). The FC reading time can be accelerated by heating the solution and halving, as a rule of thumb, the reaction time for each temperature increase of 10°C. Thus, at 40°C, readings can be taken after 30 min. Decreasing the time

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further by higher temperatures becomes problematic for manual measurements because the timing of each reading becomes more critical with each increase in temperature. The degradation of the component that absorbs at 765 nm, in particular, becomes very rapid at higher temperatures, so analyses that run just slightly too long will yield very poor results (Singleton, 1999). Thus, 40°C is usually the highest recommended temperature for manual use, but higher temperatures are described for automated analyses (Slinkard and Singleton, 1977). Precision. Because the FC method relies on reaction kinetics and not stoichiometric conversion, it is not very precise, and variations of ∼5% are typical for replicates, depending on the temperature control and timing precision of the reagent additions and spectral measurements. Spectral analysis Interferences. In wine the principal interference is sorbic acid. This is a preservative typically added to sweetened wines that have residual sugar. Because yeast would continue to ferment this sugar, spoiling the product in the bottle, such wines must be rendered sterile at bottling. This is usually done by sterile filtration, sometimes combined with the addition of sorbic acid. Sorbic acid has a very strong absorbance at 280 nm and will result in exceptionally high readings (up to 36 AU). There appears to be some dispute about the ease of dealing with the interference, as the author of this method claims that it is easy to remove the sorbic acid by isooctane extraction (Somers and Ziemelis, 1985). It has been reported, however, that an impractically large volume of isooctane would be needed to remove the sorbate (Tryon et al., 1988). Although it is likely that serial extractions would be very effective, routine multiple extractions of all samples would make the method significantly more complex when analyzing unknown wines that may contain sorbate. Background. The method also attributes 4 AU to nonphenolic substances in all samples, and so this value is subtracted to reflect the true absorbance due to phenols. The origin of this background absorbance is not clear, but is thought to be due to protein and nucleotides. The magnitude of the background absorbance does vary with a standard deviation of 1 AU (Somers and Ziemelis, 1985). Thus, for typical white wines with an uncorrected absorbance of 8 AU, the subtraction of 4 AU leaves 4 AU with

an expected standard deviation of 1 AU or 25% due to the variance in the correction factor. Standards. There is no standard or calibration with standards, so these issues are moot.

Anticipated Results The level of total phenolics in white wines varies from ∼100 to 300 mg/liter by the FC method. The levels will be on the low end of the scale if the must was subjected to oxidative treatment and the pressing was very light. Higher levels will be observed when harder pressing of the solids is utilized or if the wine was aged in new oak barrels. By spectral analysis, white wines have an average corrected absorbance of 4 AU, with a range of 1 to 11 AU. The FC results will be compromised by high levels of sulfites, but because sulfite levels are almost always measured in wines, the possibility of sulfite interference in a wine should be anticipated. The spectral method will be compromised by the presence of sorbate. In known wines this can easily be anticipated and, because its use is limited to sweet wines, it may be possible to check for it selectively. Another clue to its presence would be a very high absorbance. Sorbate can also be measured in wine (Caputi and Stafford, 1977). Red wines have total phenolic levels of 1 to 3 g/liter, with typical average of ∼1.8 g/liter. Differences are due to differing amounts of phenolics in grapes based on variety and growing conditions, with moderate to cooler climates yielding higher levels. Production techniques can have a secondary effect, and longer contact times or higher temperatures will increase the amount of phenolics extracted from the grape solids into the wine. Red wines have a reported range of 23 to 100 AU, with an average of 54 AU. Because levels are so high, interferences other than sorbate do not introduce significant error in the spectral method.

Time Considerations When the FC analysis is carried out manually, there are limits to the number of samples that can be handled at once because of the need to time the reagent mixing and spectral readings. In the author’s laboratory, single analysts can run 20 samples per day, divided into 2 sets. Since each sample is run in duplicate and 4 standards are included in each run, 50 individual results are generated in a day.

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Literature Cited Adams, D.O. and Harbertson, J.F. 1999. Use of alkaline phosphatase for the analysis of tannins in grapes and wine. Am. J. Enol. Vitic. 50:247252. AOAC (Association of Official Analytical Chemists) International. 1995. Tannin in Distilled Liquors. AOAC Official Method 952.03. In Official Methods of Analysis of AOAC International, 16th ed., (P. Cuniff, ed.) ch. 26, p. 16. AOAC Int., Arlington, Va. Baderschneider, B., Luthria, D., Waterhouse, A., and Winterhalter, P. 1999. Antioxidants in white wine (cv. Riesling): I. Comparison of different testing methods for antioxidant activity. Vitis 38:127131. Caputi, A. Jr. and Stafford, P.A. 1977. Ruggedness of official colorimetric method for sorbic acid in wine. J.A.O.A.C. 60:1044-1047. Kaur, C. and Kapoor, H.C. 2002. Anti-oxidant activity and total phenolic content of some Asian vegetables. Int. J. Food Sci. Technol. 37:153-161. Merken, H.M., Merken, C.D., and Beecher, G.R. 2001. Kinetics methods for the quantitation of anthocyanidins, flavonols, and flavones in foods. J. Agric. Food Chem. 49:2727-2732. Nigdikar, S.V., Williams, N.R., Griffin, B.A., and Howard, A.N. 1998. Consumption of red wine polyphenol reduces the susceptibility of lowdensity lipoproteins to oxidation in vivo. Am. J. Clin. Nutr. 68:258-265. OIV (Office International de la Vigne et du Vin). 1990. Indice de Folin-Ciocalteau. In Recueil des Méthodes Internationales d’Analyses des Vins et des Moûts, pp. 269-270. OIV, Paris. Pearson, D.A., Tan, C.H., German, J.B., Davis, P.A., and Gershwin, M.E. 1999. Apple juice inhibits human low density lipoprotein oxidation. Life Sci. 64:1913-1920. Sadler, N.P. and Jacobs, H. 1995. Application of the folin-ciocalteau reagent to the determination of salbutamol in pharmaceutical preparations. Talanta 42:1385-1388. Saucier, C.T. and Waterhouse, A.L. 1999. Synergetic activity of catechin and other antioxidants. J. Agric. Food Chem. 47:4491-4494. Singleton, V.L. 1974. Analytical fractionation of the phenolic substances of grapes and wine and some practical uses of such analyses. In Chemistry of Winemaking (A.D. Webb, ed.) pp. 184211. American Chemical Society, Washington, D.C. Singleton, V.L. 1988. Wine phenols. In Wine Analysis (H.F. Linskens, and J.F. Jackson, eds.) pp. 173-218. Springer-Verlag, Berlin.

Singleton, V.L. and Rossi, J.A. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16:144158. Singleton, V.L., Orthofer, R., and Lamuela-Raventos, R.M. 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteau reagent. Methods Enzymol. 299:152-178. Slinkard, K. and Singleton, V.L. 1977. Total phenol analysis: Automation and comparison with manual methods. Am. J. Enol. Vitic. 28:49-55. Somers, T.C. and Ziemelis, G. 1980. Gross interference by sulphur dioxide in standard determinations of wine phenolics. J. Sci. Food Agric. 31:600-610. Somers, T.C. and Ziemelis, G. 1985. Spectral evaluation of total phenolic components in Vitis vinifera: Grapes and wine. J. Sci. Food Agric. 36:1275-1284. Thoss, V., Baird, M.S., Lock, M.A., and Courty, P.V. 2002. Quantifying the phenolic content of freshwaters using simple assays with different underlying reaction mechanisms. J. Environ. Monit. 4:270-275. Tryon, C.R., Edwards, P.A., and Chisholm, M.G. 1988. Determination of the phenolic content of some French-American hybrid white wines using ultraviolet spectroscopy. Am. J. Enol. Vitic. 39:5-10. Vinson, J.A., Su, X.H., Zubik, L., and Bose, P. 2001. Phenol antioxidant quantity and quality in foods: Fruits. J. Agric. Food Chem. 49:5315-5321. Wiseman, S., Waterhouse, A., and Korver, O. 2001. The health effects of tea and tea components: Opportunities for standardizing research methods. Crit. Rev. Food Sci. Nutr. 41:387-412 Suppl. Yu, Z. and Dahlgren, R.A. 2000. Evaluation of methods for measuring polyphenolics in conifer foliage. J. Chem. Ecol. 26:2119-2140.

Key References Singleton and Rossi, 1965. See above. The original description for the use of the FC method for wine analysis. Singleton et al., 1999. See above. A thorough review of the FC method’s use in wine analysis by the original author. Somers and Ziemelis, 1985. See above. The key compilation of spectral methods.

Contributed by Andrew L. Waterhouse University of California, Davis Davis, California


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