Caffeinated Beverages - ACS Publications - American Chemical Society

Chapter 39

Methyluric Acids: Chemical Markers of Oxidation in Coffee, Tea, and Cocoa Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on April 26, 2016 | http://pubs.acs.org Publication Date: January 15, 2000 | doi: 10.1021/bk-2000-0754.ch039

Richard H. Stadler and Robert J. Turesky Nestlé Research Center, Nestec Ltd., Vers-chez-les-Blanc, P.O. Box 44, CH-1000 Lausanne 26, Switzerland

Recent studies have shown that C-8 hydroxylated methylxanthines, such as 8-oxocaffeine (1,3,7-trimethyluric acid), can reflect free radical mediated reactions in coffee and tea, during processing and in the finished product. In order to further extend the usage of uric acids as markers of oxidation, the C-8 hydroxylated analogue of theobromine, namely 8-oxotheobromine, is determined in unsweetened cocoa powders using LC-ESI/MS/MS. The generation of oxotheobromine is dependent on a number of factors, such as transition metal availability, oxygen, and pH. Such stable and non-volatile chemical markers of oxidation may assist in industrial quality control screening of raw materials and also aid in assessing the shelf life of methylxanthine-rich finished products.

Introduction Purine alkaloids are major dietary constituents of a number of beverages such as coffee, tea, cocoa, guarana, and cola-based refreshments, and contribute to the positive organoleptic perception enjoyed globally by these popular drinks. Raw cocoa beans are rich in methylxanthine derivatives, and theobromine - also termed 3,7dimethylxanthine - is the major alkaloid in Theobroma cacao. Its concentration is dependent on the botanical and geographical origin of the beans and ranges from 2.8 - 3.4 % w/w (/). Cocoa beans also contain a plethora of polyphenolic compounds, principally catechins and procyanidin mono-, di-, and trimers^ with a (-)-epicatechin concentration of approx. 2% on a w/w basis in the unfermented bean (2,3). The widespread public and scientific interest in health-beneficial dietary constituents in the past years has more and more focused on food commodities that are abundant in

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Parliment et al.; Caffeinated Beverages ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

385

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386 natural polyphenols, such as cocoa and tea. Flavonoid-rich extracts and pure chemical constituents of green and black tea provide antioxidant protection in vitro (4,5) and in vivo (6-8). In the case of cocoa solids, previous studies have demonstrated that the antioxidant capacity is primarily related to the flavonoid (-)-epicatechin (9). Recently, cocoa liquor constituents, identified as flavans, have been isolated and shown to be potent antioxidants in bulk oil and hydrophilic model systems (JO), probably contributing to the chemical stability of chocolate and in general decreasing the need for added preservatives (11). In fact, cocoa extracts rich in polyphenols and procyanidins have been claimed to exhibit antioxidant, antineoplastic and preservative properties (12), and inhibition of L D L oxidation by cocoa powder extracts is comparable to that of pure catechin (//). The antioxidant activity described for many foods and beverages is not only restricted to the action of polyphenolics. Model studies have demonstrated that caffeine is an efficient scavenger of hydroxyl (13) and superoxide (14) radicals. Furthermore, in situ oxidation of caffeine in soluble coffee solution procures 1,3,7trimethyluric acid, also designated 8-oxocaffeine, as the major reaction product (15), whose formation involves a C8-OH radical intermediate as recently shown by E P R spectroscopic studies (16). This chemical marker reflects the "oxidative status" of the brew and its formation is dependent on a number of physico-chemical parameters such as oxygen tension, p H , and transition metal availability (15). Analogously, theobromine subject to Fenton reagents in model systems furnishes the 8-hydroxy analogue, 3,7-dimethyluric acid, as a major reaction product (17). This study now addresses the free radical scavenging activity of inherent theobromine in cocoa powders, and the detection/quantification of the major reaction product, 8-oxotheobromine, by L C - E S I / M S / M S techniques. The impact of food additives and preservatives as well as the major physico-chemical properties that influence the in situ formation of the oxidized analogue are also discussed.

Experimental Procedures

Chemicals A l l reagents were prepared fresh before use. L(+)-ascorbate was purchased from Merck (Darmstadt, Germany). The chemicals 3,7-dimethyluric acid, theobromine, and E D T A were from Sigma (Buchs, Switzerland). Chromabond (500 mg) C18ec cartridges were from Macherey & Nagel. M n C l , FeCl .6H 0, and C u S 0 were from Aldrich (Buchs, Switzerland). PRS solid phase extraction columns (Ig) were from 1ST. The internal standard 7-methyl-3-(trideuteromethyl)uric acid was custom synthesized by Toronto Research Chemicals Inc, Canada, isotopic purity > 98%. 2

3

2

4


387 Cocoa Powders


Alkalised unsweetened cocoa powders with 12% or 20% fat were employed in this study. Theobromine contents of the powders was determined by H P L C with U V detection, showing 2.23 and 1.82 % for defatted and 20% fat powders, respectively. Iron and copper contents of the defatted powders was determined as 15.7 ppm and 5.4 ppm, respectively.

Cocoa Powder Preparation Cocoa powder solution was prepared by dissolving 1.68 g cocoa powder in 10 m L boiling distilled water. After 10 minutes under slow stirring (magnetic) at rt, aliquots were removed and incubated as described. For t samples, aliquots (1 mL) were removed and immediately acidified with 1M HC1 (50 μ ί ) in Eppendorf tubes. Deuterated 3,7-dimethyluric acid was added as an internal standard (final concentration 2 n g ^ L ) . The tubes were then centrifuged (14 000 rpm, Eppendorf systems), and the clear supernatant applied to preconditioned (each 2 bed-volumes of methanol, water, and 10 m M HC1) Isolute PRS and Chromabond C18ec columns coupled in series. After penetration of the samples under slight suction (Vasiprep, Supelco), the columns were washed with 2 bed volumes 10 m M HC1. The PRS columns were then removed and discarded, and 8-oxotheobromine eluted from the C18ec columns with 4 m L of a solvent comprised of 30% methanol acidified with acetic acid (final cone. 2 mM). The clear effluent was lyophilised and taken up in 100 of the same elution solvent and directly analysed by L C - M S / M S . 0

Oxidation Experiments with Cocoa Powder Cocoa powders were prepared as described above and an aliquot incubated in a potassium phosphate buffer (final buffer concentration 75 mM), p H 6.8 (unless otherwise stated) in a total assay volume of 1 m L (Eppendorf tubes). A typical reaction mix comprised of F e C l . 6 H 0 (100 μΜ), E D T A (500 μΜ) and 16.8 mg cocoa powder. The reactions were terminated after l h at 37°C by addition of 1 M HC1, centrifugation at 14000 rpm, 2 min. and immediate extraction over solid phase columns as described above. 3

2

Spectrosocopic analyses H P L C - M S analyses were done with a Finnigan TSQ 7000 mass spectrometer using a Vydac C-8 reverse phase column (5 μηι, 1 X 100 mm) coupled to a HewlettPackard 1100 pump. The flow rate was 0.50 mL/min and was split 1:10 with a L C Packings Acurate microflow processor prior to the injector port so that the effective flow rate through the column was 0.05 ml/min. The solvent conditions were 0.1%


388 C H C O O H and 5% C H C N at T , which increased with a linear gradient to 25% acetonitrile at 15 min, and then to 100% C H C N at 17 min, and held for 3 min prior to column re-equilibration. The electrospray interface was operated with a high voltage of 3.5 k V and a capillary temperature of 250 °C. Nitrogen was used as the sheath gas at a pressure of 80 psi. M S / M S analyses were done by collision-induced dissociation (CID) of the protonated molecular ions at a collusion energy of 40 eV. Argon was used as the collision gas at a pressure of 3 mTorr. Quantitation was done by tandem M S / M S using multiple reaction monitoring ( M R M ) mode measuring two transitions of the protonated oxomeobromine m/z 197 (M+H) at m/z 126 and 182 and the two transitions of the protonated J -oxomeobromine m/z 200 (M+H)* at m/z 129 and 185. A calibration curve was constructed at 10 concentration levels of oxotheobromine ranging from 0.05 to 37.5 ng injected on column, with the internal standard if -oxomeobromine set at 10 ng (r =0.9995). 3

3

0

3


+

3

2

3

Results

L C - M S / M S Analysis of 8-Oxotheobromine in Cocoa Powders. The L C - E S I - M S / M S daughter ion spectrum of protonated oxomeobromine m/z 197 (M+H) is shown in Figure 1, with multiple fragments which include: m/z 182 (197-CH ), 179 (197-H 0), 169 (197-CO), 151 (197- H 0 , -CO), 139 (197-CH , H N C O ) , 126 (197 - H N C O , -CO), 111 (197 - H N C O , -CO, -CH ). The daughter ions of oxomeobromine at m/z 182 and 126, and 185 and 129 for the trideuterated analgoue were chosen for M R M analyses because of their abundance and specificity. +

3

2

2

3

3

Relative Abundance 83.1

182.0 126.1

139.1 196.9

111.1

u

60

151.0

179.0 169.0 \ 80

100

120

140

160

180

200

220

240

m/z Figure 1. LC-ESIMS/MS daughter ion spectrum of 8-oxotheobromine [M+HJ+.


389 Influence of E D T A and Transition Metals on 8-Oxotheobromine Formation 3+

Supplementation of cocoa powder with F e - E D T A chelate augments the level of 8-oxotheobromine nearly 10-fold vs that of the control, whereas iron or E D T A alone had only a minor effect (Table 1). A n important property of many dietary phenolics when exposed to transition metals is the reduction of oxygen to generate superoxide radicals and hydrogen peroxide (18,19% the latter compound measured in our laboratory in the same cocoa powder and reaching up to 120 μΜ after l h . Reductive cleavage of H 0 by F e - E D T A then procures the reactive hydroxyl radical which indiscriminately reacts with cocoa constituents, including theobromine to form principally 8-oxotheobromine. A recent study with caffeine/soluble coffee has shown a similar increase in 8-oxocaffeine levels when fortified with F e - E D T A (15). Both observations - in cocoa and coffee - corroborate the ability of iron-EDTA to promote oxidative damage in situ, most probably by increasing the solubility of iron and its redox potential (20). 2+


2

2

3+

Table 1. Influence of E D T A and Transition Metals on 8-Oxotheobromine Formation in Cocoa Powder.

a

0

Conditions

8-Oxotheobromine (ppm) 4.4 7.5 6.1

ControF EDTA Fe (5.6 μg F e ^ L ) 3+

3 +

3+

3

F e - E D T A (0.56 μg Fe 7mL) F e - E D T A (2.8 μg Fe 7mL) F e - E D T A (5.6 μg Fe 7mL) 3+

3

3+

3

2+

2

C u - E D T A (5.6 μg Cu 7mL) Mn + - E D T A (5.6 μ% M n 7 m L ) 2

2

37.1 52.2 54.8 6.0 6.1

a

Defatted cocoa powder (16.8 mg) incubated for lh at 37°C, pH 6.8, as described in "Experimental Procedures". ^ Entries are averages of duplicate determinations and quantified by LC-MS/MS using deuterated 8-oxotheobromine as internal standard. Only cocoa powder. c

Fortification of cocoa powders with equivalent amounts of copper(II) or manganese(II) revealed no effect on theobromine oxidation, substantiating the minor role of C u - E D T A complexes in redox cycling (21), and inability of manganese to participate in Fenton reactions (22).


390 Influence of Ascorbate on 8-Oxotheobromine Formation Numerous cocoa-based products are fortified with the common water soluble antioxidant ascorbate, often for health-beneficial, organoleptic, and/or product stability reasons. A s depicted in Figure 2, 8-oxotheobromine formation in defatted cocoa powders that are fortified with ascorbate and F e increases substantially over time, reaching 10-fold the level of the control at a final assay concentration of 2 m M ascorbate.


3+

60 -,

u -ι 0

,

,

,

,

,

0.5

1

1.5

2

2.5

Ascorbate (mM)

Figure 2. Catalysis of 8-oxotheobromine formation in cocoa powder in the presence of ascorbate. All incubations were at pH 6.8 containing Fe^ (5.6 pg/mL). Entries are averages of duplicate determinations. Quantification of 8-oxotheobromine was by LC-MS/MS using deuterated 8-oxotheobromine as internal standard.

The augmentative action of ascorbate is even more prominent in this in situ model after addition of chelated iron (as F e - E D T A ) , reaching > 150 ppm of the methyluric acid analogue in the cocoa powder after l h (Table 2). Cocoa powder incubated under slightly alkaline conditions even further increased the level of 8oxotheobromine. This effect may be attributable to more facile autoxidation of cocoa polyphenolics, and thus more efficient redox cycling of the F e 7 F e chelates. To further demonstrate the pro-oxidant impact of ascorbate in the presence of a metal catalyst in this particular model, an equivalent amount of pure theobromine (1.97 mM) at concentrations as present in the cocoa powder under study was 3+

3

2+


391 3+

incubated in the presence of ascorbate/Fe -EDTA. Ascorbate-driven oxidation furnished 26 ppm 8-oxotheobromine at p H 6.8 after l h at 37°C. This represents a 6.7 % turnover of the total theobromine present to the C-8 hydroxylated congener.


Table 2. Influence of Ascorbate and p H on 8-Oxotheobromine Formation in Cocoa Powder.

0

8-Oxotheobromine (ppm)

0

Conditions

Control Ascorbate Ascorbate Ascorbate Ascorbate Ascorbate

5.3 6.3 8.2 51.5 155.6 21

(0.4 mM) (1 mM) (1 mM) + F e (1 mM) + F e - E D T A (1 mM) + C u - E D T A 3+

3+

2+

d

Effect of pH ; water potassium phosphate, p H potassium phosphate, p H potassium phosphate, p H potassium phosphate, p H 6

157.7 155.1 155.6 179.2 190.9

6.5 6.8 7.0 7.8

a

Defatted eoeoa powder (16.8 mg) incubated for lh at 37°C, pH 6.8, transition metals all at 5.6 μg/mL, as described in "Experimental Procedures", b Entries are averages of duplicate determinations and quantified by LC-MS/MS using deuterated 8-oxotheobromine as internal standard. Only cocoa powder, à all incubations with 1 mM ascorbate. Incubation of cocoa powder in distilled water, pH 7.5. c

e

Discussion The addition of ascorbic acid to foods is generally regarded as a health-beneficial and product stabilizing factor. However, numerous reports have highlighted the paradox action of this water soluble vitamin in foods and model systems under aerobic conditions when exposed to trace levels of catalytically active metals (20,23,24). As shown here, fortification of cocoa powders at p H 6.8 with exogenous F e - E D T A and ascorbate drastically increased levels of 8-oxotheobromine (nearly 30fold). Ascorbate alone did not have much effect, even though the particular defatted


392 cocoa powder under study contained 15 ppm natural iron load, reported to reach up to 200 ppm in certain powders (25). The major factor contributing to the pro-oxidative reactions observed i n cocoa powder solutions is the availability of catalytically active iron. Addition of ferric iron salt alone showed only a minor increase in 8-oxotheobromine, which can be explained in part by decreased solubility and more site specific interactions with cocoa constituents, and thus less accessibility of iron to the target molecule. However, fortification of cocoa powder solution with relatively low levels of F e - E D T A (0.56 μg/mL Fe ) had a significantly positive impact on C-8 hydroxylation, resulting in an 8-fold increase of 8-oxotheobromine vs the control. Thus, natural cocoa constituents, such as for example flavonoids, are able to mediate the reduction of the F e - E D T A chelate to the active ferrous state. The formation of hydrogen peroxide in the presence of catalytically active metals via polyphenol-driven autoxidation furnishes the reagents required for Fenton chemistry, procuring the highly deleterious hydroxyl radical. Determination of a major product of hydroxyl radical attack upon theobromine, i.e. 8-oxotheobromine - reflects the in situ pro-oxidative effect of various food additives and preservatives, as demonstrated here for iron and ascorbic acid. 3+


3+

3+

The employment of inherent stable and non-volatile chemical markers that reflect oxidation in methylxanthine-rich foods and beverages - either in the raw materials, intermediate, or finished products - are important tools in quality control and shelflife assessment. A recent report on cocoa bean quality criteria has identified endogenous amyl alcohols and their ratios to other volatile constituents as indicative of flavour quality and age of the beans (26). A s already demonstrated in coffee with the trimethyluric acid analogue of caffeine (15), pro-oxidative reactions in cocoa powders are indeed positively correlated to the formation of 8-oxotheobromine. Future work will focus on the levels of this chemical marker in cocoa beans of various geographical origin, and the impact of various parameters such as bean storage, degree of fermentation, roasting, and processing.

References

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393 6. Serafini, M., Ghiselli, Α., Ferro-Luzzi, A. Eur. J. Clin. Nutr. 1996, 50, 28-32 7. Ishikawa, T., Suzukawa, M., Ito, T., Yoshida, H., Ayaori, M., Nishiwaki, M., Yonemura, Α., Hara, Y., Nakamura, H. Am. J. Clin. Nutr. 1997, 66, 261-266. 8. Pietta, P., Simonetti, P. Biochem. Mol. Biol. Int. 1998, 44(5), 1069-1074. 9. Ziegleder, G., Sandmeier, D. Zucker und Süsswarenw. ZSW, 1982, 35, 217-222. 10. Sanbogi, C., Osakabe, N., Natsume, M., Takizawa, T., Gomi, S., Osawa, T. J. Agric. Food Chem. 1998, 46, 454-457. 11. Waterhouse, A.L., Shirley, J.R., Donovan, J.L. The Lancet, 1996, 348, 834. 12. Romanczyk, L.J. Jr., Hammerstone, J.F.Jr., Buck, M.M. US Patent No. 5712305, 1998. 13. Shi, X., Dalal, N.S., Jain, A.C., Food Chem. Toxicol. 1991, 29, 1-6. 14. Goodman, B.A., Glidewell, S.M., Deighton, N., Morrice, A.E. Food Chem. 1994, 51, 399-403. 15. Stadler, R.H., Fay, L.B. J. Agric. Food Chem. 1995, 43, 1332-1338. 16. Telo, J.P., Vieira, A.J.S.C. J. Chem. Soc. Perkin Trans. 2 1997, 1755-1757. 17. Stadler, R.H., Richoz, J.R., Turesky, R.J., Welti, D.H., Fay, L.B. Free Rad. Res. 1996, 24, 225-240. 18. Stadler, R.H., Turesky, R.J., Müller, O., Markovic, J., Leong-Morgenthaler, P.M. Mut. Res. 1994, 308, 177-190. 19. Inoue, S., Ito, K., Yamamoto, K., Kawanishi, S.Carcinogenesis 1992, 13, 14971502. 20. Mahoney, J.R., Graf, E. J. Food Sci. 1986, 51(5), 1293-1296. 21. McCord, J.M., Day, E.D. FEBS Lett. 1978, 86(1), 139-142. 22. Gutteridge, J.M.C., Bannister, J.V. Biochem. J. 1986, 234, 225-228. 23. Aruoma, O.I. Pro-oxidant properties: an important consideration for food additives and/or nutrient components ? In "Free Radicals and Food Additives" Eds Aruoma, O.I., Halliwell, B., Taylor & Francis, London, New York, Philadelphia, 1991, p 173-194. 24. Kitts, D.D. Trends in Food Sci & Technol. 1997, 8, 198-203. 25. Mohr, W., Zürcher, K., Knezevic, G. Gordian, 1971, 71(6), 184-188. 26. Οberparleitner, S., Ziegleder, G. Ζ. Lebensm. Unters. Forsch. A. 1997, 204, 156160.


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