HPLC-MS Analysis of Flavonoids in Foods and ... - ACS Publications

Chapter 38

HPLC-MS Analysis of Flavonoids in Foods and Beverages Downloaded by KTH ROYAL INST OF TECHNOLOGY on March 4, 2016 | http://pubs.acs.org Publication Date: January 15, 2000 | doi: 10.1021/bk-2000-0754.ch038

John F. Hammerstone and Sheryl A. Lazarus Analytical and Applied Sciences Group, Mars, Incorporated, 800 High Street, Hackettstown, NJ 07840 In the current study, we investigated the usefulness of reversed-phase and normal-phase chromatography for comparing the separation of low molecular weight flavonoids in green tea versus the oligomeric procyanidins in cocoa. The results of this study demonstrated that the reversed-phase technique was better suited for the separation of the flavan-3-ols and flavonols in green tea while the normal-phase method was superior for separation of flavan-3-ol oligomers in cocoa. Therefore, it was concluded that both techniques are required for a comprehensive survey of the flavonoid classes that are ubiquitous in nature. Since the presence of (-)-epicatechin was first described in tea (/), researchers have isolated and identified structurally similar components which led to the elucidation of the other flavan-3-ol monomers (2). Eventually the growth of this field resulted in the discovery of similar catechins in other plant materials such as cocoa (3,4). To date, detailed studies into the polyphenolic composition of plant materials have successfully elucidated more than 4000 structurally different compounds found ubiquitously in nature (5). As a result, it is not surprising that the types and quantities of polyphenols found in different plant-derived foods vary considerably. For example, in addition to the monomeric catechins (Figure 1A), other low molecular weight flavonols (Figure IB) and phenolic acids have been identified in green tea whereas a series of complex oligomers have been found in cocoa (6,7). Consequently, the need to develop separation techniques to improve upon the tedious methods historically employed to generate more efficient and effective isolation techniques such as high performance liquid chromatography (HPLC) became evident. Traditionally, reversed-phase H P L C methods have been employed for the analysis of various flavonoid classes in foods. For example, Salagoïty-Auguste and Bertrand used a C i hydrocarbon polymer column for the separation of phenolic acids in wine (8). Hertog et al. also used a similar method for the separation and quantification of low molecular weight flavonols in fruits and vegetables (9). More recently, Bronner and Beecher used a C i bonded silica column for the separation of 8

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© 2000 American Chemical Society

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

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375

Figure 1. Backbone structures for aflavan-3-ol(A) and aflavonol(B).



376 monomeric catechins in tea infusions (10). From the outset, it was shown that reversed-phase (RP) methods had limited application to flavonoids since RP methods are ineffective in separating oligomeric proanthocyanidins as demonstrated by Wilson who showed that oligomers could only be partially resolved (//). Furthermore, he also demonstrated the difficulty in determining degree of polymerization using R P methods since the oligomers elute in non-sequential order. With this limitation identified, normal-phase methodologies have been explored for the separation of discrete oligomeric classes in foods. Early efforts to separate oligomeric proanthocyanidins by Wilson in apple juice were effective only through the hexamers (//). Although showing better resolution, Rigaud et al. was only successful in separating through the pentamers in cocoa and tetramers in grape seeds (12). Recently, an improved normal-phase method has been reported by Hammerstone et al. in which a silica column was used to separate oligomers as discrete groupings through the decamers in cocoa and chocolate (13). In the current study, we investigated the usefulness of reversed-phase and normalphase chromatography for comparing the separation of low molecular weight flavonoids in green tea versus the oligomeric procyanidins in cocoa. Spectral data from a diode array detector and mass spectrometer were collected to aid in assessing the effectiveness of these two separation techniques.

Materials and Methods Reference Compounds (+)-Catechin, (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate, (-)epigallocatechin gallate, caffeine and theobromine were purchased from Sigma Chemical (St. Louis, M O ) . Samples Green tea (Camellia sinensis) was purchased commercially at a local grocery store. Cocoa (Theobroma cacao) mix for milk containing Cocoapro™ was provided by Master Foods Interamerica (Mars, Inc., Puerto Rico). Sample Preparation and Polyphenol Extraction The cocoa drink mix powder (10 g) was extracted twice with 45 mL of hexane to remove lipids. The resulting defatted material (~9 g) was extracted with 40 mL of acetone:water:acetic acid in a ratio by volume of 70:29.5:0.5, respectively, The solids were pelletized by centrifuging for 5 mins at 1500 χ g then the supernatant decanted and the organic solvent removed by rotary evaporation under partial vacuum at 40°C, As previously reported (14\ the resulting aqueous solution was loaded onto a Supelcosil Envi-18 20 mL Solid Phase Extraction (SPE) Column (Supelco, Inc., Bellefonte, P A ) previously conditioned with 3x20 mL of methanol followed by 5x20


377 mL of deionized water. After sample loading, the SPE column was rinsed with 3x20 mL of deionized water before eluting the polyphenols with 7 mL of acetone: watenacetic acid (70:29.5:0.5 v/v). Green tea beverage was prepared by boiling 1 tea bag in 250 mL of deionized water for 2 minutes. The beverage was cooled prior to loading onto a SPE column conditioned with methanol and water as described above. After sample loading, the SPE column was rinsed with 3x20 mL of deionized water before eluting the polyphenols with 5 mL of acetone: watenacetic acid (70:29.5:0.5 v/v).


Reversed-phase H P L C / M S Analysis of Proanthocyanidins Chromatographic analyses were performed using a HP 1100 Series H P L C (Hewlett Packard, Palo Alto, C A ) equipped with an auto-injector, quaternary H P L C pump, column heater, fluorescence detector and HP ChemStation for data collection and manipulation. Reversed-phase separations were perfonned using a H P (Palo Alto, C A ) 5 μηι Hypersil ODS column (200 χ 2.1 mm) at 26°C. The binary mobile phase consisted of A ) 0.5% acetic acid in water and B) 0.5% acetic acid in acetonitrile. Separations were affected by a series of linear gradients of Β into A at a flow rate of 0.4 mL/min as follows: elution starting with 8% Β in A , 0-5 mins isocratic; 8-40% Β in A , 5-60 mins; 40-100% Β in A , 60-65 mins; 100% B , 65-70 mins isocratic. Data were collected using the U V detector at 280 nm for flavan-3-ols, 320 nm and 360 nm for flavonols. Fluorescence detection (FLD) at λ = 276 nm and X = 316 nm was also used for non-gallated flavan-3-ols. Other F L D conditions included a photomultiplier tube gain of 12, frequency of 110 H z and response time of 2 seconds. H P L C / M S analyses of polyphenol extracts were performed using a H P 1100 Series H P L C as described above and interfaced to a HP Series 1100 mass selective detector (model G1946A) equipped with an API-ES ionization chamber. Methanol was delivered with a HP 1100 series H P L C pump (0.4 mL/min) via a tee into the eluant stream of the H P L C just prior to the mass spectrometer in order to aid ionization. Conditions for ionization in the negative mode include a capillary voltage of 3.5 k V and a fragmentation voltage of 100 V . Spectra were scanned over a mass range of m/z 200-2000 at 2.12 s per cycle. ε χ

tm

Normal-phase H P L C / M S Analysis of Proanthocyanidins Chromatographic analyses were performed using a H P 1100 Series H P L C (Hewlett Packard, Palo Alto, C A ) equipped with an auto-injector, quaternary H P L C pump, column heater, fluorescence detector and H P ChemStation for data collection and manipulation. Normal-phase separations of the proanthocyanidin oligomers were performed using a Phenomenex (Torrance, C A ) 5μιη Lichrosphere silica column (250 χ 4.6 mm) at 37°C. The ternary mobile phase consisted of A ) dichloromethane, B) methanol and C) acetic acid and water (1:1 v/v). Separations were achieved by a series of linear gradients of Β into A with a constant 4% C at a flow rate of 1 mL/min as previously described by Hammerstone et al. (75). In brief, elution started with 14% Β in A then increased to 28.4% Β over 30 mins; to 50% Β over 30-60 min; to 86% Β


378 over 60-65 min; and finally isocratic to 70 min. Data were collected using the U V detector at 280 nm for flavan-3-ols, 320 nm and 360 nm for flavonols. Fluorescence detection (FLD) at λ = 276 nm and % = 316 nm was also used for non-gallated flavan-3-ols. Other F L D conditions included a photomultiplier tube gain of 12, frequency of 110 H z and response time of 2 seconds. H P L C / M S analyses of polyphenol extracts were performed using a H P 1100 Series H P L C as described above and interfaced to a HP Series 1100 mass selective detector (model G1946A) equipped with an API-ES ionization chamber. Ammonium acetate (10 mM) was delivered with a HP 1100 series H P L C pump (0.1 mL/min) via a tee into the eluant stream of the H P L C just prior to the mass spectrometer in order to aid ionization. Conditions for ionization in the negative mode include a capillary voltage of 3.5 k V and a fragmentation voltage of 85 V . Spectra were scanned over a mass range of m/z 220-2020 at 2.12 s per cycle.


β χ

tm

Results and Discussion Reversed-phase H P L C techniques are most commonly employed for the separation of low molecular weight flavonoids such as the flavan-3-ol monomers. In order to develop an effective reversed-phase method, the five most predominant monomers and the two abundant xanthine alkaloids, caffeine and theobromine, found in tea and/or cocoa were purchased commercially and used as standards. A n H P L C method which was successful in separating the monomers and xanthine alkaloids was achieved using a Hypersil O D S column. Interestingly, it was observed during method development that the xanthine alkaloids, the prodelphinidin monomer, and the gallated catechins gave little or no fluorescence signal in contrast to the two procyanidin catechins. Therefore, all analyses were performed using both the U V and fluorescence (FLD) detectors. Furthermore, the mass spectrometer conditions were optimized in the negative ion mode using the catechin standards in order to aid peak identification. It should be noted that the mass range scanned excludes the xanthine alkaloids since our primary interest is in the flavan-3-ols. Next, the polyphenol extract of the cocoa drink mix was analyzed using the reversed-phase method with fluorescence detection since cocoa contains predominantly the procyanidin class and the F L D has increased sensitivity over the U V detector at 280 nm (3,4,14). The F L D trace can be seen in Figure 2 and using the extracted ion chromatogram (EIC) at m/z 289, the two monomer peaks can be identified with (+)-catechin eluting significantly earlier than (-)-epicatechin as confirmed using authentic standards. Since it is well known that cocoa contains a complex series of procyanidin oligomers (6), EICs were generated which correspond to the molecular ions for dimers through tetramers and the doubly charged ion for pentamers in order to determine their elution times. As can be seen in Figure 2, the major dimer elutes between the two monomer peaks while other dimer isomers elute after the primary pentamer peak. This non-sequential elution order along with multiple isomers for each oligomeric class makes interpretation difficult when using reversedphase techniques.



379 For comparison, the polyphenol extract of the cocoa drink mix was also analyzed using the normal-phase H P L C method previously developed with fluorescence detection and modified mass spectrometer conditions to eliminate the use of a strong base (13). Similar to previous results for cocoa and chocolate, the drink extract yielded a F L D trace in which the procyanidin oligomers elute according to degree of polymerization (Figure 3). Additionally, the multiple isomers for each oligomer class are still evident, but elute in close proximity to each other in contrast to the reversedphase method. For example, (+)-catechin elutes just prior to the (-)-epicatechin with less than a minute separating the two, whereas on the reversed-phase column, the monomers eluted approximately five minutes apart. Therefore, it can be concluded that for a sample containing complex oligomers, the normal-phase method is superior to the reversed-phase since separation as discrete oligomeric groupings can be achieved in sequential order. With the normal-phase method preferable for the cocoa polyphenol extract, green tea beverage was analyzed in a similar manner. However, since it is known that tea contains substantial quantities of gallated flavan-3-ols (7,/5), the U V detector was used exclusively since the F L D is insensitive to this class of monomers. Furthermore, U V data at 320 and 360 nm were collected in addition to the traditional 280 nm in order to selectively detect the flavonols which are also a major flavonoid class found in tea (7J5J6). The U V traces from green tea beverage extract at 280 and 360 nm can be seen in Figure 4. Using a combination of mass spectrometry and authentic standards, it is evident that not only are the monomers incompletely resolved, but also the (-)-epigallocatechin gallate elutes at the same time as the dimers in cocoa. Furthermore, the U V trace at 360 nm indicates that the flavonols elute as a large, mostly unresolved hump late in the separation. Due to these inadequacies, the more traditional technique using the reversed-phase method developed above was employed for comparison. The U V traces at 280 and 320 nm for the reversed-phase separation of the green tea beverage extract can be seen in Figure 5, Immediately, it is evident that greater resolution is achieved for green tea using the reversed-phase method compared to the normal-phase chromatography. Table 1 lists the spectral data used to tentatively identify the complex mixture of peaks observed in the reversed-phase chromatograms. As can be seen, six of the commonly described flavan-3-ol monomers were identified in the green tea beverage extract in addition to two obscure methylgallate catechins which is consistent with previous reports (7,77). Interestingly, the flavan-3-ol monomers have an unusual ionization pattern in which they tend to form the (2M-H)" ion readily. For example, in addition to the molecular ion (m/z 441) for (-)-epicatechin gallate, the (2M-H)" ion with m/z 883 was also observed. Furthermore, the spectrum was characterized by the loss of the gallic acid ester resulting in an ion with m/z 289. In addition to the monomers, oligomers through trimers were also observed in the green tea beverage which is consistent with previous results (14). While the flavan-3-ols account for the major peaks in the U V trace at 280 nm, the substantial number of peaks detected at 320 nm suggests that additional flavonoids are also present in the green tea beverage as expected (7). Traditionally, U V data at 320 nm and 360 nm are used to elucidate other classes of flavonoids such as the phenolic



380

n/r 8ft1 rofr 577

k

0

5

1

10

20

25

30

35

min

Figure 2. FLD trace (bottom panel) and EIC (top panels) of a reverse-phase separation of cocoa drink extract, m/z 289, 577, 865, 1153 and 720 correspond to monomer through pentamer, respectively^ m/z 289

/M/Z

577

m/z 865

m/z U 53

m/z 720

Figure 3. FLD trace (bottom panel) and EIC (top panel) of a normal-phase separation of cocoa drink extract, m/z 289, 577, 865, 1153 and 720 correspond to monomer through pentamer, respectively.



381

Figure 5. UV trace at 280 and 320 nm for a reversed-phase separation of tea extract. A through W corresponds to peaks identified in Table 1.



382

Table 1. Flavonoids in Green Tea by Reversed-Phase H P L C / M S . Retention Time 3.70 4.84 5.86 9.20 9.89 12.28 12.71 14.25 15.10 15.64 15.97 16.78 16.93 18.24 18.90 19.39 19.64 20.12 21.40 24.21 32.52 33.32 34.55

Peak

Major Ions (m/z)

A Β C D Ε F G H I J Κ L M N 0 P

305,611 289 577 289 457,915 593,729 457,915 897, 729, 787 479, 635, 625 479 563 943,471,287 771 593, 771 441,883,289 609, 755 577 463 755, 597 425,455 1049, 524,917 1049, 524, 1063,531 1033,516

Q R S T

u V

w

•Confirmed with authentic standard.

UV (nm) 270 278 N/A 278 274 272,335 274 274 266, 358 262, 356 270, 338 272 258, 356 256, 354 278 264, 350 272,339 256, 356 266, 346 277 268,317 266,318 266, 322 max

f

Tentative ID (-)-Epigallocatechin* ( E G C ) (+)-Catechin* Caffeine* and Dimer (-)-Epicatechin* (EC) (-)-Epigallocatechin gallate* Dimers (-f)-Gallocatechin gallate Dimers Myricetin glycoside + H T Myricetin glycoside Hydrolyzable Tannin (HT) EGC-3-(3-0-methylgaliate) Quercetin glycoside Quercetin glycoside (-)-Epicatechin gallate* Quercetin glycoside Dimer Quercetin glycoside Kaempferol glycoside EC-3-(3-0-methylgallate) Trimer Trimers Trimer

'Not applicable - signal off-scale.



383 acids and flavonols which have also been reported in tea (7,75,76). However, under the current mass spectral conditions, the phenolic acids are not detected since their molecular weights fall below the mass range scanned. Therefore, flavonol glycosides appear to account for the majority of the components detected at 320 nm as indicated in Table 1. It has been reported that the major flavonols in tea are myricetin, quercetin and kaempferol which is consistent with the U V and mass spectral data used to tentatively identify their glycosides (7,75). However, further confirmation of the aglycone would require collision-induced dissociation experiments in addition to hydrolysis or N M R experiments to elucidate the sugar moiety. Moreover, other minor phenolic constituents were detected in the U V chromatograms and tentatively identified as hydrolyzable tannins (HT) since they are known to occur in tea (7). Finally, it should be noted that the range of phenolic compounds observed in the current study may be limited since the extract was prepared from a tea infusion rather than from direct extraction of the leaves.

Conclusion This study sought to compare the effectiveness of reversed-phase and normalphase chromatography for the separation of flavonoids in tea and cocoa beverages. Since the reversed-phase technique was capable of separating the low molecular weight flavonoids, this method was found to be better suited for the tea beverage extract than the normal-phase technique. In contrast, better resolution for the cocoa extract was achieved using the normal-phase method that was capable of separating the complex series of flavan-3-ol oligomers as discrete groups in increasing order of polymerization. In conclusion, a combination of reversed-phase and normal-phase chromatography along with multiple detection systems (e.g., fluorescence, D A D , M S ) are necessary for a comprehensive survey of the various classes of flavonoids in foods and beverages.

References

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Tsujimura, M. Sci. Papers Inst. Phys. Chem. Res. Tokyo. 1929, 10, 253-261. Bradfield, A. E.; Penney, M. J. Chem. Soc. 1948, 2249-2254. Forsyth, W. G. C. Biochem. J. 1955, 60, 108-111. Quesnel, V. C. Phytochemistry. 1968, 7, 1583-1592. Flavonoids in Health andDisease;Rice-Evans, C.; Packer, L., Eds.; Marcel Dekker: New York, NY, 1997. Porter, L. J.; Ma, Z.; Chan, B. G. Phytochemistry. 1991, 30, 1657-1663. Finger, Α.; Kuhr, S.; Engelhardt, U. H. J. of Chromat. 1992, 624, 293-315. Salagoïty-Auguste, M.-H.; Bertrand, A. J. Sci. Food. Ag. 1984, 35, 1241-1247. Hertog, M. G. L.; Hollman, P. C. H.; Katan, M. B. J. Agric. Food Chem. 1992, 40, 2379-2383. Bronner, W. E.; Beecher, G. R. J. of Chromat. A. 1998, 805, 137-142.



384 11. Wilson, E. L. J.Sci.Food Agric. 1981, 32, 257-264. 12. Rigaud, J.; Escribano-Bailon, M. T.; Prieur, C.; Souquet, J. M.; Cheynier, V. J. of Chromat. A. 1993, 654, 255-260. 13. Hammerstone, J. F.; Lazarus, S. Α.; Mitchell, A. E.; Rucker, R.; Schmitz, H. H. J. Agric. Food Chem. 1999, 47, 490-496. 14. Lazarus, S. Α.; Adamson, G. E.; Hammerstone, J. F.; Schmitz, H. H. Submitted for publication in J. Agric. Food Chem. 1998. 15. Balentine, D. A. In Phenolic Compounds in Food and Their Effects on Health I. Ho, C.-T.; Lee, C. Y.; Huang, M.-T., Eds.; ACS Symposium Series 506; ACS: Washington, DC, 1992; 1, pp 102-117. 16. Bailey,R.G.; McDowell, I.; Nursten, H. E. J.Sci.Food Agric. 1990, 52, 509525. 17. Saijo, R. Agric. Biol. Chem. 1982, 46, 1969-1970.


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