RP HPLC and RP TLC in the Analysis of Flavonoids

Transcription

1 Chem. Anal. (Warsaw), 50, 301 (2005) RP HPLC and RP TLC in the Analysis of Flavonoids by Ryszard Baranowski, Joanna Kabut and Irena Baranowska* Department of Analytical and General Chemistry, The Silesian University of Technology, ul.strzody 7, Gliwice, Poland Key words: flavonoids, RP TLC, RP HPLC, fruit, vegetables, wine, tea, urine Effect of mobile phase composition on the separation of flavonoids from five major classes (catechins, flavonols, flavanones, flavones and anthocyanidins) has been investigated on RP 18 TLC plates. Water acetonitrile mixtures and trifluoroacetic acid in water acetonitrile mixtures have been used as the mobile phases. An RP HPLC system for these compounds has also been developed. Interferences from vitamins and methylxanthines in the new RP HPLC system have been investigated. Flavonoids were determined in fruit, vegetables, wine, tea and urine samples. Rozdzielano flawonoidy z piêciu g³ównych klas (katechiny, flawony, flawanony, flawonole i antocyjanidyny) metod¹ chromatografii cienkowarstwowej na p³ytkach RP 18, stosuj¹c jako fazê ruchom¹ mieszaniny wody i acetonitrylu z dodatkiem kwasu trifluorooctowego. Dla tej samej grupy flawonoidów opracowano uk³ad RP HPLC. W opracowanym uk³adzie badano wp³yw interferencji ze strony witamin i metyloksantyn. znaczono zawartoœæ flawonoidów w próbkach: owoców, warzyw, win, herbat i w moczu. * Corresponding author. irena.baranowska@polsl.pl; Tel./fax:

2 302 R. Baranowski, J. Kabut and I. Baranowska Flavonoids are one of ten classes of plant polyphenols. Polyphenols are the product of metabolism of plants. The main structure of flavonoids is a skeleton of 2- phenylbenzopyrone. Among flavonoids we distinguish 13 classes and 5000 compounds [1]. The classes differ in the main skeleton of these compounds. The main classes of flavonoids which occur in food are: catechins, flavones, flavonols, flavanones and anthocyanidins [2,3]. Within each class there are many compounds which differ in quantity and quality of their substituents (hydroxy and methoxy groups or sugar radicals) [1,4]. The quantity, type and place of substituents in a molecule determine the properties of these compounds [1]. Flavonoids have the antioxidant capacity [3 5] due to which they are significant in prevention of many diseases, mainly tumours [2,4,6]. HPLC and TLC are often used for separation and quantitative determination of flavonoids. TLC is the most often used technique for research of flavonoids as it is simple and cheap [1]. It is used to avoid more expensive techniques, e.g. HPLC [5]. Silica gel and cellulose were used as stationary phases in normal phase system and silica gel modified by CN and NH 2 was used in the case of RP TLC. Mixtures of solvents such as: methanol with ethyl acetate and water with methanol were used [1,2,5,7]. HPLC is also used to isolate, identify and determine flavonoids. Separation is usually carried out in a reversed phase system using RP 18 stationary phase [1 4]. Methanol and acetonitrile containing a small number of modifiers such as tetrahydrofuran (THF), trifluoroacetic acid (TFA), acetic and phosphoric acids are the most frequently employed mobile phases. [1,3]. Reversed phase system is preferable for flavonoids studies because they are polar substances with a very different polarity. This difference depends on the form in which flavonoids occur i.e. aglycones or glycosides [8]. The following studies were carried out by TLC and HPLC techniques in the reversed phase system. n the basis of TLC analysis the HPLC gradient system, we had worked out earlier [19], was changed in such a way that it allowed separation of more complex compositions of flavonoids. The modified system was used for the analysis of plant and urine samples. EXPERIMENTAL Reagents Standard methanol solutions of flavonoids: (±)-catechin, ( )-epicatechin, ( )-epigallocatechin gallate, ( )-epicatechin gallate, rutin, hesperedin, apigenin-7--neohesperidoside, quercetrin, neohesperedin, luteolin, apigenin, chrysin, (±)-naringenin, hesperetin, pinocembrin, quercetin, myricetin, kaempferol, pelargonidin (Sigma Aldrich) (Tab. 1).

3 RP HPLC and RP TLC in the Analysis of Flavonoids 303 Table 1. List and structures of studied flavonoids Group of substances Common name Atom number Catechins H Flavonols H Flavones Flavanones (±)-catechin H H H H H ( )-epicatechin H H H H H ( )-epigallocatechin gallate gal H H H H H ( )-epicatechin gallate gal H H H H kaempferol H H H H quercetin H H H H H myricetin H H H H H H rutin RhGlu H H H H quercetrin Rh H H H H chrysin H H apigenin H H H luteolin H H H H apigenin-7-neohesperidoside H Neoh H H pinocembrin H H (±)-naringenin H H H hesperetin H H H CH 3 neohesperedin H Neoh H CH 3 hesperedin H RhGlu H CH 3 Glu glucose; Neoh neohesperidose; Rh rhamnose; Gal gallic acid 2' 3' 4' ' 2 3 6' 5' Standard water solutions of methylxanthines: theophylline, theobromine (Sigma), caffeine, 1,7-dimethylxanthine, 3-methylxanthine, 7-methylxanthine (Aldrich).

4 304 R. Baranowski, J. Kabut and I. Baranowska Standard methanol solutions of vitamins: B 1 (thiamine), B 6 (pyridoxine), C (ascorbic acid), B 2 (riboflavin) (Sigma Aldrich). Acetonitrile (gradient grade for liquid chromatography), water for chromatography, trifluoroacetic acid (Merck), methanol (PCh, Gliwice). Apparatus TLC was performed on cm glass plates precoated with DS stationary phase (Merck, Darmstadt, Germany). Mixtures of water and acetonitrile (1:2, 1:1, 2:1) and mixtures of water and acetonitrile (1:1) containing 0.05%, 0.1%, 0.2% trifluoroacetic acid (TFA) were used as mobile phases. The visualization of chromatograms was performed using mixture of 3% boric acid and 10% oxalic acid (3:1) with inspection under UV illumination. A Merck Hitachi L 4500A chromatograph equipped with DAD detector was used in the HPLC investigations. Stationary phase: LiChrosorb RP 18 (4 125 mm, 7 µm). Mobile phase: system of two solvents: 0.05% TFA in water (solvent A) and 0.05% TFA in acetonitrile (solvent B); gradient (0 min 90% A and 10% B; 25 min 80% A and 20% B, 35 min 75% A and 25% B, 60 min 25% A, 75% B). The flow rate was 1 ml min 1, t = 21 C. Absorption spectra were registered in the range of nm. Recording of chromatograms was carried out at the wavelengths 210 nm, 250 nm, 270 nm, 360 nm. Solid phase extraction of flavonoids was carried out by means of Baker-spe system (J.T Baker Inc. Set Phillipsburg, USA) Extraction procedure 2 g of freeze-dried samples of pear, banana, cherry, peach, carrot, celery, corn, fennel, horseradish, parsley leaves, leek, parsley root, green pea, green pepper, yellow pea, soy sprouts, (Elena, Kokanin Poland) were extracted in 20 ml of 50% methanol over 2 h, filtered and evaporated to 2 ml. 20 µl of these extracts were introduced on the chromatographic column. Preparation of tea samples 2 g of black tea such as: Lipton, Saga, Posti, Pickwick and green tea were poured over with 20 ml of boiling water and were brewed under the lid for about 10 min, cooled, filtered and washed with 5 ml of water. The analyzed compounds were extracted from tea samples using the Bakerbond spe C 8, 3 ml columns (Yunan, Saga, green tea) and C 18, 1 ml columns (Lipton, Pickwick). In Bakerbond spe for both cases before the introduction of 25 ml of samples the column was conditioned by 3 ml of MeH and 3 ml of water. Flavonoids were eluted with 10 ml of MeH 25% NH 4 H (10:0.2), evaporated until dry, dissolved in 1 ml of MeH and 20 µl were introduced on the chromatographic column. Preparation of wine samples The analyzed compounds from wine samples were extracted using the Bakerbond spe C 18, 1 ml columns and Bakerbond spe SDB 1, 3 ml columns. In both cases before the introduction of 10 ml of samples the columns were conditioned by 3 ml of MeH and 3 ml of water. Flavonoids were eluted with 10 ml of MeH 25% NH 4 H (10:0.2), evaporated until 2 ml and 20 µl were injected into chromatographic column.

5 RP HPLC and RP TLC in the Analysis of Flavonoids 305 Preparation of urine samples Urine samples were collected from healthy people whose diet is rich in flavonoids. The analyzed compounds from urine samples were extracted using the Bakerbond spe C 18, 1 ml columns. Before the introduction of 2 ml of samples the column was conditioned by 3 ml MeH and 10 ml of water. Flavonoids were eluted successively with: 3 ml water, 3 ml of 50% MeH, 3 ml of 100% MeH, evaporated until dry, dissolved in 0.2 ml of MeH and then 20 µl were injected in the chromatographic column. RESULTS AND DISCUSSIN In order to improve the conditions of separation of flavonoids, belonging to such classes as: catechins, flavones, flavonols, flavanones and anthocyanidins, using HPLC, worked out earlier [19], by the TLC technique in a reversed phase system was carried out, prior to HPLC. Separations were made on RP 18 plates. Water with different acetonitrile percentage was used as a mobile phase. In the whole range of the percentage content of acetonitrile in the mixture we observed a good separation of flavonoids in the form of aglycones within particular classes: flavones (chrysin, luteolin, apigenin), flavanones (hesperetin, pinocembrin, (±)-naringenin), flavonols (kaempferol, myricetin, quercetin), catechins ((±) catechin, ( )-epicatechin, ( )-epigallocatechin gallate, ( )- epicatechin gallate) and anthocyanidins (pelargonidin). For some flavonoids, within the same class of compounds, significant differences in the values of R M factors of aglycones and glycosides can be observed. This was especially noted when they differ in sugar substituents, e.g.: quercetin and its two glycoside forms: rutin and quercetrin, hesperetin and its glycosides: hesperedin and neohesperedin and apigenin and apigenin-7--neohesperidoside. It is much more difficult to separate flavonoids belonging to different classes and having similar polarity. Within studied flavonoids in the glycoside form it concerns mainly such compounds as rutin and hesperidin which have the same sugar as one of the substituents. Examples of aglycones, being difficult to separate, are the following: quercetin with luteolin, both apigenin with kaempferol, and pinocembrin with chrysin. Pinocembrin and chrysin both have two hydroxy groups and insignificant differences in the structure of the main skeleton, which means that in the case of flavones exist a double bond between second and third carbon atom and in the case of flavanones this bond is a single one. Kaempferol, quercetin, luteolin, apigenin differ in the number of hydroxy groups and their arrangement (Tab. 1). Howewer difficulties with their separation are due to the fact that hydroxy group in C3 position can react with carboxyl group in C4 position. Then we have seemingly the same number of H groups within pairs: kaempferol, apigenin and luteolin, quercetin. nly with the use of water acetonitrile mixture (45:55,

6 306 R. Baranowski, J. Kabut and I. Baranowska v/v) the separation of apigenin and kaempferol may be achieved. Pinocembrin and chrysin may be separated when the mobile phase contains 60% of acetonitrile. Mobile phases containing 0.05%, 0.1% and 0.2% trifluoroacetic acid in the mixture of water and acetonitrile (1:1) have been investigated. The obtained R M values were similar trifluoroacetic acid does not improve the separation of the analysed compounds. According to the data obtained in TLC method a gradient system worked out by the authors earlier and used for flavonoids analysis in real samples was modified [19]. In the worked out HPLC conditions the separation of 18 flavonoids was achieved, namely: (±)-catechin, ( )-epicatechin, ( )-epigallocatechin gallate, ( )-epicatechin gallate, rutin, hesperedin, quercetrin, neohesperedin, apigenin-7--neohesperidoside, myricetin, quercetin, luteolin, (±)-naringenin, hesperetin, kaempferol, apigenin, pinocembrin, chrysin (Fig. 1). Intensity, AU Retention time, min Figure 1. Chromatogram a mixture of standards; detection at l = 270 nm, 1. (±)-catechin, 2. ( )-epicatechin, 3. ( )-epigallocatechin gallate, 4. ( )-epicatechin gallate, 5. rutin, 6. hesperedin, 7. apigenin-7- --neohesperidoside, 8. quercetrin, 9. neohesperedin, 10. myricetin, 11. quercetin, 12. luteolin, 13. (±)-naringenin, 14. hesperetin, 15. kaempferol, 16. apigenin, 17. pinocembrin, 18. chrysin Retention times of the flavonoids are given in Table 2. In the worked out HPLC procedure separation was carried out on RP 18 column. The separation took place at 21 C, the mobile phase flow rate was 1 ml min 1.

7 RP HPLC and RP TLC in the Analysis of Flavonoids 307 Table 2. Retention times of flavonoid standards Flavonoid standards t r,min (±)-Catechin 6.96 ( )-Epicatechin ( )-Epigallocatechin gallate ( )-Epicatechin gallate Rutin Hesperedin Apigenin-7--neohesperidoside Quercetrin Neohesperedin Myricetin Quercetin Luteolin (±)-Naringenin Hesperetin Kaempferol Apigenin Pinocembrin Chrysin Table 3. Retention times of methylxanthine standards Methylxanthines t r, min Caffeine 7.49 Theophylline 3.83 Theobromine ,7-Dimethylxanthine Methylxanthine Methylxanthine 1.77

8 308 R. Baranowski, J. Kabut and I. Baranowska In the mobile phase the following compounds were used: A: 0.05% TFA in H 2 : B: 0.05% TFA in acetonitrile. The following gradient was applied: 0 min 10% B, 25 min 20% B, 35 min 25% B, 60 min 75% B. Changes in the initial phase (10% B) caused an extention of retention times of catechins. This enables simultaneous marking of methylxanthines: caffeine, theophylline, theobromine and 1,7-dimethylxanthine and their metabolites 3-,7-methylxanthines- (Tab. 3). The separation of methylxanthines from flavonoids prevents their interference in the case of real samples in which there are both groups of compounds. The differences in retention times are significant for these groups because methylxanthines show the absorption maximum at 270 nm, similarly as catechins. In the worked out procedure interferences from vitamins were also examined. Vitamins B 1 (thiamine), B 6 (pyridoxine), C (ascorbic acid) have similar retention times to 3-,7-methylxantines and vitamin B 2 (riboflavin) retention time to ( )-epicatechin. However the differences in absorption spectra of these compounds allow their differentiation and possibilities of detection at different wavelengths. The newly worked out procedure additionally enables the separation of rutin hesperidin, kaempferol apigenin and chrysin pinocembrin. The dependence of absorbance on the flavonoids concentration was determined at l = 270 nm. Flavonoids were introduced into the column in the amount of 10 ng to 1 µg. The values of a and b in the linear dependence of peak area on concentration expressed by the equation y = ax + b, limits of detection and limits of determination are shown in Table 4. The worked out procedure was used for the analysis of real samples: tea, wine, freeze-dried fruit and vegetable and urine. The results of these analyses are shown in Tables 5 7. In freeze-dried fruit and vegetable samples we identified catechins ((±)-catechin, ( )-epicatechin, ( )-epicatechin gallate), flavonols (rutin, quercetrin, myricetin, quercetin, kaempferol), flavones (luteolin, apigenin, chrysin, apigenin-7-neohesperidoside) and flavanones ((±)-naringenin, hesperetin) (Tab. 5). The main classes of flavonoids identified in wines are: anthocyanidins, catechins and flavonols [9,10]. Table 4. Coefficients of linear regression for flavonoids Flavonoid (Continuation on the next page) LD Limit of detection ng LQ Limit of quantitation ng (±)-Catechin ( )-Epicatechin ( )-Epigallocatechin gallate ( )-Epicatechin gallate Rutin < a b

9 RP HPLC and RP TLC in the Analysis of Flavonoids 309 Table 4 (Continuation) Hesperedin < Apigenin-7--neohesperidoside < Quercetrin < Neohesperedin Pelargonidin Quercetin Luteolin (±)-Naringenin < Hesperetin < Kaempferol Apigenin < Pinocembrin < Chrysin < Table 5. Flavonoids contents in freeze-dried fruit and vegetable samples Sample Flavonoid (content µg g -1 ) Banana (±)catechin (6.23); ( )epicatechin (56.13) Pear (±)catechin (82,94); hesperedin (45.76); hesperetin (1.73); (±)catechin (65.33); ( )epicatechin (36.12); ( )epigallocatechin gallate (15.86); Leek rutin (55.91); apigenin-7--neohesperidoside (52.36); quercetrin (43.20); quercetin (21.51); kaempferol (7.25); chrysin (3.33) Peach ( )epicatechin gallate (15.17); rutin (1.1) Carrot ( )epicatechin gallate (1.18) Parsley root quercetrin (95.19); apigenin (1.61) ( )epicatechin (16.43); rutin (7.41); apigenin-7--neohesperdo-side (21.06); Parsley leaves quercetin (38.44); ( )epicatechin (54.27); ( )epigallocatechin gallate (47.49); Cherry ( )epicatechin gallate (78.70); quercetrin (387.08); neohespere-din (181.08); (±)naringenin (419.81); chrysin (0.75) Yellow pea ( )epicatechin (43.28); rutin (0.07); (±)naringenin (19.65) Corn pinocembrin (2.18) (Continuation on the next page)

10 310 R. Baranowski, J. Kabut and I. Baranowska Table 5. (Continuation) Parsley root quercetrin (95.19); apigenin (1.61) ( )epicatechin (16.43); rutin (7.41); apigenin-7--neohesperdo-side (21.06); Parsley leaves quercetin (38.44); ( )epicatechin (54.27); ( )epigallocatechin gallate (47.49); Cherry ( )epicatechin gallate (78.70); quercetrin (387.08); neohespere-din (181.08); (±)naringenin (419.81); chrysin (0.75) Yellow pea ( )epicatechin (43.28); rutin (0.07); (±)naringenin (19.65) Corn pinocembrin (2.18) Green pea Soy sprouts Celery root Horseradish Green pepper Fennel (±)catechin (55.56); ( )epicatechin (15.41); ( )epigallocatechin gallate (0.23); ( )epicatechin gallate (17.13); rutin (0.07); (±)naringenin (55.22); hesperetin (15.3); chrysin (7.2) (±)catechin (57.87); ( )epicatechin gallate (34.65); rutin (15.23); neohesperedin (112,31); (±)naringenin (23.15); hesperetin (17.14); chrysin (1.29) (±)catechin (23.17); ( )epicatechin (54.12); (-)epicatechin gallate (15.87); quercetrin (34.78); luteolin (20.08); (±)naringenin (15.12); hesperetin (16.34) ( )epicatechin (78.98); ( )epigallocatechin gallate (12.03); ( )epicatechin gallate (16.19); rutin (3.32); hesperedin (0.7): quercetrin (23.14); (±)naringenin (13.22); hesperetin (14.87) (±)catechin (56.22); ( )epicatechin (67.17); ( )epigallocatechin gallate (24.16); ( )epicatechin gallate (25.11); rutin (23.16); quercetrin (47.13); luteolin (34.57);(±)naringenin (16.78); hesperetin (15.22) (±)catechin (325.9); ( )epicatechin (56.17); ( )epigallocatechin gallate (34.54); ( )epicatechin gallate (14.22); rutin (21.21); apigenin-7--neohesperidoside (25.16); quercetrin (205.43); quercetin (119.13); kaempferol (12.43); chrysin (15.15) In this paper the presence of flavonoids from such groups as: catechins ((±)-catechin, ( )-epicatechin, ( )-epigallocatechin gallate, ( )-epicatechin gallate) in the analysed wine samples has been confirmed, likewise in other investigations. Besides flavones (apigenin-7--neohesperidoside, chrysin), flavanones (hesperedin) and flavonols (rutin, quercetrin, quercetin, myricetin) have been found in these samples (Tab. 6). Table 6. Flavonoids contents in wines Wine sample Baron de Lestac Bordeaux (France) chrysin (Continuation on the next page) Flavonoids (±)catechin ( )epicatechin ( )epigallocatechin gallate ( )epicatechin gallate rutin hesperedin apigenin-7--neohesperidoside quercetrin neohesperedin quercitin Content by RP 18, µgg Content by SDB, µg g

11 RP HPLC and RP TLC in the Analysis of Flavonoids 311 Table 6 (Continuation) Cabernet- Sauvignon (France) Pueblo Viejo Rioja Crianza (Spain) Porto Don Pablo (Portugal) ReservedeMonsieur Louis Bordeaux (France) y ( )epicatechin ( )epicatechin gallate hesperedin quercetrin neohesperedin apigenin-7--neohesperidoside hesperetin ( )epicatechin ( )epigallocatechin gallate ( )epicatechin gallate rutin hesperedin quercitrin neohesperedin ( )epicatechin ( )epigallocatechin gallate rutin hesperedin quercetrin ( )epicatechin ( )epicatechin gallate rutin quercetrin neohesperedin apigenin-7--neohesperidoside In tea analysis mainly catechins [9,11 17] or catechins and flavonols have been mentioned [18]. Using the worked out procedure it was discovered that tea additionally possesses flavonoids from such groups as flavones (apigenin-7--neohesperidoside, chrysin, luteolin, apigenin), flavanones (hesperedin, neohesperedin, hesperetin, (±)-naringenin), catechins ((±)-catechin, ( )-epicatechin, ( )-epigallocatechin gallate, ( )- epicatechin gallate), flavonols (rutin, quercetrin, quercetin, myricetin, kaempferol) (Tab. 7). Table 7. Flavonoids contents in tea samples Yunan Saga Tea sorts Flavonoids (contents µg g -1 ) Green tea (Ahmed) Lipton Pickwick nd not detected. (±)catechin (20.33); ( )epicatechin (117.08); quercetrin (89.15); luteolin (22.55) ( )epicatechin (10.77); ( )epigallocatechin gallate (10.50); rutin (1.61); quercetrin (3.48); quercetin (6.89); hesperetin (11.18) (±)catechin (76.49); ( )epicatechin (138.51); ( )epicatechin gallate (149.04); rutin (51.40); quercetrin (37.28); apigenin-7--neohesperidoside (36.48); quercetin (0.91); luteolin (5.37); (±)naringenin (6.27); hesperetin (11.94); kaempferol (33.68); apigenin (3.04); chrysin (4.97) ( )epigallocatechin gallate (14.47); rutin (298.37); hesperedin (279.75); quercetin (52.10); chrysin (0.33) ( )epigallocatechin gallate (14.19); ( )epicatechin gallate (109.23); rutin (12.41); quercetrin (58.16); neohesperedin (5.57); myricetin (ND); luteolin (9.98); chrysin (0.38)

12 312 R. Baranowski, J. Kabut and I. Baranowska The developed HPLC procedure has also been applied to the analysis of urine samples. The samples have been prepared with the use of a modification of an earlier described method [20], used for extraction of flavonoids from Gingko Biloba. The modification consisted in an introduction of additional wash-step (3 ml of mixture of chloroform 2- propanol, 8:2, v/v) to the SPE procedure. The aim of this modification was the disposal of methylxanthines from the urine sample. The mixture of chloroform 2-propanol (8:2, v/v) has been used before in the SPE and liquid-liquid extraction of methylxanthines from body fluids in the presence of neurotransmitters. Methylxanthines can be also removed from an extraction column using dichloromethane or chloroform because flavonoids have a limited solubility in these solvents [11]. Urine samples were collected from healthy people whose diet was rich in flavonoids. The samples were also collected immediately after drinking tea. In the samples we identified flavonoids such as aglycones: ( )-epicatechin ( µg ml 1 ), (±)-catechin (0.72 µg ml 1 ), chrysin ( µg ml 1 ), hesperetin ( µg ml 1 ), apigenin (0.21 µg ml 1 ). Thus it was assumed that also appear small quantities of flavonoid glycosides: rutin ( µg ml 1 ), quercetrin (1.9 µg ml 1 ), apigenin-7-neohesperidoside ( µg ml 1 ), hesperedin (1.64 µg ml 1 ), neohesperedin (1.04 µg ml 1 ), which were not metabolised by the organism. It can be concluded that the chromatographic procedure proposed in this paper enables the analysis of complex mixtures of flavonoids belonging to different compound classes both in glycoside and aglycone forms. Acknowledgements This work was supported by the State Committee for Scientific Research (Poland), project No.4 T09A REFERENCES 1. Wollegast J. and Anklam E., Food Res. Intern., 33, 423 (2000). 2. Marken H.M. and Beecher G.R., J. Agric. Food. Chem., 48, 577 (2000). 3. Crozier A. and Jensen E., J. Chromatogr. A, 761, 315 (1997). 4. Merken H.M. and Beecher G.R., J. Chromatogr. A, 897, 177 (2000). 5. Matysik G. and Soczewiñski E., Chromatographia, 52, 357 (2000). 6. Nogata Y., hta H., J. Chromatogr., A, 667, 59 (1994). 7. Sherma J., J. Chromatogr. A, 880, 129 (2000). 8. Castelle K.V., J. Chromatogr., A, 240, 81 (1982). 9. Hertog M.G.L., Hollman P.C.H. and Putte B., J. Agric. Food. Chem., 41, 1242 (1993). 10. Lapidot T., Harel S. and Akiri B., J. Agric. Food. Chem., 47, 67 (1999). 11. Shao W., J. Sci. Food Agric., 69, 535 (1995). 12. Arts I.C.W., Putte B. and Hollman P., J. Agric. Food. Chem., 48, 1746 (2000).

13 RP HPLC and RP TLC in the Analysis of Flavonoids Bronner W.E. and Beecher G.R., J. Chromatogr. A, 805, 137 (1998). 14. Dalluge J.J. and Nelson B.C., J. Chromatogr. A, 881, 411 (2000). 15. Goto T. and Yoshida Y., J. Chromatogr. A, 749, 295 (1996). 16. Zuo Y., Chen H. and Deng Y., Talanta, 57, 307 (2002). 17. Lin J.K., Lin C.L. and Liang Y.C., J. Agric. Food. Chem., 46, 3635 (1998). 18. Price K.R., Rhodes M.J. and Barnes K.A., J. Agric. Food. Chem., 46, 2517 (1998). 19. Baranowski R., Kabut J. and Baranowska I., Anal. Lett., 37, 175 (2004). 20. Pietta P.G., Gardana C. and Mauri P.L., J. Chromatogr. B, 693, 249 (1997). Received December 2004 Accepted January 2005