Analytical Methods Rapid tea catechins and caffeine determination by HPLC using microwave-assisted extraction and silica monolithic column A.A. Rahim a,⇑ , S. Nofrizal a , Bahruddin Saad a,b a School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia b Doping Control Center, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia a r t i c l e i n f o Article history: Received 11 October 2011 Recei ved in revised form11 Septe mbe r 2013 Accep ted 25 Septe mbe r 2013 Avail able onli ne3 October 2013 Keywords: Catechin Monolith ic column High performance liquid chromatography Green tea Oolong tea Black tea a b s t r a c t A rapid reversed-p hase high performance liquid chroma tographic method using a monolith ic column for the determinati on of eight catechin monomers and caffeine was developed . Using a mobile phase ofwa ter :aceto nit ril e: me tha no l (83 :6: 11) at a flowrate of 1.4 mL mi n 1 , thecatec hi ns an d ca ffe ine we re iso - cratically separa ted in about 7 min. The limits of detection and quantifi cation were in the range of 0.11– 0.29 a nd 0 .33–0 .87 mg L1 , respectively . Satisfactory recov eries were obta ined (94.2–10 5.2 ± 1.8%) for all sample s wh en spike d at thre e concent rati ons (5, 40 and 70 mg L1 ). In combination with microwave- assisted extraction (MAE), the method was applied to the determination of the catechins and caffeine in eleven tea samples (6 green, 3 black and 2 oolong teas). Relatively high levels of caffeine were found in black tea, but higher levels of the catechins, especially epigallocatechin gallate (EGCG) were found in green teas. 2013 Elsevier Ltd. All rights reserved. 1. Introduction Tea is one of the most frequently consumed beverages in the world, d atin g back to more tha n 500 0 yea rs ag o. N umero us studies have recorded the beneficial effects of tea, e.g., anti-oxidant ( Vin- son & Da bb agh, 1998; Ye n & Ch en, 1995 ), anti-ca rcin oma (Sadzuka, Sug iyam a, & Son obe, 2000) and arte rios cler osis prev ent ion (Kr itz & Sinzing er, 1997). The majo r nut raceuti cals in teas are the cate- chins. There is already growing evidence that tea polyphenols re- duce the risk of heart diseases and cancer in humans (Crespy & Williamson, 2004 ). In some studies, tea has been associated with ant ialle rgi c acti on (San o, Suzu ki, Mi ya se, Yo shino, & Ma ed a- Yamamoto, 1999 ) and an ti microbial pr op er ties (Greenwalt, Led ford , & Ste inkr aus, 1998; Vaq uer o, Albe rto & Nan dra, 200 7). Further studies have demonstrated that the co-administration ofdru gs wit h cate chin s (C), epic atec hin (EC ) and epig allo cate chin gal - late (EGCG) inhibits glucuronidation and sulfation of orally admin- istered drugs thereby increasing the bioavailability of such drugs (Prasain & Barnes, 2007). Moreover some epidemiological studies have linked the consumption of tea with a lower risk of several typ es of can cer including those of th e st omach , or al cav ity , oesoph- agus and lungs (Hakim, Harris, Chow, Dean, Brown & Ali, 2004). Therefo re, tea appears to be an effective chemopreven tive agent for tox ic chem icals and carc inogens (Karori, Wachira, Wanyo ko, & Ngure, 2010). The weight percentage of the soluble ingredients account for as much as 30% of tea, which differs according to variety, production area, climate, and processin g condition s ( Bronn er & Beeche r, 1998; Han, Tian, & Chen, 1997; Lu, Chu, Yan, & Chen, 2009). The content of sol ub le ingr ed ien ts in gr een te a is ge ne ra lly gr ea ter than those in bla ck tea as the lat ter is com ple tel y ferme nt ed . Po lyp he no ls co ns ti- tute the major portion of the soluble ingredients and are also the essential compone nts of tea which have physiological functions. Cat echi ns are the primary poly phe nols in the tea, and accounts for 75–80% of the soluble ingredients (Lu et al., 2009). Caf feine is the major alkaloid of tea, presen t in the ran ge of3.0– 4.0 % (Fer nandez, Ma rtin, Gon zale z, & Pablos, 200 0; Naik & Nagalakshmi, 1997; Zhang, Lin, He, & Petteruti, 2002). In humans, caffe ine stim ula tes the hea rt (Amm on, 1991; Ash ihar a, Sano, & Crozier, 2008), central nervous system (Davis, Zhao, Stock, Mehl, Buggy & Hand, 2003; Nehlig, Daval, & Debry, 1992), and the respi- ratory system (Doher ty & Smith, 2005; Richmon d, 1949). It is a diuretic and has the effect of delaying fatigue ( Grandhi, Donnelly, & Rogers, 2007; Haskell, 1926). The structu res of the vari ous catechin monomers and caffeine are shown inTable 1. High performance liquid chromatography (HPLC) is by far the most popular method for the analysis of tea catechins, gallic acid, purine alkaloids, theanine, etc. ( Peng, Song, Shi, Li, & Ye, 2008 ). In these methods, the stationary phase used is based on particulate pa cki ng ma te rials (main ly C1 8 wi th 5 lm par ticle size ). Par ticu late 0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.131 ⇑ Corresponding author. Tel.: +60 4 653388; fax: +60 4 6574854. E-mail address: afi[email protected](A.A. Rahim). Food Chemistry 147 (2014) 262–268 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Rapid tea catechins and caffeine determination by HPLC using
microwave-assisted extraction and silica monolithic column
A.A. Rahim a,⇑, S. Nofrizal a, Bahruddin Saad a,b
a School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysiab Doping Control Center, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia
a r t i c l e i n f o
Article history:
Received 11 October 2011
Received in revised form11 September 2013
Accepted 25 September 2013
Available online 3 October 2013
Keywords:
Catechin
Monolithic column
High performance liquid chromatography
Green tea
Oolong tea
Black tea
a b s t r a c t
A rapid reversed-phase high performance liquid chromatographic method using a monolithic column for
the determination of eight catechin monomers and caffeine was developed. Using a mobile phase of
water:acetonitrile:methanol (83:6:11) at a flowrate of 1.4 mL min1, thecatechins and caffeine were iso-
cratically separated in about 7 min. The limits of detection and quantification were in the range of 0.11–
0.29 and 0.33–0.87 mg L 1, respectively. Satisfactory recoveries were obtained (94.2–105.2 ± 1.8%) for all
samples when spiked at three concentrations (5, 40 and 70 mg L 1). In combination with microwave-
assisted extraction (MAE), the method was applied to the determination of the catechins and caffeine
in eleven tea samples (6 green, 3 black and 2 oolong teas). Relatively high levels of caffeine were found
in black tea, but higher levels of the catechins, especially epigallocatechin gallate (EGCG) were found in
green teas.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
Tea is one of the most frequently consumed beverages in the
world, dating back to more than 5000 years ago. Numerous studies
have recorded the beneficial effects of tea, e.g., anti-oxidant (Vin-
son & Dabbagh, 1998; Yen & Chen, 1995), anti-carcinoma (Sadzuka,
Sugiyama, & Sonobe, 2000) and arteriosclerosis prevention (Kritz &
Sinzinger, 1997). The major nutraceuticals in teas are the cate-
chins. There is already growing evidence that tea polyphenols re-
duce the risk of heart diseases and cancer in humans (Crespy &
Williamson, 2004). In some studies, tea has been associated with
as shorter analysis times and improved resolution can be realised.
The superiority of our proposed method based on monolithic col-
umn due to the short separation time (7 min) is evident. Separation
times rivaling to those of UPLC systems can be realised by employ-
ing suitable gradient programmes.
3.4. Optimisation of microwave assisted extraction (MAE)
Several parameters viz., type of solvent, pressure, power and
time for the microwave irradiation were studied and optimisedin order to obtain suitable extraction conditions of the interested
tea components. Supporting information is given as a Supplemen-
tary file.
3.4.1. Effect of extracting solvents
The effect of (i) acetonitrile:water (1:1), (ii) methanol:water
(1:1) and (iii) water:acetonitrile:methanol (83:6:11) as extracting
solvents were studied. It is clear that the third extracting solvents
(i.e., the HPLC mobile phase) resulted in the highest extraction of
the components of interest. Hu et al. (2009) also obtained high
extraction efficiency for catechin monomers and caffeine whenmethanol:acetonitrile (1:1) were used. On the other hand, our
Fig. 1. Typical chromatograms for the separation of catechins and caffeine standards using particulate DBS Hypersil gold C-18 (a), monolithic column (b) and green tea (c),oolong tea (d), black tea (e) samples on monolithic column; isocratic elution with water:acetonitrile:methanol (83:6:11); flow rate,1.4 mL min1. Assignment of peaks: (1)
Arce, L., Rios, A., & Valcarcel, M. (1998). Determination of anti-carcinogenic
polyphenols present in green tea using capillary electrophoresis coupled to a
flow injection system. Journal of Chromatography A, 827 (1), 113–120.
Ashihara, H., Sano, H., & Crozier, A. (2008). Caffeine and related purine alkaloids:
Biosynthesis, catabolism, function and genetic engineering. Phytochemistry,69(4), 841–856.
Bonoli, M., Pelillo, M., Toschi, T. G., & Lercker, G. (2003). Analysis of green tea
catechins: Comparative study between HPLC and HPCE. Food Chemistry, 81(4),
631–638.
Bronner, W., & Beecher, G. (1998). Method for determining the content of catechins
in tea infusions by high-performance liquid chromatography. Journal of Chromatography A, 805(1–2), 137–142.
Buqing, L., Huiling, Z., Qingling, S., Buchang, Z., & Shengfang, G. (1996). Studies on
the extraction of polysaccharide from middle and lower middle grade green tea
and the effectiveness on blood-glucose depressing. Journal of Tea Science, 1–7.
Crespy, V., & Williamson, G. (2004). A review of the health effects of green tea
catechins in vivo animal models. Journal of Nutrition, 134(12), 3431S–3440S.
Davis, J. M., Zhao, Z., Stock, H. S., Mehl, K. A., Buggy, J., & Hand, G. A. (2003). Central
nervous system effects of caffeine and adenosine on fatigue. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 284(2),
R399–R404.
Dawidowicz, A. L., & Wianowska, D. (2005). PLE in the analysis of plant compounds:
Part I. The application of PLE for HPLC analysis of caffeine in green tea leaves.
Journal of Pharmaceutical and Biomedical Analysis, 37 (5), 1155–1159.
Doherty, M., & Smith, P. (2005). Effects of caffeine ingestion on rating of perceived
exertion during and after exercise: A meta-analysis. Scandinavian Journal of Medicine and Science in Sports, 15(2), 69–78.
Fernandez, P., Martin, M., Gonzalez, A., & Pablos, F. (2000). HPLC determination of
catechins and caffeine in tea. Differentiation of green, black and instant teas.
Analyst, 125(3), 421–425.
Fernández-Cáceres, P. L., Martín, M. J., Pablos, F., & González, A. G. (2001).
Differentiation of tea (Camellia sinensis) varieties and their geographical origin
according to their metal content. Journal of Agricultural and Food Chemistry,49(10), 4775–4779.
Grandhi, S., Donnelly, L. E., & Rogers, D. F. (2007). Phytoceuticals: The new ‘physic
garden’ for asthma and chronic obstructive pulmonary disease. Expert Review of Respiratory Medicine, 1(2), 227–246.
Greenwalt, C., Ledford, R., & Steinkraus, K. H. (1998). Determination and
characterization of the antimicrobial activity of the Fermented
Sadzuka, Y., Sugiyama, T., & Sonobe, T. (2000). Efficacies of tea components on
doxorubicin induced antitumor activity and reversal of multidrug resistance.
Toxicology Letters, 114(1–3), 155–162.
Sano, M., Suzuki, M., Miyase, T., Yoshino, K., & Maeda-Yamamoto, M. (1999). Novel
antiallergic catechin derivatives isolated from oolong tea. Journal of Agriculturaland Food Chemistry, 47 (5), 1906–1910.
Song, G., Lin, J., Qu, F., & Huie, C. (2003). Extraction of catechins and caffeine from
different tealeaves and comparison with micellar electrokinetic
chromatography. Chinese Science Bulletin, 48(22), 2438–2443.
Spigno, G., & De Faveri, D. (2009). Microwave-assisted extraction of tea phenols: A
phenomenological study. Journal of Food Engineering, 93(2), 210–217.
Vaquero, M. J. R., Alberto, M. R., & Maca de Nandra, M. R. (2007). Antibacterial effect
of phenolic compound from different wines. Food Control, 18, 93–101.Vinson, J. A., & Dabbagh, Y. A. (1998). Tea phenols: Antioxidant effectiveness of teas,
tea components, tea fractions and their binding with lipoproteins. NutritionResearch, 18(6), 1067–1075.
Wang, H., Helliwell, K., & You, X. (2000). Isocratic elution system for the
determination of catechins, caffeine and gallic acid in green tea using HPLC.
Food Chemistry, 68(1), 115–121.
Wang, D., Lu, J., Miao, A., Xie, Z., & Yang, D. (2008). HPLC–DAD–ESI–MS/MS analysis
of polyphenolsand purinealkaloids in leaves of 22tea cultivarsin China. Journalof Food Composition and Analysis, 21(5), 361–369.
Wang,H., Provan, G. J., & Helliwell, K. (2003). HPLC determination of catechinsin tea
leaves and tea extracts using relative response factors. Food Chemistry, 81(2),
307–312.
Yang, X. R., Ye, C. X., Xu, J. K., & Jiang, Y. M. (2007). Simultaneous analysis of purine
alkaloids and catechins in Camellia sinensis, Camellia ptilophylla and Camelliaassamica var. kucha by HPLC. Food Chemistry, 100(3), 1132–1136.
Yen, G. C., & Chen, H. Y. (1995). Antioxidant activity of various tea extracts in
relation to their antimutagenicity. Journal of Agricultural and Food Chemistry,43(1), 27–32.
Zhang, H., Lin, L. Z., He, X. G., & Petteruti, M. P. (2002). Analytical methods for theactive components in tea products (p. 803). American Chemical Society (ACS)
Symposium series.
Zuo, Y., Chen, H., & Deng, Y. (2002). Simultaneous determination of catechins,
caffeine and gallic acids in green, Oolong, black and pu-erh teas using HPLC
with a photodiode array detector. Talanta, 57 (2), 307–316.
268 A.A. Rahim et al./ Food Chemistry 147 (2014) 262–268