Simultaneous determination of polyphenols and major purine alkaloids in Greek Sideritis species, herbal extracts, green tea, black tea, and coffee by high-performance liquid chromatography-diode array detection

Herein, a high-performance liquid chromatography-diode array detection method has been developed for the simultaneous determination of 15 phenolic antioxidants: flavan-3-ols, (-)-epigallocatechin, (+)-catechin, (-)-epigallocatechin gallate, (-)-epicatechin, (-)-epicatechin gallate, (-)-gallocatechin, a phenolic acid (gallic acid), a hydroxycinnamic acid (chlorogenic acid), flavones (apigenin), flavonols (kaempferol, quercetin, and myricetin), and purine alkaloids (caffeine theophylline, theobromine) in different herb extracts, tea, and coffee varieties. The developed method was validated and successfully applied in order to determine the polyphenolic content to estimate the antioxidant activity of the Sideritis species commonly known as Greek mountain tea. To the best of our knowledge, this is the first report on the quantitative determination of catechins and other polyphenols in Greek mountain tea. Acidic hydrolysis was necessary for the simultaneous determination of the aglycones of the target analytes. According to our results, chlorogenic acid, myricetin, apigenin, catechin, and epicatechin gallate are found in the Sideritis species.     

Simultaneous analysis of tea catechins, caffeine, gallic acid, theanine and ascorbic acid by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatography (MEKC) method for the simultaneous analysis of five tea catechins, theanine, caffeine, gallic acid and ascorbic acid has been developed. The catechins are (-)-epicatechin, (+)-catechin, (-)-epigallocatechin, (-)-epicatechin gallate and (-)-epigallocatechin gallate. p-Nitrophenol serves as both reference and internal standard. All the components are separated within 13 min with a 57 cm uncoated fused-silica column. On-column detection was carried out at 200 nm. This method has been used to measure these compounds in fresh tea leaves and tea liquor. The limit of detection for all analytes ranged from 1 to 20 microg/ml.     

Development of HPLC Method for Catechins and Related Compounds Determination and Standardization in Miang (Traditional Lanna Fermented Tea Leaf in Northern Thailand)

High performance liquid chromatography (HPLC) for catechins and related compounds in Miang (traditional Lanna fermented tea leaf) was developed to overcome the matrices during the fermentation process. We investigated a variety of columns and elution conditions to determine seven catechins, namely (+)-catechin, (−)-gallocatechin, (−)-epigallocatechin, (−)-epicatechin, (−)-epigallocatechin gallate, (−)-gallocatechin gallate, (−)-epicatechin gallate, as well as gallic acid and caffeine, resulting in the development of reproducible systems for analyses that overcome sample matrices. Among the three reversed-phase columns, column C (deactivated, with extra dense bonding, double endcapped monomeric C18, high-purity silica at 3.0 mm × 250 mm and a 5 µm particle size) significantly improved the separation between Miang catechins in the presence of acid in the mobile phase within a shorter analysis time. The validation method showed effective linearity, precision, accuracy, and limits of detection and quantitation. The validated system was adequate for the qualitative and quantitative measurement of seven active catechins, including gallic acid and caffeine in Miang, during the fermentation process and standardization of Miang extracts. The latter contain catechins and related compounds that are further developed into natural active pharmaceutical ingredients (natural APIs) for cosmeceutical and nutraceutical products.     

Simultaneous determination of catechins, caffeine and gallic acids in green, Oolong, black and pu-erh teas using HPLC with a photodiode array detector

A simple and fast HPLC method using a photodiode array detector was developed for simultaneous determination of four major catechins, gallic acid and caffeine. After multiple extractions with aqueous methanol and acidic methanol solutions, tea extract was separated within 20 min using a methanol-acetate-water buffer gradient elution system on a C(18) column. The sample extraction data demonstrated that the single extraction used in the previous studies with aqueous acetonitrile or methanol is not sufficient; the multiple extraction procedure is essential for the quantitative analysis of catechins, phenolic acids and caffeine in teas. Several green, Oolong, black and pu-erh teas were successfully analyzed by this method. The analytical results obtained indicated that green teas contain higher content of catechins [(-)-epigallocatechin gallate, (-)-epigallocatechin, (-)-epicatechin gallate, and (-)-epicatechin] than both Oolong, pu-erh and black teas because fermentation process during the tea manufacturing reduced the levels of catechins significantly. The fermentation process also remarkably elevated the levels of gallic acid in full-fermented pu-erh and black teas. Another interesting finding is the low level of caffeine in Oolong teas, especially in Fujian Oolong tea.     

Simultaneous determination of catechins, rutin, and gallic acid in Cistus species extracts by HPLC with diode array detection

A simple high-performance liquid chromatography method using a diode array detector (DAD) is developed for the simultaneous analysis of five major catechins: (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GCT), (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), and the phenolic plant metabolites gallic acid (GA) and rutin (RT) in lyophilized extracts of Cistus species. The optimal analytical conditions are investigated to obtain the best resolution and the highest UV sensitivity for the quantitative detection of catechins. The optimized conditions (acetonitrile-phosphate buffer 50mM, pH 2.5, gradient elution system on a C(18) reversed-phase column with a flow rate of 1 mL/min and UV absorbance at 210 nm) allowed a specific and repeatable separation of the studied analytes to be achieved. All compounds are successfully separated within 32 min. Calibration curves are linear in the 2-50 microg/mL range for GCT, C, and EGCG and in the 5-50 microg/mL range for GA, EGC, EC, and RT. The limit of detection values ranged from 0.24 to 0.74 microg/mL. The limit of quantitation limit values ranged from 0.77 to 1.94 microg/mL. The validated method is applied to the determination of the specific phytochemical markers GA, GCT, C, and RT in Cistus incanus and Cistus monspeliensis lyophilised extracts. The recovery values ranged between 78.7% and 98.2%. The described HPLC method appears suitable for the differentiation and determination of the most common catechins together with the glycoside rutin and the phenolic compound gallic acid and can be considered an effective and alternative procedure for the analyses of this important class of natural compounds.     

Simultaneous determination of twelve tea catechins by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection was developed for the determination of twelve tea catechins including four major catechins: epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG); four of their epimers at the C-2 position, C, GC, CG and GCG; and four methylated catechin derivatives, epigallocatechin-3-O-(3-O-methyl)gallate, gallocatechin-3-O-(3-O-methyl)gallate, epigallocatechin-3-O-(4-O-methyl)gallate and epicatechin-3-O-(3-O-methyl)gallate. These catechins were separated on an ODS C18 reversed-phase column by isocratic elution with 0.1 M NaH2PO4 buffer (pH 2.5)-acetonitrile (87:13) containing 0.1 mM EDTA.2Na. The detection limits (S/N = 3) of these catechins were approximately 10-40 pmol ml-1 at an applied voltage of 600 mV. Extracting these catechins from tea leaf powder with H2O-acetonitrile (1:1) at 30 degrees C for 40 min inhibited the epimerization at C-2 significantly from these epicatechins compared to extraction with hot water at 90 degrees C. This analytical method is sensitive to and appropriate for the simultaneous determination of various biologically active catechins in green tea.     

Full Validation of a Simple Method for Determination of Catechins and Caffeine in Brazilian Green Tea (Camellia sinensis var. assamica) Using HPLC

This paper describes the validation of an HPLC method for the assay of a green tea brew. The method employs a RP-18 column with water:methanol:ethyl acetate elution and UV detection at 280 nm. Specificity was evaluated using a photodiode array detector. The validation data showed that the assay is specific, accurate, precise, and reproducible for determination of six catechins and caffeine simultaneously. The response was linear over a range of 37–185 μg mL^?1 for caffeine, 99–500 μg mL^?1 for (?)-epigallocatechin (EGC), 20–100 μg mL^?1 for (+)-catechin (C), 30–150 μg mL^?1 for (?)-epicatechin (EC), 150–800 μg mL^?1 for (?)-epigallocatechin gallate (EGCG), 20–105 μg mL^?1 for (?)-gallocatechin gallate (GCG) and 40–205 μg mL^?1 for (?)-epicatechin gallate (ECG) (r > 0.9999 for all compounds). The range of recoveries was 96.12–110.48% according to substances. The RSD values for intra- and inter-day precision studies were <2.07 and <6.65%, respectively. The composition of samples assayed suggests that the summer is the best season for extract a major content of EGCG and caffeine. This assay can be readily utilized as quality controlled method for major green tea compounds.     

Determination of catechins and catechin gallates in biological fluids by HPLC with coulometric array detection and solid phase extraction

Tea polyphenols are well known for their beneficial health effects that involve their anti-carcinogenic, anti-mutagenic, anti-pathogenic and anti-oxidative properties. The main polyphenols of green tea are favan-3-ols (catechins) and their corresponding gallate compounds, which constitute about one-third of the dry weight of tea leaves. Their main ingredients are (+)-catechin (C), (−)-epicatechin (EC), (−)-gallocatechin (GC), (−)-epigallocatechin (EGC), (−)-catechin gallate (CG), (−)-epicatechin gallate (ECG), (−)-gallocatechin gallate (GCG) and (−)-epigallocatechin gallate (EGCG). Each has slightly different biological properties. We have developed a method to simultaneously analyze all these compounds in plasma and urine. The samples were first incubated with β-d-glucuronidase and sulfatase to release the catechin residues from their corresponding conjugates for subsequent extraction by selective solid phase column, Waters Oasis HLB extraction cartridges. The extracted molecules were resolved by reversed phase HPLC and monitored by coulometric chemical detection on a CoulArray detector. All eight catechin compounds were analyzed in a single chromatogram within 25 min. For plasma and urine analyses, good linearity (>0.9950) was validated in the range 10-2000 and 10-5000 ng/ml, respectively. The coefficients of variance (CV) were less than 5%. Absolute recovery was greater than 85% and detection limit was 5 ng/ml. The chromatogram exhibited minimal interference as a result of the highly selective solid phase extraction and CoulArray detection.     

高效液相色谱法分析元宝枫叶中儿茶素类物质

本文建立了元宝枫树叶中儿茶素种类及其含量的高效液相色谱(HPLC)测定方法。采用反相C18色谱柱,以甲醇/水(含0.5%乙酸)=25/75(V/V)为流动相,对没食子儿茶素(GC)、表没食子儿茶素(EGC)、儿茶素(C)、表没食子儿茶素没食子酸酯(EGCG)、表儿茶素(EC)和没食子儿茶素没食子酸酯(GCG)进行定性、定量分析;以甲醇/水(含0.5%乙酸)=35/65(V/V)为流动相,对表儿茶素没食子酸酯(ECG)和儿茶素没食子酸酯(CG)进行定性分析,柱温均为35℃,检测波长为278 nm,流速为1.0mL/min。结果表明:元宝枫叶中有EGC、EC和GCG,其它五种则无。EGC平均含量为0.0389 mg/g,方法精密度(RSD)为0.42%(n=6);EC平均含量为0.0289 mg/g,方法RSD为1.5%(n=6);GCG平均含量为0.284 mg/g,方法RSD为0.32%(n=6)。该方法简便、准确、分离效果好,为元宝枫叶开发成茶叶、饮料以及医疗保健品提供重要依据。     

Microemulsion electrokinetic chromatography for the analysis of green tea catechins: effect of the cosurfactant on the separation selectivity

Microemulsion electrokinetic chromatography (MEEKC) was applied to the separation of six catechins and caffeine, the major constituents of the green tea. The developed methods involved the use of sodium dodecyl sulfate (SDS) as surfactant, n-heptane as organic solvent and an alcohol as cosurfactant. The separations were performed under acidic conditions (pH 2.5 phosphate buffer, 50 mM) to ensure good stability of the catechins, with reversed polarity (anodic outlet). The effect of the alcohol nature on the MEEKC selectivity was evaluated; nine alcohols were used as cosurfactant: 1-butanol, tert-butanol, 1-pentanol, 2-pentanol, 3-pentanol, cyclopentanol, 1-hexanol, 2-hexanol, and cyclohexanol. The migration order of (+)-catechin (C), (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-gallocatechin (GC), (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG), caffeine and theophylline was significantly affected by the alcohol used as cosurfactant. Using nine microemulsions, four different selectivities were achieved: A (cyclohexanol); B (2-pentanol, 3-pentanol, 1-hexanol, 2-hexanol); C (1-butanol, 1-pentanol, cyclopentanol); D (tert-butanol). MEEKC methods, based on 2-hexanol and cyclohexanol as cosurfactant were validated and successfully applied to the analysis of catechins and caffeine in commercial green tea products.     

Densitometric determination of (+)-catechin and (−)-epicatechin by 4-dimethylaminocinnamaldehyde reagent

We report the optimization of a sensitive, selective and robust derivatization method using 4-dimethylaminocinnamaldehyde (DMACA) for densitometric determination of (+)-catechin and (−)-epicatechin. The separation of these compounds was achieved by thin-layer chromatography (TLC) on cellulose plates developed with water. With DMACA in HCl, both compounds gave blue bands, while under the same conditions, vanillin produced a fast fading red coloration of bands. Quantitation at 655 nm showed that for both compounds the calibration curve was linear from 2 to 12 ng and polynomial from 2 to 30 ng, and the repeatability of chromatography of 20 ng was 3.5% (RSD, n = 6). The visible limit of detection of both standards was 1 ng, but the densitometric limit of detection was lower (0.2 ng). The optimized DMACA reagent is superior to the more frequently used vanillin reagent and is applicable also for determination of mixtures containing other catechins ((−)-catechin, (−)-epicatechin gallate, (−)-epigallocatechin gallate, procyanidin A2, procyanidin B1 and procyanidin B2).     

A new norisoprenoid and other compounds from Fuzhuan brick tea

Fuzhuan brick tea, a kind of dark tea consumed mainly in the border regions of Southwestern and Northwestern China since the 1860s, is produced from the leaves of Camellia sinensis var. sinensis by microbial fermentation. From this special fermented tea, a new norisoprenoid, 3R,9R-oxido-5-megastigmene, was isolated, together with α-linolenic acid, strictin, isovitexin, astragalin, (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, (+)-gallocatechin, (-)-epigallocatechin, (-)-epigallocatechin gallate and gallic acid. The structures of the compounds were identified by spectroscopic means. The new compound didn't show any inhibition activity against the tested enteric pathogenic microorganisms at a concentration of 800 μg/mL by the hole plate diffusion method.     

Direct enantioseparation of catechin and epicatechin in tea drinks by 6-O-alpha-D-glucosyl-beta-cyclodextrin-modified micellar electrokinetic chromatography

Cyclodextrin-modified micellar electrokinetic chromatography was applied to the enantioseparation of catechin and epicatechin using 6-O-alpha-D-glucosyl-beta-cyclodextrin together with sodium dodecyl sulfate and borate-phosphate buffer. Factors affecting chiral resolution and migration time of catechin and epicatechin were studied. The optimum running conditions were found to be 200 mM borate-20 mM phosphate buffer (pH 6.4) containing 25 mM 6-O-alpha-D-glucosyl-beta-cyclodextrin and 240 mM sodium dodecyl sulfate with an effective voltage of +25 kV at 20 degrees C using direct detection at 210 nm. Under these conditions, the resolution (Rs) of racemic catechin and epicatechin were 4.15 and 1.92, respectively. With this system, catechin and epicatechin enantiomers along with other four catechins ((-)-catechin gallate, (-)-epicatechin gallate, (-)-epigallocatechin, (-)-epigallocatechin gallate) and caffeine in tea samples were analyzed successfully. The difference of migration time between catechin and epicatechin is discussed.     

Efficient procedure for isolating methylated catechins from green tea and effective simultaneous analysis of ten catechins,three purine alkaloids,and gallic acid in tea by high-performance liquid chromatography with diode array detection

Monomers of (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate (EGCG), (−)-epicatechin (EC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3″Me) and (−)-3-O-methyl epicatechin gallate (ECG3′Me) (purity, >97%) were successfully prepared from extract of green tea by two-time separation with Toyopearl HW-40S column chromatography eluted by 80% ethanol. In addition, monomers of (−)-catechin (C), (−)-gallocatechin (GC), (−)-gallocatechin gallate (GCG), and (−)-catechin gallate (CG) (purity, >98%) were prepared from EC, EGC, EGCG, and ECG by heat-epimerization and semi-preparative HPLC chromatography. With the prepared catechin standards, an effective and simultaneous HPLC method for the analysis of gallic acid, tea catechins, and purine alkaloids in tea was developed in the present study. Using an ODS-100Z C₁₈ reversed-phase column, fourteen compounds were rapidly separated within 15 min by a linear gradient elution of formic acid solution (pH 2.5) and methanol. A 2.5–7-fold reduction in HPLC analysis time was obtained from existing analytical methods (40–105 min) for gallic acid, tea catechins including O-methylated catechins and epimers of epicatechins, as well as purine alkaloids. Detection limits were generally on the order of 0.1–1.0 ng for most components at the applied wavelength of 280 nm. Method replication generally resulted in intraday and interday peak area variation of <6% for most tested components in green, Oolong, black, and pu-erh teas. Recovery rates were generally within the range of 92–106% with RSDs less than 4.39%. Therefore, advancement has been readily achievable with commonly used chromatography equipments in the present study, which will facilitate the analytical, clinical, and other studies of tea catechins.     

Sample stacking for the analysis of catechins by microemulsion EKC

In this study, an on-line concentration method, ASEI (anion-selective exhaustive injection)-sweeping technology which was coupled with microemulsion EKC (MEEKC), was used to analyze and detect six catechins ((-)-epicatechin, (+)-catechin, (-)-epigallocatechin gallate, (-)-epicatechin gallate, (-)-epigallocatechin, and (-)-gallocatechin). In addition to the effects of the buffer pH and electrolyte concentration on stacking, the compositions of microemulsion (types of oil phase, and types and levels of cosurfactant) also dominated the stacking effect of catechins. In MEEKC, the effect of the type of oil in microemulsion on separation mechanism is often unclear. This study had demonstrated that the oil type in microemulsion indeed altered the affinity of oil droplets with analytes. Finally, this proposed ASEI-sweeping MEEKC method was able to detect trace level of catechins in food products that was not previously possible by a normal MEEKC method.     

Uniformly-sized, molecularly imprinted polymers for (-)-epigallocatechin gallate, -epicatechin gallate and -gallocatechin gallate by multi-step swelling and polymerization method

Uniformly-sized, molecularly imprinted polymers (MIPs) for (-)-epigallocatechin gallate (EGCg), -epicatechin gallate (ECg) and -gallocatechin gallate (GCg) were prepared by a multi-step swelling and polymerization method using 2-vinylpyridine as a functional monomer, ethylene glycol dimethacrylate as a cross-linker and cyclohexanol as a porogen. Molecular recognition abilities of the obtained MIPs were evaluated in liquid chromatography using a mixture of ethanol and water, or ethanol as the eluent. Each MIP gave the highest molecular recognition ability for the respective template molecule. In addition, (-)-EGCg and -ECg had the same configuration (2R,3R) at positions 2 and 3, and therefore resulting in high cross reactivity each other. However, (-)-GCg, which has different configuration at position 2 with (-)-EGCg and -ECg, showed low cross reactivity with them. On the other hand, those MIPs showed no molecular recognition against (-)-epigallocatechin and -epicatechin, which have no gallate group at position 3. These results indicate that the MIPs prepared can recognize configuration at position 2 and a gallate group at position 3. Furthermore, the MIP for (-)-GCg could be successfully used for isolating (-)-EGCg and -ECg from green tea extract.     

Comparative analysis of tea catechins and theaflavins by high-performance liquid chromatography and capillary electrophoresis

This paper describes the simultaneous determination of catechins and theaflavins in green and black teas, using reversed-phase high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The tea polyphenols analyzed included (+)-catechin, catechin gallate, (-)-epicatechin, epicatechin-3-gallate, epigallocatechin, epigallocatechin-3-gallate, theaflavin, theaflavin-3-monogallate, theaflavin-3'-monogallate and theaflavin-3,3'-gallate. These polyphenols together with six other tea ingredients such as caffeine, adenine, theophylline, quercetin, gallic acid and caffeic acid were separated within 27 min by HPLC and in less than 10 min by CE. The optimal analytical conditions of both chromatographic methods were investigated for the convenience and reliability for routine analysis. Both HPLC and CE were found to be reliable and compatible. The reproducibility of the within-day assay using both methods was generally >90%. The day-to-day variation of retention time was <5% for HPLC, while the variation of migration time for CE was <2%. The analysis time of CE was three-times faster, however it is five-times less sensitive than HPLC, which has detection limits of 0.05 microg/ml and 0.5 microg/ml for catechins and theaflavins, respectively.     

Analysis of oxidized epigallocatechin gallate by liquid chromatography/mass spectrometry

(-)-Epigallocatechin gallate (EGCG) of catechins changes from non-colored at around neutral pH to yellow at higher pH region in aqueous solution. The pH-dependent oxidation of EGCG was analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). LC/MS/MS analysis of EGCG and its related compounds, (-)-epicatechin gallate (ECG) and (-)-epigallocatechin (EGC), successfully elucidated the structure relationship of EGCG solution involving the color change reaction at different pH conditions. The oxidation species produced at alkaline pH was detected at a different retention time from EGCG in the chromatograms of the EGCG sample. The oxidation species was found to correspond to M+14 (where M is the molecular weight of EGCG), which has two hydrogen atoms removed and addition of one oxygen atom to the gallyl moiety in the B-ring of EGCG.     

Effect of tea catechins on the structure of lipid membrane and beta-ray induced lipid peroxidation

Inhibiting effect of four tea catechins, (−)-epicatechin (EC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate (EGCG), on the lipid peroxidation induced by β-ray in tritiated water was examined using a spin probe method. 16-Doxylstearic acid (16NS) was incorporated into the liposome prepared from egg yolk phosphatidylcholine and the rate of the decrease of ESR intensity of 16NS was used as a measure of the inhibiting effect. In the low concentration region below 10⁻⁵M, catechins showed their inhibitions on the lipid peroxidation according to the order of ECG>EGCG>EC>EGC. This result was explained by a model that the initiator of the peroxidation is the hydroxyl radical (·OH) and the catechins adsorbed on the lipid membrane surface acting as scavengers of ·OH. In the high concentration range, however, the effect was diverse and it decreased with the increase of it in the case of EGCG. EGCG in this range was considered to enter into the interior of the membrane and break the structure, which causes the decrease of 16NS. Observation with transmission electron microscope (TEM) revealed that the size of the liposome became larger with the increasing concentration of EGCG and finally it was broken into fragments, showing that EGCG broadened the area of the liposome as expected from the result of ESR.     

Improving sensitivity by large-volume sample stacking combined with sweeping without polarity switching by capillary electrophoresis coupled to photodiode array ultraviolet detection

An easy, simple, and highly efficient on-line preconcentration method for polyphenolic compounds in CE was developed. It combined two on-line concentration techniques, large-volume sample stacking (LVSS) and sweeping. The analytes preconcentration technique was carried out by pressure injection of large-volume sample followed by the EOF as a pump pushing the bulk of low-conductivity sample matrix out of the outlet of the capillary without the electrode polarity switching technique using five polyphenols as the model analytes. Identification and quantification of the analytes were performed by photodiode array UV (PDA) detection. The optimal BGE used for separation and preconcentration was a solution composed of 10 mM borate-90 mM sodium cholate (SC)-40% v/v ethylene glycol, without pH adjustment, the applied voltage was 27.5 kV. Under optimal preconcentration conditions (sample injection 99 s at 0.5 psi), the enhancement in the detection sensitivities of the peak height and peak area of the analytes using the on-line concentration technique was in the range of 18-26- and 23-44-fold comparing with the conventional injection mode (3 s). The detection limits for (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (+)-catechin (C), (-)-epigallocatechin gallate (EGCG), and (-)-epicatechin gallate (ECG) were 4.3, 2.4, 2.2, 2.0, and 1.6 ng/mL, respectively. The five analytes were baseline-separated under the optimum conditions and the experimental results showed that preconcentration was well achieved.