High-throughput analysis of catechins and theaflavins by high performance liquid chromatography with diode array detection

Journal of Chromatography A, 1132 (2006) 132–140

High-throughput analysis of catechins and theaflavins by high performance liquid chromatography with diode array detection Andrew P. Neilson a , Rodney J. Green a , Karl V. Wood b , Mario G. Ferruzzi a,∗ a

Department of Food Science, Purdue University, 745 Agriculture Mall Dr. West Lafayette, IN 47907, United States b Campus-wide Mass Spectrometry Center, Purdue University, West Lafayette, IN 47907, United States Received 26 April 2006; received in revised form 20 July 2006; accepted 25 July 2006 Available online 17 August 2006

Abstract Increased interest in potential health-protective activities of flavonoid-rich tea has created the need to take advantage of HPLC column and system advances in order to optimize methodologies for flavonoid analysis. Two new RP-C18 methods for HPLC-DAD analysis of tea flavonoids were developed to facilitate separation of catechins within 5 min and separation of catechins and theaflavins within 10 min total analysis time. Calibration results indicate that these methods have on-column limits of detection on the order of 1–10 pmol for most tea catechins, and method replication generally resulted in intraday and interday peak area variation of <5% for catechins and <9% for theaflavins in green and black tea infusions. These new methods are therefore sensitive, reproducible, and represent a 2–4-fold reduction in HPLC analysis time from existing analytical methods. These improvements are readily achievable with commonly used HPLC equipment, thus facilitating increased sample throughput and efficiency across a broad range of experimental applications. © 2006 Elsevier B.V. All rights reserved. Keywords: Catechins; Theaflavins; Flavonoids; Tea; HPLC-DAD; LC-ESI-MS

1. Introduction Tea brewed from the leaves of Camellia sinensis is one of the most widely consumed beverages worldwide. Green and oolong teas are preferred in Japan and China, whereas black tea is predominantly consumed in India, Europe, and Africa. Tea is a rich source of flavonoids, which are effectively extracted into tea infusions when brewed with hot water. Monomeric catechins (flavan-3-ols), including epigallocatechin (EGC), epicatechin (EC), epigallocatechin gallate (EGCG), and epicatechin gallate (ECG) (Fig. 1) are the major flavonoids in green tea, and typically constitute 2–8% of the dry weight of green tea leaves [1–3]. Enzyme-catalyzed fermentation of green tea to oolong and black teas results in extensive oxidation and polymerization of the catechins into theaflavins (Fig. 1) and more complex, poorly characterized thearubigins. Depending on the extent of the fermentation process, oolong tea and black tea usually have significantly lower levels of catechins than green tea. Oolong tea

∗

Corresponding author. Tel.: +1 765 494 0625 E-mail address: [email protected] (M.G. Ferruzzi).

0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.07.059

undergoes a less extensive fermentation than black tea, and thus has higher levels of catechins and lower levels of theaflavins than black tea [3,4]. Major theaflavins in oolong tea and black tea include theaflavin (TF), theaflavin 3-monogallate (TF 3-MG), theaflavin 3 -monogallate (TF 3 -MG), and theaflavin 3,3 -digallate (TFDG), which typically constitute 1–5% of the dry weight of oolong tea and black tea leaves [3–6]. Catechins constitute 0.5–5% of the dry weight of oolong tea and black tea leaves, and thearubigins are also present in significant amounts [2,3,6,7]. The high concentration of catechins and theaflavins in tea, coupled with its broad consumption, make tea a significant dietary source of these flavonoids. Epidemiological and experimental evidence have highlighted potential therapeutic and health-protective activities of tea catechins and related derivatives, including protection against oxidative stress and inflammation, reduced risk of cardiovascular disease and cancer, and improved markers of bone health [8–18]. Interest in designing and carrying out more extensive in vitro, animal, and human intervention studies to investigate the health-promoting activities of tea has created a need for

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Fig. 1. Structures of the major tea flavonoids. Epicatechin monomers present in green tea undergo oxidation and condensation to yield theaflavins in fermented black and oolong teas.

improved, high-throughput analytical methods which are suitable across a wide range of research applications. Separation by high performance liquid chromatography (HPLC) followed by UV or electrochemical detection is the most widely used method for analysis of tea flavonoids. The majority of recently published analytical HPLC methods have focused on optimization of the conditions used to extract, detect, and characterize tea flavonoids found in infusions and biological matrices. These methods appear to have not taken full advantage of HPLC column and system advances in order to optimize chromatographic performance as well [2,4,19–28]. These studies all report separation methods that require 20–80 min HPLC analysis time per sample, with the average being 20–40 min, depending on the analytes measured. While providing excellent resolution, these existing methods have several shortcomings. First, long analysis times dictate that some samples may not be analyzed for several hours following extraction, potentially leading to sample degradation under certain conditions and thus introducing error [29–31]. Second, for extensive experiments with numerous samples, HPLC analysis can become a limiting factor that seriously reduces analytical throughput. Third, long analysis times result in inefficiency, as they require the use of large volumes of solvents and instrument time that could alternatively be used for other analyses. A rapid, high-throughput method with significantly shorter HPLC analysis time is required to facilitate larger, more extensive studies with large sample sets. The specific objective of this study was to develop an improved method for the analysis of catechins and theaflavins using commonly available HPLC equipment in order to reduce total analysis time, thereby improving efficiency, reducing analytical costs, and increasing sample throughput.

2. Experimental 2.1. Reagents, standards, and samples Methanol (MeOH) (HPLC grade), acetonitrile (ACN) (HPLC grade) and glacial acetic acid were obtained from Mallinckrodt Baker (Phillipsburg, NJ, U.S.A.). Trifluoroacetic acid (TFA) (99%) was obtained from Aldrich (Milwaukee, WI, U.S.A.). Caffeine (CF), (−)-epigallocatechin (EGC) (>98%), (±)-catechin hydrate (C), (+)-epicatechin (EC) (>98%), (−)epigallocatechin gallate (EGCG) (>95%), and (−)-epicatechin gallate (ECG) (>98%) standards were purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.). Theaflavin (TF) (primary grade) standard was obtained from Chromadex (Santa Ana, CA, U.S.A.). Commercially available green tea and black tea (R. Twining and Co., Ltd., London, England), decaffeinated green tea (Upton Tea Imports, Hopkinton, MA, U.S.A.), and oolong tea (R.C. Bigelow Inc., Fairfield, CT, U.S.A.) were obtained from local vendors. Distilled deionized water (ddH2 O) was produced using a Barnstead MegaPure MP-1 system (Dubuque, IA, U.S.A.). Acetic acid solution was prepared by diluting glacial acetic acid in ddH2 O (2:98, v/v). Mixtures of standards representing major green tea and decaffeinated green tea components (CF, EGC, C, EC, EGCG, and ECG) and major oolong and black tea components (CF, EGC, C, EC, EGCG, ECG, and TF) were prepared in acetic acid solution in order to assess the effectiveness of HPLC separation. For tentative peak identification, standard curves, and limits of detection (LOD), solutions of CF, EGC, C, EC, EGCG, and ECG standards were prepared in acetic acid solution and TF standard was prepared in acetic acid solution/MeOH (80:20, v/v). For linear dynamic range (LDR) evaluation, concentrated (approxi-

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mately 0.05 M) stock solutions of CF and EGCG were prepared in acetic acid solution/MeOH (80/20, v/v) utilizing sonication to facilitate solubilization. Serial dilutions were performed with acetic acid solution to produce the following standard solutions: 1 mM to 10 ␮M for CF, EGC, C, EC, EGCG, and ECG standard curves; 0.5 mM to 10 ␮M for TF standard curves; 1 mM to 5 nM for LOD of CF, EGC, C, EC, EGCG, and ECG; 0.1 mM to 50 fM for LOD of TF; and 50 mM to 5 nM for LDR of CF and EGCG. 2.2. Sample preparation Green tea, decaffeinated green tea, black tea, and oolong tea aqueous infusions were used as the source of catechins and theaflavins for HPLC-diode array detection (HPLC-DAD) analysis, and were prepared in bulk for use throughout the study. Each infusion was made by pouring 250 mL boiling ddH2 O over 2.2 g tea and brewing for 5 min with mild stirring. After brewing, 20 mL of each infusion was diluted with 10 mL acetic acid solution in order to lower pH and stabilize polyphenol components prior to HPLC-DAD analysis. Acidified solutions were centrifuged for 10 min at 1950 × g on a Thermo IEC Centra CL2 centrifuge (Needham Heights, MA, U.S.A.). Aliquots of each supernatant were then collected and stored at −80 ◦ C under a blanket of nitrogen until analysis. Representative green tea and black tea infusions used to confirm peak identity by HPLC followed by electrospray ionization coupled to mass spectrometry (HPLC-ESI-MS) experiments were prepared similarly but with 5 g tea instead of 2.2 g. 2.3. HPLC-DAD analysis HPLC-DAD analysis was performed on a Waters Alliance 2695 separation module (Milford, MA, U.S.A) which employs a low-pressure quaternary proportioning valve to achieve gradient mixing. Separations were achieved using a 3.9 mm × 100 mm (3.5 ␮m particle size) XTerra RP-C18 column (Waters) thermostated to 35 ◦ C with a Waters column temperature control module. In order to minimize system void volume (Vo ), the column was not fitted with a guard column; however, samples were filtered through a 0.45 ␮m PTFE filter (National Scientific, Rockwood, TN, U.S.A.) prior to injection. The HPLC-DAD system was further optimized by employing a minimum length of PEEK tubing (1/16 in OD × 0.005 in ID) (Upchurch Scientific, Oak Harbor, WA, U.S.A.) to connect the injector port, column, and detector. The system dwell volume was 650 ␮L, and the system Vo was 965 ␮L (which corresponds to a void time of approximately 0.8 min). Samples were thermostated at 5 ◦ C prior to injection. Sample analysis was competed within 5 h with no observable precipitation in sample vials. Injection volume was 10 ␮L. System flow rate was 1.2 mL/min. Typical system operating pressure range was 20–25 MPa, which is within the allowable system operating pressure for the Waters 2695 module (approximately 34 MPa). DAD was performed with a Waters 2996 photodiode array detector with wavelength scan between 200 and 600 nm. Chromatographic data was collected and integrated using Waters’ Empower software. Calibration plots were constructed with

authentic catechin standards by plotting peak areas from the DAD absorbance signal at 280 nm versus catechin concentration. Preparation of catechin standard solutions is described in Section 2.5. Two distinct HPLC-DAD elution methods were employed. Green tea infusions, which contain primarily catechins and caffeine, were analyzed using a binary solvent system [24] comprised of the following solutions: Phase A-ddH2 O, ACN, and TFA (919/80/1, v/v) and Phase B-ddH2 O, ACN, MeOH, and TFA (699/270/30/1, v/v). Prior to each injection, the system was equilibrated to 95/5 (A/B). Following sample injection, the phase composition changed according to the following gradient: 95/5 at 0 min, 30/70 at 1.5 min (convex), 1/99 at 3 min (convex), and 95/5 from 3 to 5 min (step, immediate) for a total chromatographic run time of 5 min. A longer 10 min method employing a ternary solvent system was utilized to analyze oolong and black tea infusions, in order to resolve slower-eluting theaflavins in addition to catechins. For the10 min ternary method, Phases A and B were identical to the 5 min binary elution method; Phase C was MeOH (100%). Prior to each run, the system was equilibrated to 95/5/0 (A/B/C). Following sample injection, the phase composition changed according to the following gradient: 95/5/0 at 0 min, 30/70/0 at 1.5 min (convex), 1/99/0 at 3 min (convex), 0/15/85 at 6 min (concave), 95/5/0 at 7 min (convex), and 95/5/0 from 7 to 10 min (constant). The last step of both the 5 min binary and 10 min ternary gradient elution methods reequilibrated the column to the initial gradient conditions (95/5), and no further equilibration was required between chromatographic runs. Thus, the chromatographic run time was equal to the total analysis time for samples analyzed by both methods. 2.4. HPLC-ESI-MS analysis HPLC-ESI-MS analysis was performed on selected representative infusions (green and black tea only) in order to verify tentative peak identification obtained by retention time and absorption spectra in HPLC-DAD analysis. HPLC-ESI-MS separation was achieved using a Dionex P580 liquid chromatography system (Dionex Corp., Sunnyvale, CA, U.S.A.), equipped with a Dionex UVD340U UV detector set at 280 nm. Samples were injected into a 20 ␮L injection loop prior to separation on the XTerra column previously described. The column was maintained at ambient temperatures (approx. 23 ◦ C). System flow rate was 1.2 mL/min. The Dionex P580 LC employed for HPLC-ESI-MS experiments was unable to duplicate the low void volume, ternary solvent system, and convex/concave gradient profiles that were critical parameters of the Waters Alliance 2695 LC employed for HPLC-DAD experiments. Due to these unavoidable differences in system capabilities, the elution methods employed for HPLCDAD analysis were modified for HPLC-ESI-MS experiments. A single 18 min elution scheme based on the 5 min binary gradient used in HPLC-DAD analysis of green tea and decaffeinated green tea infusions was employed for all HPLC-ESI-MS analyses. The mobile phases were identical to those used for binary HPLC-DAD elution. Prior to injection, the system was equilibrated to 95/5 (A/B). Following sample injection, the phase

A.P. Neilson et al. / J. Chromatogr. A 1132 (2006) 132–140

composition changed according to the following gradient: 95/5 at 0 min, 30/70 at 3 min, 1/99 at 6 min and held constant until 15 min, 95/5 at 16 min and held constant until 18 min (all transitions linear). Following HPLC separation, the post-column effluent was split 17/83 (waste/retained): 0.2 mL/min was discarded as waste and 1.0 mL/min was introduced into an LCQ Classic (ThermoFinnigan Corp., San Jose, CA, U.S.A.) mass spectrometer system where ESI-MS was performed in positive mode. For ESI, the electrospray needle voltage was 4 kV, and the capillary temperature was 205 ◦ C. Typical background source pressure was 1.2 × 10−5 Torr as read by an ion gauge. The drying gas was nitrogen. Pre- and post-ion lens multipole voltages were −3 and −7 V, respectively, while the lens voltage was −16 V. Ion trap DC offset voltage was −10 V, and the ion trap temperature was slightly above ambient (roughly 27 ◦ C). Following ion trapping, ions were mass analyzed and detected in the electron multiplier at 850 V. The LCQ mass analyzer was scanned to 2000 m/z in positive mode to confirm peak identity by observation of the corresponding ionized molecule ([M + H]+ ). 2.5. HPLC-DAD calibration and measurement of variability Calibration curves for all authentic standards were prepared by performing HPLC-DAD analysis in triplicate on five incremental dilutions of each standard, ranging from 1 mM to 10 ␮M for CF, EGC, C, EC, EGCG, and ECG, and from 0.5 mM to 10 ␮M for TF. LODs were determined by performing HPLCDAD analysis in triplicate on incrementally diluted solutions of each standard, ranging from 1 mM to 5 nM for CF, EGC, C, EC, EGCG, and ECG and from 0.1 mM to 50 fM for TF. LDR was determined for CF and EGCG by performing HPLC-DAD analysis in triplicate on 12 incrementally dilutions of each solution, ranging from 50 mM to 5 nM. All calibration analyses for CF, EGC, C, EC, EGCG, and ECG were performed using the 5 min binary elution method, and all calibration analyses for TF were performed using the 10 min ternary elution method. Intraday variation (repeatability) of analytical response was assessed by performing HPLC-DAD analysis on aliquots of each infusion nine times on the same day. Interday variation (reproducibility) of analytical response was assessed by performing HPLC-DAD analysis on freshly thawed aliquots of each infusion in triplicate on 3 consecutive days. 2.6. Data analysis Statistical analysis was performed on SAS 8.2 (SAS Institute Inc., Cary, NC, U.S.A.). Percent coefficient of variation (CV) was defined as sample standard deviation divided by sample mean, multiplied by 100%. 3. Results and discussion 3.1. HPLC-DAD separation and peak identification HPLC-DAD separation of standard mixtures representing major green tea and decaffeinated green tea components by

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Fig. 2. Elution profiles of mixed authentic standards representing major green tea components by a 5 min binary HPLC-DAD method (A) and major black and oolong tea components by a 10 min ternary HPLC-DAD method (B) using a Waters XTerra RP-C18 3.9 mm × 100 mm (3.5 ␮m particle size) column. Elution conditions: binary gradient elution with Phase A-ddH2 O, ACN, and TFA (919/80/1, v/v) and Phase B-ddH2 O, ACN, MeOH, and TFA (699/270/30/1, v/v) and ternary gradient elution with identical Phases A and B from binary elution in addition to Phase C–MeOH; column temperature: 35 ◦ C; flow rate: 1.2 mL/min; injection volume: 10 ␮L; DAD detection λ: 280 nm; typical operating pressure range: 20–25 MPa. Refer to Table 1 for peak identities. The initial 3 min of both methods were identical in order to provide similar elution of weakly retained solutes (caffeine and epicatechin derivatives, peaks 1–6), while also providing elution of strongly retained solutes such as theaflavin (peak 7) by the ternary method.

binary elution was achieved in 5 min (Fig. 2A). Separation of standard mixtures representing major black tea and oolong tea components by ternary elution was achieved in 10 min (Fig. 2B). Both HPLC-DAD methods exhibited similar elution profiles of weakly retained solutes (CF and all catechins, peaks 1–6), as initial elution parameters (for the first 3 minutes after injection) of both binary and ternary elution methods were identical. Conditions of the ternary method allow for elution of more strongly retained solutes present in oolong and black teas (such as catechin oxidation and dimerization products, including theaflavins) over 10 min. HPLC-DAD analysis of green, decaffeinated green, black and oolong tea infusions (Figs. 3 and 4) resulted in elution profiles of CF, EGC, C, EC, EGCG, ECG, and TF similar to those observed from analysis of mixed standards (Fig. 2). Preliminary assignment of peak identities in sample infusions was based on comparison of peak retention time (tR ) and online DAD spectra with those obtained by HPLC-DAD analysis of authentic standards (Table 1). Based on these observations, the following peak assignments were made: 1-CF, 2-EGC, 3-C, 4-EC, 5-EGCG, 6-ECG, 7-TF. Preliminary peak identification by HPLC-DAD was subsequently confirmed by HPLC-ESIMS experiments identifying the ionized molecules ([M + H]+ ) corresponding to the tentatively identified compounds in representative infusions (Table 1). To account for potential peak identification errors caused by unavoidable differences between the HPLC-DAD and HPLC-ESI-MS chromatographic condi-

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Fig. 4. Representative elution profiles of an oolong tea infusion (A) and a black tea infusion (B) by a 10 min ternary HPLC-DAD method. Refer to Fig. 2 for elution conditions (response at 280 nm). Refer to Table 1 for peak identities.

Fig. 3. Representative elution profiles of a decaffeinated green tea infusion (A) and a green tea infusion (B) by 5 min binary HPLC-DAD method. Refer to Fig. 2 for elution conditions (response at 280 nm). Refer to Table 1 for peak identities.

tions, representative infusions and mixed authentic standards were analyzed on the HPLC-DAD instrument using the same conditions employed for HPLC-ESI-MS experiments. Separation profiles and spectra achieved using the HPLC-ESI-MS chromatographic conditions (18 min binary method) were iden-

Table 1 Summary of data used to identify and quantify major components of tea infusions eluted by 5 min and 10 min HPLC-DAD methods: HPLC-DAD observed retention time (tR ) and adjusted retention time (t R ), wavelength of maximal absorption (λMAX ) observed in HPLC-DAD detection, molecular weight (MW), ionized molecule ([M + H]+ ) observed in HPLC-ESI-MS at stated HPLC-DAD retention time, calibration curve slope and intercept, calibration curve coefficient of determination (R2 ), limit of detection (LOD), and linear dynamic range (LDR) Peak

Identification Compounda

1 2 3 4 5 6 7 8 9 10

CF EGC C EC EGCG ECG TF TFMG1f TFMG2f TFDG

Calibration b

t R b,c

λMAX

(min)

(min)

(nm)

1.8 1.9 2.2 2.3 2.7 3.4 5.5 6.1 6.3 6.4

1.0 1.1 1.4 1.5 1.9 2.6 4.7 5.3 5.5 5.6

tR

273 271 280 279 275 278 268, 375, 447 272, 378, 447 274, 372, 450 277, 376, 460

MW

[M + H]+

Sloped,e

Interceptd,e

(g mol−1 )

(m/z)

(␮V s pmol−1 )

(␮V s)

194.2 306.3 290.3 290.3 458.4 442.4 564 716 716 868

195 307 291 291 459 443 565 717 717 869

352 62.6 0.226 127 438 572 589 – – –

191 473 33.1 −905 −3830 −3320 −4030 – – –

R2d,e

>0.999 >0.999 0.941 >0.999 >0.999 >0.999 >0.999 – – –

LODe

LDRe

(pmol)

(ng)

(pmol)

0.910 20.9 1.83 7.91 3.69 2.78 31.9 – – –

0.177 6.40 0.531 1.54 1.69 1.23 18.0 – – –

87.3–13600 – – – 7.33–28600 – – – – –

Refer to Fig. 2 for elution conditions employed for HPLC-DAD analysis. HPLC-ESI-MS elution conditions: 18 min modified binary gradient elution employing identical column and solvents used for binary HPLC-DAD elution; column temperature: ambient (approx. 23 ◦ C); flow rate: 1.2 mL/min; injection volume: 20 ␮L; UV detection λ: 280 nm. ESI-MS conditions: mode: positive; electrospray needle voltage: 4 kV; background source pressure: 1.2 × 10−5 Torr; drying gas: nitrogen; ion trap DC offset voltage: −10 V; ion mass analysis/detection: electron multiplier at 850 V; mass analyzer scanned to 2000 m/z. Refer to Section 2 for more complete HPLC-ESI-MS details. a Identification and calibration data for CF, EGC, C, EC, EGCG, ECG, and TF were obtained using authentic standards. Identification data for TFMG1, TFMG2, and TFDG are based on DAD spectra and observed [M + H]+ from tea infusions only; therefore, no calibration data is given for these compounds. b Observed and adjusted retention times for CF, EGC, C, EC, EGCG, and ECG represent values for both 5 min and 10 min HPLC-DAD methods; values for TF, TFMG1, TFMG2, and TFDG represent 10 min HPLC-DAD method only. c Adjusted retention times were obtained by subtracting system void time, t (0.8 min), from observed retention times. o d Calibration slope, intercept, and coefficient of determination were obtained by HPLC-DAD, and represent the least-squares regression line relating absorbance peak area at 280 nm (␮V s) as the dependent variable with concentration (pmol on-column) as the independent variable. e Calibration curve, LOD, and LDR represent total moles on-column, and represent HPLC-DAD detection values. LOD is also represented as total ng on-column. f TFMG1 and TFMG2 refer collectively to TF 3-MG and TF 3 -MG. Due to similar DAD spectra and identical observed [M + H]+ , these two peaks could not be definitively assigned.

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tical when performed on both HPLC-ESI-MS and HPLC-DAD instruments (chromatograms not shown), facilitating the association between tR and spectra obtained from HPLC-DAD analysis and [M + H]+ observed in HPLC-ESI-MS experiments. Three peaks, labeled as 8–10, with tR greater than that of TF were detected in black tea. These peaks were tentatively identified as theaflavins based on similarities between their DAD absorption spectra and that of TF (peaks 8–10 all exhibited characteristic theaflavin absorption spectra with three distinct λMAX ). Peaks 8–10 were subsequently confirmed as theaflavins by observation of the corresponding [M + H]+ in HPLC-ESI-MS (Table 1). Peaks 8 and 9 both exhibited [M + H]+ of 717 m/z, and were identified collectively as TF 3-MG (716 g mol−1 ) and TF 3 -MG (716 g mol−1 ). Due to the fact that TF 3-MG and TF 3 -MG are positional isomers with identical molecular masses and similar DAD spectra, the identity of these peaks could not be conclusively assigned to either isomer. Peaks 8 and 9 were therefore designated as theaflavin monogallate 1 (TFMG1) and theaflavin monogallate 2 (TFMG2), solely on the basis of LC elution order (Table 1). Peak 10 was identified as TFDG (MW 868 g mol−1 ) by observation of [M + H]+ 869 m/z (Table 1). Caffeine, EC, EGC, EGCG, and ECG were found in all infusions. C was not detected in any of the infusions. TF, TF 3-MG, TF 3 -MG, and TFDG were detected in black tea (extremely small peaks with tR s corresponding to the theaflavins, which were not confirmed by spectra nor HPLC-ESI-MS, were also detected in oolong tea).

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defined as the total moles-on-column, calculated by multiplying the solution concentration by the injection volume (10 ␮L). Mean peak areas from triplicate HPLC-DAD analyses of a range of solution concentrations were used to determine calibration curves for each standard, defined as the least-squares regression line relating absorbance peak area at 280 nm (␮V s) to sample concentration (Table 1). Coefficients of determination (R2 ) were >0.999 for calibration curves of all standards except C. In order to calculate the LOD, HPLC-DAD analysis was performed on incrementally diluted concentrations of each standard until the signal (peak height):noise ratio at 280 nm was reduced to below 3:1. LOD for each standard was then defined as the concentration (moles-on-column) that resulted in a signal:noise ratio of 3:1. LODs for CF and all catechins were approximately on the order of 1–10 pmol; EGC had the highest LOD at 20.9 pmol. The LOD for TF was 31.9 pmol (Table 1). The LDR for CF and EGCG was taken to be the concentration range over which the least-squares regression line relating peak area to standard concentration exhibited R2 ≥ 0.999 (obvious deviations from linearity at extremely low concentrations that did not cause R2 < 0.999 were not included in the calculation). The LDR was determined to be 3 orders of magnitude for CF and 4 orders of magnitude for EGCG (Table 1). 3.3. Repeatability and reproducibility Intraday variation (repeatability) of analytical response was quantified by performing HPLC-DAD analysis on aliquots of each infusion nine times on the same day. For each infusion, peak areas of the nine runs were used to calculate the CV for each compound within the same day. Interday variation (reproducibility) of analytical response was quantified by performing HPLC-DAD analysis on freshly thawed aliquots of each infusion in triplicate on 3 consecutive days. For each infusion, the mean peak areas of triplicate analyses on each day were treated

3.2. Standard curves, limits of detection, and linear dynamic range The calibration and reproducibility results were generally consistent with HPLC-DAD and HPLC-UV methods for the analysis of catechins reported in the literature [21,23,27,32,33]. For all calibration calculations, analyte concentration was

Table 2 Percent coefficient of variation (CV) of peak areas for intradaya and interdayb replication of HPLC-DAD analysis of tea infusions Peak

1 2 3 4 5 6 7 8 9 10

Compound

CF EGC C EC EGCG ECG TF TFMG1d TFMG2d TFDG

Green tea

Decaffeinated green tea

Black tea

Oolong tea

Intraday CV

Interday CV

Intraday CV

Interday CV

Intraday CV

Interday CV

Intraday CV

Interday CV

0.5 1.4 – 1.3 0.3 0.4 – – – –

4.1 2.3 – 8.0 3.8 3.7 – – – –

1.0 1.4 – 4.5 0.9 0.4 – – – –

1.6 2.4 – 6.9 1.3 2.0 – – – –

0.6 15.4c – 5.5c 10.7 2.3 4.7 5.8 7.1 8.6

1.9 11.5c – 3.4c 6.5 3.0 4.0 6.7 7.3 8.7

0.3 2.9c – 3.5c 0.5 2.6 – – – –

1.6 1.9c – 1.9c 1.0 0.6 – – – –

Refer to Fig. 2 for HPLC-DAD elution conditions. a Intraday CVs represent data from nine analyses of each infusion within the same day. b Interday CVs represent averaged data from triplicate analyses of each infusion performed on 3 consecutive days; triplicate analyses on the same day were averaged to give single data points used to assess variation between days. c Due to the small peak areas of EGC and EC in black and oolong tea infusions and the gradient conditions employed for HPLC analysis, spectral purity was difficult to verify for these peaks. CVs for EGC and EC peak areas in these infusions are estimates. d TFMG1 and TFMG2 refer collectively to TF 3-MG and TF 3 -MG. Due to similar DAD spectra and identical [M + H]+ in HPLC-ESI-MS, these two peaks could not be definitively assigned.

Refer to Fig. 2 for HPLC-DAD elution and detection conditions. Refer to Table 1 for identification, calibration and standard curve data. a Values reported as mean ± SD (n = 9) obtained from intraday variation (repeatability) assessment of analytical response for each infusion. b Infusions were prepared by pouring 250 mL boiling ddH O over 2.2 g tea leaves and brewing for 5 min with mild stirring. 2 c Due to the small peak areas of EGC and EC in black and oolong tea infusions and the gradient conditions employed for HPLC analysis, spectral purity was difficult to verify for these peaks. Concentrations of EGC and EC in these infusions are estimates.

1.11 0.640c 0.253c 0.614 0.211 77.1 ± 21.8 ± 8.30 ± 66.24 ± 14.7 ± – 0.0228 0.083c 0.00348c 0.00536 0.00191 1.59 ± 0.285 ± 0.114 ± 0.578 ± 0.133 ± – 1.05 1.21c 1.32c 2.02 0.382 0.277 ± ± ± ± ± ± 1.30 0.0884 0.210 0.128 0.113 0.0314 0.0634 0.517 1.01 0.526 0.147 4.30 ± 20.6 ± 13.1 ± 40.3 ± 7.11 ± – CF EGC EC EGCG ECG TF

0.0503 0.00936 0.0196 0.0305 0.00738

2.44 0.716 1.42 3.49 0.817

0.0885 ± 0.269 ± 0.180 ± 0.352 ± 0.0643 ± –

0.00131 0.00675 0.0139 0.0459 0.00133 68.4 ± 26.8 ± 20.0 ± 105 ± 25.2 ± – 1.41 ± 0.350 ± 0.276 ± 0.918 ± 0.227 ± –

mg/250 mL

mM

± ± ± ± ± ±

0.0216 0.0158c 0.0183c 0.0176 0.00345 0.00197

63.0 6.77 15.2 14.6 12.5 4.43

mM mg/250 mL

Oolong tea Black tea

mM

These HPLC methods were designed to minimize peak retention times and optimize peak resolution through the use of convex and concave gradient transitions. These nonlinear elution gradients are now attainable on several commercially available HPLC systems, and are useful for achieving moderate performance improvements beyond the capabilities of linear gradients. However, if the instrument to be used is incapable of providing nonlinear gradients, the methods can readily be adapted to

mg/250 mL

3.5. Practical application

mM

Calibration data was used to quantify the catechin and theaflavin content of each aqueous tea infusion. Quantification was based on mean absorbance peak areas for each compound obtained for assessment of intraday variability (n = 9, see Section 3.3). Values for catechin and theaflavin content of each infusion are given in Table 3. Levels of catechins were generally lower in black and oolong teas than in green tea and decaffeinated green tea. These values are consistent with published data regarding catechin and theaflavin contents of commercially available teas [1–7].

Decaffeinated green tea

3.4. Composition of tea infusions

Green tea

as single data points to calculate the CV for each compound between days. Both intraday and interday CVs were generally <5% for catechins in green, decaffeinated green, and oolong teas, but were significantly higher in black tea (Table 2). Both intraday and interday CVs were <9% for theaflavins in black tea (Table 2). Intraday CVs were generally smaller than interday CVs in green and decaffeinated green teas, but no such pattern was apparent in oolong and black teas. Compounds with low responses (EC in all infusions; EGC, EGCG, and theaflavins in black tea) had the highest CVs. The relatively low CVs for most catechins in both intraday and interday experiments indicate that this method is both repeatable and reproducible. Several factors may account for the higher CVs observed for small peaks. First, unavoidable random inconsistencies introduced by manually setting peak integration limits will exert a proportionally larger influence on small peaks than on large peaks. Also, the small peaks may have been close to the lower limit of the LDR, and small decreases in peak area between analyses may have fallen into areas of nonlinear response, and hence wider variation. Lastly, small amounts of sample degradation between samples or days may have a proportionally greater influence on the areas of small peaks, especially if working near the lower limit of the LDR. This explanation is supported by the fact that black tea generally had the highest CVs for EGC and EGCG, and relatively high CVs for all four theaflavins. Black tea also had the smallest peak areas for all analytes (data not shown). Due to its extensive fermentation, black tea has much lower levels of catechins (and, hence, smaller catechin peaks) than green or oolong teas. The theaflavins peaks in black tea were also much smaller than the catechin peaks in the other samples. Thus, these higher CVs may be a function both of analytical response and other confounding factors.

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Table 3 Concentrationsa of major components of tea infusionsb measured by HPLC-DAD analysis

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linear gradients with slight modifications, such as the use of non-continuous (step) transitions or multiple linear transitions which approximate the curved gradient profiles provided by convex and concave elution methods. Similarly, if the instrument to be used is incapable of providing mobile phases composed of >2 solvents, the ternary method employed to resolve catechins and theaflavins within 10 min can be adapted to a binary method by increasing the percent organic modifier in one or both of the first two solvents and eliminating 100% MeOH as the third solvent. These modifications can be made with minimal loss of separation efficiency but result in moderate increases in retention times (data not shown), and thus make these methods applicable for most HPLC equipment. In order to separate catechins and theaflavins within 5 and 10 min total analysis time (t), respectively, the HPLC system was optimized to minimize void volume (Vo ) and void transit time (to ). System to was estimated by the retention time of the nonretained solvent front and was approximately 0.8 min (roughly one sixth of t for the 5 min method). Given the desired t, the theoretical on-column separation time (t ) is found by subtracting to : t = t–to . For to = 0.8 min, the 5 min method provides 4.2 min actual on-column separation time. The time each compound actually spends on-column is indicated by adjusted retention times (t R ) found by subtracting to from observed retention times (tR ): t R = tR –to . For HPLC separations with t > 15–20 min, to is not a major factor as it is usually much smaller than t and tR , and thus tR ≈ t R . However, for rapid methods with t ≤ 10 min, to becomes significant: compounds spend an appreciable fraction of t simply traversing Vo, and thus tR > t R . When duplicating the separation, only those compounds with tR < t (t R < t ) will elute within t. Minimization of to is crucial for achieving sufficient t for a given separation when t is fixed, and as a result, separation on non-optimized HPLC systems may be impossible without increasing t. A practical first step when duplicating this method would be to assess system to , after which the desired separation may be achieved by either adjusting t to account for the existing to or optimizing to in order to achieve separation in t. 4. Conclusion The HPLC methods described represent roughly a 2–4fold reduction in total analysis time (5–10 min, as opposed to 20–40 min, on average) and, by extension, reduction in solvent usage associated with typical HPLC methods used to measure catechins and theaflavins. Calibration and reproducibility data indicate that our rapid, high-throughput HPLC methods are repeatable, reproducible, and sensitive. Use of these rapid methods will likely reduce sample degradation between extraction and analysis, and also increase analytical throughput required for complex studies investigating biological activities of catechins and theaflavins. It is important to note that such improvement is readily achievable with commonly used HPLC equipment and does not require specialized ultra performance LC (UPLC) instrumentation, thus increasing the feasibility of applying these methods across a broad spectrum of research applications. These methods therefore warrant serious consideration for application

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