Introduction Interest in tea and tea extracts has increased with reports of health benefits associated with their consumption1. The high

levels of catechins, particularly in green tea, have been investigated as contributing to the health benefits, possibly due to their antioxidant activity. Table 1 lists catechins found in tea, while Figure 1 shows the structures of the most highly expressed epi-structured catechins. Tea is prepared from leaves and buds of the evergreen shrub Camellia sinensis. Green tea is produced by drying tea leaves soon after harvest while black tea is produced by allowing tea leaves to oxidize, also known as fermentation, prior to drying. Oolong tea is produced by allowing partial oxidation before drying2. Oxidation of tea reduces the levels of catechins due to the formation of condensation products such as theaflavins and thearubigins which contribute to the flavor profile of black and Oolong teas. This report outlines the use of LC/TOF MS to analyze the levels of catechins in infusions of green, Oolong, and black teas.

Analysis of Catechins in Brewed Tea using LC/TOF Mass Spectrometry

A P P L I C A T I O N N O T E

Author:

LC/MS Applications Team

PerkinElmer, Inc. Waltham, MA

Liquid Chromatography/ Mass Spectrometry

2

Experimental

Green, oolong, and black tea samples analyzed in this study are listed in Table 2. Catechin standards were obtained from Cerilliant® (Round Rock, TX). Tea infusions were prepared as outlined in Table 3. Liquid chromatography and mass spectrometry analysis was performed as outlined in Tables 4 and 5 respectively. The retention times for all catechins were determined using standard reference materials (data not shown). Chromera® and TOF MS Driver software was used for acquisition and data analysis. The lockmass calibration solution was 0.0005% (v/v) trifluoroacetic acid and 15 µg/mL leucine-enkephalin in methanol. Lockmass ions were m/z 112.9856 and m/z 554.262 with a search window of 50 mmu. Extracted ion chromatograms were generated with an m/z ±0.04 window. All samples were analyzed in triplicate. Calibrator levels 1 to 7 were: 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, and 5.0 µg/mL. Calibration curves were generated using non-weighted linear regression. The catechin (C) standard was used for quantitation of epigallocatechin (EGC) in tea samples.

O

OH

OH

HOOH

OH

EC

OH

O

OH

OH

OH

HO

OH

O

O

O

OH

OHOH

OH

OHHO

OH

ECG

EGC

EGCG

O

O

O

OH

OHOH

OH

OH

OH

HO

OH

Figure 1. Epi-structured catechins.

Table 1. Catechins examined in this study.

Compound Abbreviation

(+)-Catechin C

(-)-Epicatechin EC

(-)-Gallocatechin GC

(-)-Epigallocatechin EGC

(-)-Catechin-3-gallate CG

(-)-Epicatechin-3-gallate ECG

(-)-Gallocatechin-3-gallate GCG

(-)-Epigallocatechin-3-gallate EGCG

Table 2. Tea samples.

Sample Tea Type Origin

G1 Green Not Listed

G2 Green China

G3 Green China

G4 Green U.S.

G5 Green Japan

G6 Green Japan

O1 Oolong Taiwan

B1 Black Blend

B2 Black U.S.

B3 Black Blend

B4 Black Blend

B5 Black Blend

Table 4. Liquid Chromatography.

Flexar FX-15 UHPLC System Parameters

Gradient (min.) Column: Brownlee SPP, PFP, 2.1 x 100 mm

Time % B Column temperature: 45 °C

0 5 Pre-column filter: 0.5 µm stainless steel

5.0 30 Flow rate: 0.4 mL/min.

5.1 95 Mobile phase A: water plus 0.1% formic acid

6.9 95 Mobile phase B: acetonitrile plus 0.1% formic acid

7.0 5 3 min. equilibration at 5% B

Table 3. Sample Preparation.

Procedure

200 ± 1 mg tea leaves per sample

20 mL water per sample

15 min. incubation at 80 °C

15 min. incubation at RT

Centrifuge 5 min. at 1,500 RCF

1:85 dilution w/0.1% formic acid in water

20 µL injection for LC/TOF MS analysis

Table 5. Mass Spectrometry.

AxION 2 TOF MS Parameters

Negative Pulse mode

Ultraspray 2 ESI source

Nebulizer: Left 80 psi, Right 20 psi

Capillary exit: -100V

Skimmer: -25V

Drying gas: 12 L/min at 350 °C

Endplate heater: Medium

Lockmass Calibration

3 spectra/sec. acquisition rate

3

Results

The upper panel of Figure 2 shows an extracted ion chromatogram (EIC) demonstrating baseline resolution of all catechin calibration standards. Calibration curves for all catechin standards are shown in Figure 3. These results show good linearity (r2 0.9983 to 0.9992) over the tested concentration range. Imprecision of replicate analysis of all calibrator levels was determined (Table 6). These results demonstrated low imprecision (average 3.6 %CV, range 0.7 to 9.6 %CV). The lower panel of Figure 2 shows EICs from analysis of the G1 green tea sample. All tea samples were analyzed in triplicate and are summarized in Figure 4. These results showed that the epi-structured catechins (EC, ECG, EGC and EGCG) were more abundant than the non-epi-structured catechins (C, CG, GC and GCG), consistent with published literature3. In addition, all catechin levels were lower in black tea compared to green tea. This is due to condensation reactions of catechins which occur during oxidation of tea leaves in the production of black tea. Oolong tea production involves a lower level of oxidation than that of black tea. The O1 Oolong tea sample is a low oxidation level tea (approximately 18%) and the level of catechins in this sample was intermediate between the green and black tea samples, consistent with the intermediate level of oxidation. For example the EGCG concentration of the Oolong sample was 171.9 µg/mL, a level between the average EGCG levels for the green (318.1 µg/mL) and black (38.6 µg/mL) teas.

Figure 2. Representative extracted ion chromatograms. Upper panel, calibration curve L7 first replicate. Lower panel, tea sample G1 first replicate.

Figure 3. Calibration curves. Peak area counts (y-axis) and calibrator concentration in µg/mL (x-axis). Lower left panel, formulas and calculated m/z for molecular ions for all analytes.

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Conclusion

This work demonstrated that analysis of catechins levels in tea is readily accomplished using LC/TOF MS. This analysis may be applied to monitoring catechin levels in different tea varietals. In addition, the technique is applicable to the analysis of green tea extracts, a popular type of dietary supplement.

References

1. Cabrera C, Artacho R, Gimenez R, Journal of the American College of Nutrition, 25 (2) pp79-99 (2006), Beneficial Effects of Green Tea – A review.

2. Dou J, Lee VS, Tzen JTC, Lee MR, Journal of Agricultural and Food Chemistry, 55 (18) 7462-7468 (2007), Identification and Comparison of Phenolic Compounds in the Preparation of Oolong Tea Manufactured by Semifermentation and Drying Processes.

3. Astill C, Birch MR, Dacombe C, Humphrey PG, Martin PT, Journal of Agricultural and Food Chemistry, 49 (11) 5340-5347 (2001), Factors Affecting the Caffeine and Polyphenol Contents of Black and Green Tea Infusions.

C

CG

GCGCG

ECECG

EGCEGCG

0

100

200

300

400

500

600

G1 G2 G3 G4 G5 G6 O1 B1 B2 B3 B4B5

Analyte

Conc

entr

atio

n (u

g/m

L)

Sample

Figure 4. Summary of quantitative results for all tea samples. Each bar represents the average value for three replicate measurements.

Table 6. Imprecision of calibration curve levels (%CV, n=3).

Level EGCG ECG EC GCG GC CG C

L1 3.6 0.7 4.9 9.6 3.6 6.8 3.7

L2 6.7 3.6 1.9 2.1 4.9 3.8 1.1

L3 7.7 3.0 2.7 4.5 2.4 3.0 3.2

L4 6.6 5.3 3.2 6.7 4.6 6.8 3.0

L5 1.8 2.8 3.9 4.8 4.7 1.8 3.4

L6 2.6 1.9 2.9 4.3 3.9 3.0 4.5

L7 2.3 1.9 0.8 1.1 1.0 1.1 0.9