Skip to Content
Merck
HomeChemical Analysis for Food and Beverage TestingAnalysis of Caffeine and Catechins in Green Tea

HPLC-UV Analysis of Caffeine and Catechins in Green Tea (Decaffeinated)

Dr. Ajay Kaparwan
Mumbai Application Laboratory, India

INTRODUCTION

Tea from the leaves of Camellia sinensis is used to make green tea, black tea, white tea, and oolong tea. The different teas are possible due to the different processing employed. Green tea is not fermented so its polyphenolic components of which the majority are catechins are not oxidized. As such, this decaffeinated green tea assay focuses on catechins and caffeine.

Here, we describe and validate an HPLC method as per Ph. Eur. Monograph 2668 (04/2018)1 as a reference method for the separation and quantification of catechins and caffeine in decaffeinated green tea using a Chromolith® High Resolution RP-18e 150 x 4.6 mm column (Table 1). The monolithic silica column, due to its bimodal pore structure, provides advantages regarding column backpressure and matrix robustness for the analysis of such natural matrix samples.

Chemical structures of catechins and caffeine in two rows. The first row from left to right shows catechin, epicatechin, epigallocatechin, and epigallocatechin-3-O-gallate. The second row from left to right shows, gallocatechin, epicatechin-3-O-gallate, gallocatechin-3-O-gallate, and caffeine.

Figure 1.Chemical structures of catechins and caffeine

EXPERIMENTAL CONDITIONS

Column:

Chromolith® High Resolution RP-18e, 150 x 4.6 mm (1.52023)

Mobile Phase:

[A] 0.01% v/v formic acid prepared by adding 0.1 mL to 1000 mL water;

[B] 0.01% v/v formic acid prepared by adding 0.1 mL to 1000 mL methanol;

[C] acetonitrile;

All mobile phases were filtered through a 0.45 µm membrane filter.

Gradient:

 

Time

A(%)

B(%)

C(%)

  0

97

  0

3

  5

97

  0

3

23

67

30

3

29

67

30

3

30

30

67

3

31

30

67

3

33

97

  0

3

37

97

  0

3

Flow rate:

0.5 mL/min

Back pressure:

40 bar (576 psi)

Column temp.:

41° C

Detector:

UV @ 210 nm

Injection:

2 µL

Sample(s) & Sample Preparation

Extraction solution:

Water:Methanol (30:70 v/v) at 70°C.

Stabilizing solution:

Weigh 250 mg of (ethylenedinitrilo) tetra-acetic acid and 250 mg of ascorbic acid into a 1000 mL volumetric flask. Add 500 mL water and mix to dissolve. Add 100 mL of acetonitrile and top to mark with water.

Test solution:

Weigh 0.2 g of finely crushed decaffeinated green tea sample into a test tube. Add 5 mL of extraction solution to this and stopper it. Vortex to mix and place this in a 70°C water bath. At 5 min, remove from bath and vortex. Return to bath and after 10 min, remove to vortex again and allow to cool. Centrifuge at 2500 rpm for 10 minutes. Decant the supernatant to 10 mL of a volumetric flask. Repeat the extraction with a 5 mL extraction solution and combine the extracts into the 10 mL flask. Top up with cold extraction solution and mix well. Further, dilute 1 mL of this solution to 20 mL with stabilizing solution before filtering through a 0.45 µm PVDF syringe filter.

Catechin standards:

Weigh 7 mg of the catechin into a 20 mL volumetric flask. Add diluent and swirl, sonicate for 20 minutes. Dilute 920 µL of this solution to 10 mL with diluent. Final conc. is 32 µg/mL.

Diluent: 5 mL of extracting solution to 100 mL volumetric flask and top to mark with stabilizing solution. Mix well and sonicate for 5 minutes before use.

Reference solution A:

Weigh 20 mg of Epigallocatechin-3-O-gallate RS and 20 mg of Caffeine RS into a 25 mL volumetric flask. Add water to dissolve and top to mark. Dilute 2 mL of this solution to 50 mL with stabilizing solution. Filter through 0.45 µm PVDF syringe filter. Final conc. is 32 mg/mL.

RESULTS

Chromatogram of blank sample plotted between intensity and retention time with a peak at 4.9 minutes

Figure 2.Blank chromatogram.

Chromatogram of reference solution A plotted between intensity and retention time showing two peaks labeled Epigallocatechin-3-O-gallate and Caffeine

Figure 3.Chromatogram of reference solution A.

Chromatogram of catechin standard mixture plotted between intensity and retention time showing different labeled peaks of catechins and caffeine

Figure 4.Chromatogram of catechin and caffeine standard mixture. See Table 4 for retention times (determined with single solution standards).

Chromatogram of green tea sample 1 plotted between intensity and retention time showing labeled peaks of gallocatechin, epigallocatechin, catechin, caffeine, epigallocatechin-3-O-gallate, epicatechin, gallocatechin-3-O-gallate, and epicatechin-3-O-gallate

Figure 5.Chromatogram of green tea sample 1

Chromatogram of green tea sample 2 between intensity and retention time showing labeled peaks of gallocatechin, epigallocatechin, catechin, caffeine, epigallocatechin-3-O-gallate, epicatechin, gallocatechin-3-O-gallate, and epicatechin-3-O-gallate

Figure 6.Chromatogram of green tea sample 2.

Sr. No.

Green Tea

% Caffeine*     as Per EP Monograph

% Catechin expressed as Epigallocatechin-3-O-gallate as per EP Monograph

% Water Content by KF

1

Sample 1

0.17

10.66

8.2

2

Sample 2

0.09

6.25

10.5

Table 2.Results for 2 green tea samples

*Decaffeinated tea would still have minor amounts of caffeine as observed in the tea sample chromatograms. The decaffeination process can reduce the caffeine amount to <2.5% of the original caffeine load.

System Suitability and Repeatability Data

 

Area in (mAU)

Standard

Gallocatechin

Epigallocatechin

Catechin

Caffeine

Epigallocatechin-3-O-Gallate

Epicatechin

Gallocatechin-3-O-Gallate

Epicatechin-3-O-Gallate

Standard_1

1437880

1458772

1221835

842610

1528633

1238253

1499736

991214

Standard_2

1433198

1471379

1232416

849415

1541607

1247795

1511801

999639

Standard_3

1447688

1489368

1248150

861672

1562190

1263432

1537666

1012880

Standard_4

1438758

1459020

1222586

845011

1530189

1238082

1501816

992558

Standard_5

1444325

1472992

1233040

850780

1543571

1249197

1518150

1000504

Mean

1440369.8

1470306.2

1231605

849897.6

1541238

1247351.8

1513833.8

999359

Standard Deviation

5688.40

12571.12

10644.83

7359.28

13467.85

10378.96

15279.98

8613.96

% RSD

0.4

0.9

0.9

0.9

0.9

0.8

1

0.9

Table 3.Reproducibility of area counts (mAU) of catechin and caffeine standard (32 µg/mL each compound)
 

Retention Time (minutes)

Standard

Gallocatechin

Epigallocatechin

Catechin

Caffeine

Epigallocatechin-3-O-Gallate

Epicatechin

Gallocatechin-3-O-Gallate

Epicatechin-3-O-Gallate

Standard_1

8.516

15.037

15.955

17.949

19.911

20.466

21.844

24.605

Standard_2

8.511

15.033

15.954

17.95

19.914

20.468

21.847

24.61

Standard_3

8.512

15.036

15.956

17.954

19.919

20.474

21.853

24.615

Standard_4

8.507

15.029

15.948

17.946

19.906

20.461

21.84

24.603

Standard_5

8.512

15.046

15.964

17.963

19.923

20.479

21.858

24.621

Mean

8.5116

15.0362

15.9554

17.9524

19.9146

20.4696

21.8484

24.6108

Standard Deviation

0.00321

0.00630

0.00573

0.00658

0.00666

0.00702

0.00716

0.00736

% RSD

0.04

0.04

0.04

0.04

0.03

0.03

0.03

0.03

Table 4.Retention Time of Caffeine and Catechin standards (32 ug/mL)

Component

Ph. Eur.  Specification

Observed Value

% RSD

2%

<2%

Retention Time

1%

<1%

Tailing Factor

2

<2

Resolution, Reference Solution A

Minimum 1.5

>2

Table 5.System Suitability Criteria set in Ph. Eur and performance observed

Method Sensitivity and Linearity

Compound

STYX

Slope

LOD (µg/mL)

LOQ (µg/mL)

Correlation Coefficient

Gallocatechin

57107.43385

41551.7194

4.54

13.74

0.997

Epigallocatechin

54498.80895

43955.46957

4.09

12.4

0.998

Catechin

52338.64729

38664.96098

4.47

13.54

0.997

Caffeine

33376.35649

24990.34237

4.41

13.36

0.997

Epigallocatechin-3-O-gallate

36394.676

46555.82273

2.58

7.82

0.999

Epicatechin

38574.65905

38499.92522

3.31

10.02

0.998

Gallocatechin-3-O-gallate

47867.18528

47943.76143

3.29

9.98

0.998

Epicatechin-3-O-gallate

31694.71884

31686.72165

3.3

10

0.998

Table 6.Determined LOD, LOQ and linearity of method using 10 standard solutions spanning the concentration range 0.08 – 48 µg/mL for each compound.
Calibration curves plotted between intensity and concentration (µg/mL) obtained for caffeine, epigallocatechin-3-O-gallate (purple color), gallocatechin-3-O-gallate (pink color), epigallocatechin (blue color), gallocatechin (dark purple color), catechin (light green color), epicatechin (dark green color), epicatechin-3-O-gallate (dark blue color), and caffeine (yellow color) with equations of the form y=mx+c for each of the obtained lines.

Figure 7.Calibration curves for the catechins and caffeine.

% Recovery values of the Catechins and Caffeine spiked into decaffeinated green tea sample are displayed in Table 7.

No.

Spike Level

Added (µg/mL)

Found (µg/mL)

Recovery (%)

Gallocatechin

1

LOQ

14

13.6

97.6

2

50%

16

16.9

105.9

3

100%

32

31.2

97.6

4

150%

48

51.6

107.5

Epigallocatechin

1

LOQ

13

11.8

91.1

2

50%

16

16.1

100.6

3

100%

32

29.4

92

4

150%

48

47.6

99.1

Catechin

1

LOQ

14

13.5

96.5

2

50%

16

14.6

91.2

3

100%

32

34

106.4

4

150%

48

47.6

99.1

Caffeine

1

LOQ

14

13.8

98.3

2

50%

16

14.4

90.1

3

100%

32

30.2

94.3

4

150%

48

47.9

99.7

Epigallocatechin-3-O-Gallate

1

LOQ

8

7.7

96.4

2

50%

16

15.6

97.3

3

100%

32

30.6

95.5

4

150%

48

45.8

95.4

Epicatechin

1

LOQ

10

9.2

91.9

2

50%

16

14.5

90.6

3

100%

32

31.3

97.8

4

150%

48

51.8

107.9

Gallocatechin-3-O-Gallate

1

LOQ

10

10.2

101.5

2

50%

16

15.1

94.5

l3

100%

32

33.5

104.8

4

150%

48

45.9

95.7

Epicatechin-3-O-Gallate

1

LOQ

10

9.1

91.3

2

50%

16

16.3

102.1

3

100%

32

30.2

94.2

4

150%

48

50.5

105.1

Table 7.Recovery levels at different spike levels

CONCLUSION

A gradient reverse phase HPLC method for the green tea assay using the monolithic HPLC column following the Ph. Eur. Monograph 2668 (04/2018)has been modified and validated. It was shown that the Chromolith® High resolution column offers good resolution (>2) between each of the catechins present in decaffeinated green tea.

The assay method as per Ph. Eur. (edition 10) is simple, linear, and specific. The Limits of Detection (LOD) ranged from 2.58 to 4.54 µg/mL and the Limits of Quantification (LOQ) was from 7.82 to 13.74 µg/mL with good linearity up to 48 µg/mL for the determined catechins and caffeine.

The % RSD for system precision of the catechins and caffeine was <2%. The analyte recovery of the catechins and caffeine ranged from 90.1 to 107.9%.

The Chromolith® High Resolution RP-18e column is an ideal column for this application as it offers due to its bimodal pore structure the advantage of low back pressure and matrix robustness for a longer lifetime compared to the traditional porous particulate columns without sacrificing resolution, accuracy, and precision.

RELATED PRODUCTS
Loading

REFERENCES

1.
Ph. Eur. (edition 10) 2668 (04/2018) GREEN TEA Camelliae sinensis non fermentata folia. [Internet].
Sign In To Continue

To continue reading please sign in or create an account.

Don't Have An Account?