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.
Figure 1.Chemical structures of catechins and caffeine
EXPERIMENTAL CONDITIONS | |||||||||||||||||||||||||||||||||||||||||
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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: |
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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. |
Figure 2.Blank chromatogram.
Figure 3.Chromatogram of reference solution A.
Figure 4.Chromatogram of catechin and caffeine standard mixture. See Table 4 for retention times (determined with single solution standards).
Figure 5.Chromatogram of green tea sample 1
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 |
*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 |
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 |
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 |
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 |
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 |
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.
REFERENCES
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