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

Dr. Ajay Kaparwan
Mumbai Application Laboratory, India

• Introduction
• Experimental Conditions
• Results
• Conclusion

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

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

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.

Table 2. Results for 2 green tea samples

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

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

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

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

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

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.

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.

Table 7. Recovery levels at different spike levels

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

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