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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

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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

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.

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

Table 3.Reproducibility of area counts (mAU) of catechin and caffeine standard (32 µg/mL each compound)
Table 4.Retention Time of Caffeine and Catechin standards (32 ug/mL)
Table 5.System Suitability Criteria set in Ph. Eur and performance observed

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.
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.

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.

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REFERENCES

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