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Monitor Anitoxidants in Tea Extract

Using an Ultra Aqueous C18 HPLC Column and Unique TOFMS


by Julie Kowalski, Ph.D., Innovations Chemist
  • Use highly aqueous mobile phases without collapsing the stationary phase.
  • Extract data for specific compounds and manually inspect spectra for other compounds.
  • Simple sample preparation.

Much focus has been given to the health benefits of foods and beverages that contain antioxidant compounds. By reacting with free radical-forming compounds before they can cause cell damage, antioxidants protect the body against oxidative stress.[1] Some foods and beverages naturally contain antioxidants, but supplementing foodstuffs has been on the rise due to demands by health conscious consumers. Recently, green tea has been successfully promoted as a health drink because it contains antioxidant phenolic compounds.

Using LC/TOFMS, we show a straightforward method for determining the presence of antioxidant compounds in commercial tea formulations. Samples were prepared by adding approximately 15g of dry tea product to 200mL of methanol which was cooled to approximately 20°C. The mixture was stirred for 5 minutes and decanted. The tea product was rinsed with an additional 20mL of cooled methanol. The 200mL and 20mL solutions were combined, then filtered through a 0.45µm syringe filter to capture particles. The filtered solution was used directly for analysis.

We used a 150 x 2.1mm Ultra Aqueous C18 HPLC column for the analysis and, because a tea extract is a complex matrix, we used a gradient elution and mobile phases with a high water content. The Ultra Aqueous C18 stationary phase is ideal for such an application: the phase is specifically designed to prevent collapse of the C18 alkyl chains in highly aqueous mobile phases.[2]

The Ultra Aqueous C18 phase proved ideal for resolving the complex tea matrix, as shown by the large number of peaks in Figure 1. The resolving power of this chromatographic system, in combination with the LECO Unique TOF Mass Spectrometer, allow the analyst to both extract data for specific compounds of interest and manually inspect spectra for other compounds, including phenolic glycosides and esters of phenolic acid.

If you are analyzing antioxidants in tea, or other complex mixtures of compounds, an Ultra Aqueous C18 column gives you the reliable results you need, without restricting your ability to use the mobile phase composition that works best for your application.

Table I  Phenolic compounds of interest.

Compound
[M-H]-
gallocatechin-3-gallate
catechin
epigallocatechin-3-methyl gallate
epicatechin di-gallate
epicatechin-3-gallate
catechin gallate
457.206
289.154
471.208
609.318
441.208
Note: m/z 441.2 can be either epicatechin-3-gallate or catechin gallate.


Figure 1  A complex mix of tea extract components is best separated on an Ultra Aqueous C18 column with a highly aqueous mobile phase (total ion chromatograms of Table 1 compounds).


LC_FF0425

For conditions see Figure 3.


Figure 2  (-)-Epicatechin produced by infusion of a standard (A) and spectrum of (-)-epicatechin created from an extracted ion chromatogram (B).


LC_FF0425A & LC_FF0425B

For conditions see Figure 3.


Figure 3  Spectra of phenolic compounds identified in the tea extract.


A)  quercitin dicoumaryl glycoside


LC_FF0425G


B)  epigallocatechin 3-methyl gallate


LC_FF0425F


C)  caffeic acid ester


LC_FF0425E


D)  gallocatechin-3-gallate


LC_FF0425C


E)  catechin


LC_FF0425D


Sample:

Inj.:

10µL

Conc.:

15g tea extracted w/ 200mL + 20mL methanol

Sample diluent:

methanol

Column:

Ultra Aqueous C18

Cat.#:

9178562

Dimensions:

150 x 2.1 mm

Particle size:

5µm

Pore size:

100Å

Conditions:

Mobile phase:

A: 0.15% formic acid in water;
B: 0.15% formic acid in acetonitrile (v/v)

Time (min.)

%B

0
60

5
9.5

Flow:

0.3mL/min.

Temp.:

ambient

Det.:

Leco Unique LC-TOFMS

ESI voltage:

-3500 V

Desolvation temp.:

300°C

Nebulizer pressure:

375 kPa

Desolvation gas:

7000 cc/min.

Interface temp.:

100°C

Nozzle:

-80 V

Data acquisition:

3.13 spectra/sec.



For information about the LECO Unique TOFMS, please visit the LECO website: www.leco.com

References

[1] Free radical damage is implemented in many disease models, including cancer, in many degenerative illnesses, and in the aging process.
[2] When the long, hydrophobic alkyl chain of a conventional C18 stationary phase is exposed to a highly aqueous mobile phase it folds down on itself, causing loss of retention. A prolonged equilibration time in a high organic solution is needed to restore the phase. The Ultra Aqueous C18 stationary phase is not susceptible to phase collapse — not even in mobile phases with very highly aqueous content.

RELATED SEARCHES

phenolic glycosides

,

esters of phenolic acid

,

LECO Unique TOF Mass Spectrometer

,

Ultra Aqueous C18