Application
News
No. L543A
First Edition: Jul. 2019
Second Edition: Oct. 2019
For Research Use Only. Not for use in diagnostic procedures.
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Reproducibility
Table 3 shows the relative standard deviation (%RSD) for
retention time and area in analyses repeated six times using
1 mg/L of the standard solution, which is the lowest
concentration of the calibration curve. In both analyses,
relative standard deviation of 1% or less were obtained for
both retention time and area.
Relative Standard Deviation in Analyses Repeated
Six Times (%RSD)
Analytical condition Column
Retention
time
Area
JAS method-compliant
Shim-pack GIST C18
(particle size: 5 μm)
0.05 0.79
High-speed analysis
Shim-pack GIST-HP C18
(particle size: 3 μm)
0.02 0.53
Analysis of Benifuuki Green Tea
The test specimen was a commercially-available Benifuuki
green tea. Pretreatment was carried out referring to the JAS
method.
(2),(3)
Fig. 3 shows the pretreatment protocol. Extraction from the
powdered specimen was performed using a mixed solution
of phosphoric acid and ethanol at 30 °C. Next, extracts were
filtrated with a filter paper and a 0.2 μm membrane filter, and
diluted 10 times with water prior to HPLC injection.
Fig. 4 shows the chromatograms of the Benifuuki green tea.
The upper part of Fig. 4 shows the chromatogram using a
Shim-pack GIST C18 analytical column (particle size: 5 μm)
according to the JAS method, and the bottom portion shows
that using a Shim-pack GIST-HP C18 column (particle size:
3 μm) for high-speed analysis in the same series. Methylated
catechin was separated from the other compounds in both
analyses.
The contents of methylated catechin were calculated from
the quantitative values. As a result, the specimen contained
12 g/kg of methylated catechin in both the JAS method-
compliant analysis and the high-speed analysis.
Pretreatment Protocol
Chromatograms of Benifuuki Green Tea
Upper: JAS Method-Compliant Analysis,
Bottom: High-Speed Analysis
Conclusion
Analyses of methylated catechin in Benifuuki green tea were
carried out using the Nexera Series and Shim-pack GIST C18
columns. Using a high-speed analytical column in the same
series, it was possible to reduce analysis time and mobile
phase consumption to approximately 1/4 respectively while
maintaining the same separation performance.
In both of the analyses described above, the Benifuuki green
tea used in this experiment was found to contain 12 g/kg of
methylated catechin.
[References]
(1) Mari Maeda-Yamamoto, Mitsuaki Sano, Nahomi Matsuda, Toshio
Miyase, Keiko Kawamoto, Naoko Suzuki, Masayasu Yoshimura,
Hirofumi Tachibana and Katsuhiro Hakamata, The Change of
Epigallocatechin-3-O-(3-O-methyl) gallate Content in Tea of
Different Varieties, Tea Seasons of Crop and Processing Method,
Nippon Shokuhin Kagaku Kogaku Kaishi (published by Japanese
Society for Food Science and Technology), 48, 64-68 (2001).
(2) Japanese Agricultural Standard, Determination of the O-
methylated Catechin in ‘Benifuuki’ Green Tea (Camellia sinensis L.)
- High-performance liquid chromatographic method (JAS 0002),
Ministry of Agriculture, Forestry and Fisheries (March 29, 2018).
(3) Yuji Homura, Yusuke Hiejima, Takashi Kodama, Masumi Tanaka,
Hideki Horie, Tadanao Suzuki and Akemi Yasui, Validation of
Method for Determining O-methylated Catechin in ‘Benifuuki’
Green Tea (Camellia sinensis L.) by Interlaboratory Study, Nippon
Shokuhin Kagaku Kogaku Kaishi, 63(7), 312-318 (2016).
Nexera and Shim-pack are trademarks of Shimadzu Corporation in Japan and/or other countries.
Weigh 250 mg of Benifuuki green tea (homogenized) in 25 mL of volumetric flask
Add 20 mL of phosphoric acid/ ethanol/ water (1:50:49), then stir
Stand in a water bath (60 min, 30 ˚C)
Allow to stand to R.T.
Add water to make 25 mL
Swing roll mixing
Filtrate with filter paper (discard initial flow)
Filtrate with membrane filter (discard initial flow)
Dilute ten times with water
HPLC
■Peak
1. EGCG3”Me
1
0.0 0.5 1.0 1.5 2.0 2.5 3.0 min
0
10
20
30
40
50
60
70
80
90
100
110
120
130
mAU
■Peak
1. EGCG3”Me
1
0.0 2.5 5.0 7.5 10.0 min
0
25
50
75
100
125
150
175
mAU
