Determination of Theanine in Commercial Tea by Liquid Chromatography with Fluorescence and Diode Array Ultraviolet Detection

Abstract

Two liquid chromatographic methods that involve precolumn derivatization with o-phthaladehyde (OPA) and phenylisothiocyanate (PITC) with fluorescence and diode array UV detection for the determination of theanine have been developed. The chromatographic separations were achieved by reverse-phase high-performance liquid chromatography using octadecyl columns and gradient elution. The methods were applied to evaluate the theanine content of commercial tea leaves. The coefficient of variation of the peak area repeatability for within day (n = 8) and between day (n = 8 over 10 days) was lower than 3% for both of the methods. The estimated limit of detection (LOD) and limit of quantitation (LOQ) for the OPA method was 0.12 and 0.35 microg theanine, respectively. The PITC method was 500-fold more sensitive with LOD and LOQ values of 0.25 and 0.75 ng, respectively. The theanine content of the commercial tea samples varied from 2-5 mg/g leaf. The overall % recoveries for these methods ranged from 93-99.3. The sensitivity and simplicity of the method render them suitable for use in quality control laboratories.

Pentachlorophenol removal by Rhizopus oryzaeCDBB‐H‐1877 using sorption and degradation mechanisms

“…Samples were centrifuged for 10 min at 12 000 × g at 4 °C, and diluted 20 times in 0.4 mol L −1 borate buffer, pH 10.5. Five microliters of diluted samples was mixed with 52.2 µL of 0.4 mol L −1 borate buffer, pH 10.5, and 2.5 µL of o ‐phthalaldehyde, the derivatization reagent . Reaction was maintained at 27 °C during 2 min at 600 rpm and samples were injected into HPLC using a Nova‐Pak Waters C 18 reverse‐phase column and detected in a diode array detector at 335 nm.…”

“…The A mobile phase was 0.14 mol L −1 sodium acetate containing 0.05% triethylamine adjusted to pH 6.8 with acetic acid–methanol (90:10), the B mobile phase was acetonitrile–water (60:40) as reported by Thippeswamy et al . ; elution was performed at a flow rate of 0.5 mL min −1 .…”

Rapid and selective quantification of l-theanine in ready-to-drink teas from Chinese market using SPE and UPLC-UV

“…Many studies have reported the contents of L-theanine in different teas (Alcázar et al, 2007;Chen et al, 2003;Feldheim, Yongvanit, & Cummings, 1986;Liang et al, 2006;Keenan et al, 2011;Peng Syu et al, 2008;Thippeswamy et al, 2006;Wang et al, 2010;Ying et al, 2005). However, there are no literature reports for the L-theanine level in RTD teas.…”

“…These include capillary electrophoretic (Aucamp, Hara, & Apostolides, 2000;Chen et al, 2003;Horie, Mukai, & Kohata, 1997;Kvasnička & Krátká, 2006) and chromatographic methods. L-theanine can be analysed simultaneously with amino acids by high performance liquid chromatographic (HPLC) methods involving pre-column derivatization with o-phthaladehyde, phenylisothiocyanate or dabsyl chloride, and fluorescence and diode array UV detection (Alcázar et al, 2007;Liang, Ma, Lu, & Wu, 2006;Syu, Lin, Huang, & Lin, 2008;Thippeswamy et al, 2006;Van der Pijl, Chen, & Mulder, 2010;Ying, Ho, Chen, & Wang, 2005). Other HPLC methods employing different columns and detectors were reported for the quantitative and qualitative analysis of L-theanine in different teas without derivatization (Bedner, Sander, & Sharpless, 2010;Desai & Armstrong, 2004;Ding, Yu, & Mou, 2002;Keenan, Finnie, Jones, Rogers, & Priestley, 2011;Peng, Song, Shi, Li, & Ye, 2008;Wang et al, 2010;Zhu et al, 2004).…”

A novel colorimetric determination of free amino acids content in tea infusions with 2,4-dinitrofluorobenzene

“…sasanqua (Tsushida and Takeo, 1984), theanine also demonstrated the same chemical characters with most amino acids, such as color reaction with ninhydrin (Li and Hu, 1989;Nakagawa and Anan, 1979;Selvendran and Selvendran, 1973), derivatization by 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl-Cl) (Otsuki et al, 1979), o-phthalaldehyde (OPA) (Goto et al, 1993;Tsushida and Takeo, 1983;Ying et al, 2005), 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) (Zhu et al, 2001), 9-fluoromethoxycarbonylglycine chloride (FMOC-Cl) (Desai and Armstrong, 2004), phenylisothiocyanate (PITC) (Thippeswamy et al, 2006), 2,4-dinitrofluorobenzene (DNFB) (Friedman et al, 2007), etc. These properties have been applied to analyze the compositions of free amino acids and theanine content in tea products, often combined with isolation and quantification by paper chromatography, thinlayer chromatography, automatic amino acid analyzer, highperformance liquid chromatography (HPLC) or capillary electrophoresis.…”