Journal of Food Composition and Analysis 22 (2009) 137–141

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Journal of Food Composition and Analysis

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Original Article

A novel colorimetric determination of free amino acids content in tea

infusions with 2,4-dinitrofluorobenzene
Lin Chen a,b, Qi Chen a, Zhengzhu Zhang a, Xiaochun Wan a,*
a
Key Laboratory of Tea Biochemistry & Biotechnology, Ministry of Agriculture & Ministry of Education, Anhui Agricultural University,
West 130 Changjiang Road, Hefei, Anhui 230036, China
b
Tea research institute, Fujian Academy of Agricultural Sciences, Fu’an, Fujian 355015, China

A R T I C L E I N F O A B S T R A C T

Article history: A simple procedure is presented for the estimation of free amino acids content in tea infusions based on
Received 6 December 2007 the reaction of amino groups with 2,4-dinitrofluorobenzene (DNFB) and subsequent measurement of
Received in revised form 23 July 2008 the dinitrophenyl (DNP)-amino acids extracted from reaction mixtures into ethyl acetate at 420 nm.
Accepted 2 August 2008
The contents of free amino acids in various tea infusions determined by the proposed method are almost
consistent with an existing ninhydrin colorimetric method, but the derivatization reaction with DNFB is
Keywords: more sensitive, and the spectral curves of every resulting product will be completely overlapped in the
Tea infusions
range of 420–480 nm during a week of storage. The absorbance values of DNP-derivatives (Y) are linear
Amino acids
with the concentrations of amino acids (X) in the range of 0.02–1.00 mg/mL, and their linear regression
Colorimetric method
2,4-dinitrofluorobenzene equations are Y = 0.9920X + 0.0036 (R2 = 0.9998) for L-theanine and Y = 0.9403X0.0008 (R2 = 0.9995)
Ninhydrin for L-glutamic acid, respectively. A complete reaction system contains 1.0 mL of 0.2 mol/L sodium
Food composition bicarbonate solution, 1.0 mL of 1% DNFB (a 1.0-mL portion is dissolved in 100 mL of 1,4-dioxane) and
1.0 mL of each sample, incubated at 60 8C for 40 min in the dark. Under the described standard
conditions, the average recoveries of amino acids in infusions of black tea, green tea, Oolong tea, yellow
tea, white tea and dark tea are 99.194–101.250% with R.S.D. of 0.507–1.299% estimated by L-theanine
and 98.947–101.840% with R.S.D. of 0.500–1.271% estimated by L-glutamic acid.
ß 2009 Published by Elsevier Inc.

1. Introduction sasanqua (Tsushida and Takeo, 1984), theanine also demonstrated

Free amino acids are regarded as important taste components in
terms of tea quality, especially for green tea (Horie and Kohata,
2000). Although 26 varieties of amino acids could be found and
identified in tea plants (Wang, 1984), most of them in tea leaves
were theanine, glutamic acid, aspartic acid, serine, glutamine,
alanine, arginine, threonine (Takeo, 1980), and theanine accounted
for about 60–70% of total contents of free amino acids in spring
shoots (Takeo, 1981). Theanine is primarily responsible for umami (a
brothy or savory) taste of green tea, similar to that of sodium
glutamate (Juneja et al., 1999). Newly interests have been peaked at
its positive effects on immune system responsiveness to infection,
blood-pressure reduction, relaxation, neuroprotection, and mod-
ulation of chemotherapy (Eschenauer and Sweet, 2006). As a unique
amino acid identified only in the tea plants (Sakato, 1950), with the
exception of a kind of mushroom, Xerocomus badius (Casimir et al.,
1960), and certain species of genus Camellia, C. japonica and C.
* Corresponding author. Tel.: +86 5515786401; fax: +86 5515786765.
E-mail address: tealab@ahau.edu.cn (X. Wan).
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-hydroxysucci-
nimidyl carbamate (AQC) (Zhu et al., 2001), 9-fluoromethoxycar-
bonylglycine 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, thin-
layer chromatography, automatic amino acid analyzer, high-
performance liquid chromatography (HPLC) or capillary electro-
phoresis. However, when it came to measure total content of free
amino acids in tea extracts, the ninhydrin colorimetric method was
preferably adopted just as for determination of compounds
containing amino groups in food and pharmaceutical products
(Sun et al., 2006). Teas are usually categorized into six types, black
tea, green tea, Oolong tea, yellow tea, white tea and dark tea in China.

0889-1575/$ – see front matter ß 2009 Published by Elsevier Inc.

doi:10.1016/j.jfca.2008.08.007
138 L. Chen et al. / Journal of Food Composition and Analysis 22 (2009) 137–141
Because of predominant content of L-theanine in various tea in-
fusions, L-theanine and its precursor, L-glutamic acid were com-
monly used to prepare standard curves to estimate total free amino
acids content with this colorimetric determination (Liang et al.,
2003; Sasaoka et al., 1963).
Since the description by Sanger of the easy reactivity of 2,4-
dinitrofluorobenzene with free amino groups (Sanger, 1945), many
applications of this reagent have been reported. It has become a
routine tool in the analysis of the amino end-groups of proteins,
and of amino acids in protein hydrolysates (Dubin, 1960). Because
the fluorine and nitro groups withdraw electron density from the
benzene ring, a carbon atom in the ring undergoes nucleophilic
attack by the N-terminal nitrogen of an amine group, forming
compounds (dinitrophenyl (DNP)-derivatives) that will strongly
absorb light in the UV and/or visible spectrum. As a commercial
product, the appearance of DNFB is colorless liquid or light yellow
crystal. Absorption spectrum of DNFB will change in solutions with
different pH conditions, but not to its derivatives of amino acids,
and these phenomena have not been reported yet. Moreover, DNP-
amino acids can be completely extracted into ethyl acetate from
acidified mixtures of water and 1,4-dioxane. Thus, compared
with the existing colorimetric determination based on ninhydrin
reaction, a novel analytical procedure was developed to estimate
free amino acids content in tea infusions after derivatization with
DNFB in this paper.
2. Materials and methods
2.1. Samples and chemicals
Black tea (Qimen Gongfu), green tea (Yuexi Cuilan), Oolong tea
(Fujian Tieguanyin), yellow tea (Huoshan Huangdacha), white tea
(King of Mudan) and dark tea (Guangxi Liubaocha) were collected
from their original regions. L-Theanine with purity of 99.3% was
obtained from Jintan Qianyao Pharmaceutical Material Factory
(Jiangsu province, China). L-Glutamic acid was a biochemical
reagent with purity of 98.5%. DNFB was a product of Tokyo
Chemical Industry Co. (Japan). Trifluoroacetic and acetonitrile
were of HPLC grade purchased from Tedia Company (USA).
Ninhydrin and other chemicals used in this experiment were of
analytical grade obtained in China. Unless otherwise specified, all
reagents were prepared with demineralized water purified by Pall
PL5243 PURELAB Classic water purification system (USA).
2.2. Preparation of tea infusions
All tea samples were ground into minute particles by an
analytical mill (IKA All Basic, German). Each of them (0.300 g) was
placed into a 50-mL tube to which added with 40 mL of water. The
tubes covered with stoppers were heated on a water bath (100 8C)
for 60 min, and the cooled samples were adjusted to 40 mL with
water. Filtered through two layers of Double-ring no.102 filter
paper (Xinhua Paper Industry Co. Ltd., Hangzhou, China) by
reduced pressure, these tea infusions were used for measuring the
contents of free amino acids and L-theanine.
2.3. Determination of free amino acids by DNFB derivatization
In a 7-mL plastic centrifugation tube, 1.0 mL of each tea
infusions was added (to avoid disturbance from components
soluble in ethyl acetate, all tea infusions were pre-extracted
with five volumes of ethyl acetate before analysis.). This was
followed by addition of 1.0 mL of 0.2 mol/L sodium bicarbonate
solution and 1.0 mL of 1% DNFB (a 1.0 mL portion was dissolved
in 100 mL of 1,4-dioxane). The probes in the sealed tubes were
then placed on a water bath (60 8C) and left to stand for 40 min
in the dark. All derivative reactions were stopped by addition of
0.5 mL of 1 mol/L HCl. The resulting DNP derivatives (0.75 mL)
were extracted with 5 mL ethyl acetate. After they were stand
for 10 min, the UV spectrum characters of upper phases were
analyzed with ethyl acetate as reference (HITACHI U-3010
Spectrophotometer coupled with UV Solution 2.0 workstation,
10 mm light path length.).
2.4. Determination of free amino acids by ninhydrin reaction
In a 25-mL volumetric flask, 1.0 mL of each tea infusions was
added. This was followed by addition of 0.5 mL of 1/15 mol/L
phosphate buffer solution (pH 8.04) and 0.5 mL of 2% ninhydrin
solution containing 0.8 mg/mL of SnCl22H2O. The mixtures in the
volumetric flasks were then placed on a boiling water bath for
15 min. The probes were quickly cooled with cold water, and
adjusted to 25 mL with water. After they were left to stand still for
10 min, the absorbance values of these blue-purple products were
measured against a reagent blank (tea infusions replaced by the
equal volume of water was carried through the procedure). The
other details referred to GB/T 8314-2002, a national standard
method used to determine free amino acids content of tea
infusions in China.
2.5. Determination of L-theanine content by HPLC
Contents of L-theanine in the various tea infusions were
determined with SHIMADZU Prominence HPLC consisting of LC-
20AD pump, DGU-20A5 degasser, and SPD-M20A diode array
detector (200 nm), operated with LC solution workstation. The
chromatographic analysis was performed on a C18 reverse phase
column (Spherigel, 250 mm  4.6 mm id, particle size 5 mm) at
25 8C. The mobile phase consisted of 0.05% trifluoroacetic acid–
water (v/v) (A) and 50% acetonitrile–water (v/v) (B). 5 ml of each
tea infusions was manually injected, followed by filtration
through a 0.45-mm filter. Solvent A had been maintained at
flow rate of 1.0 mL/min for 15 min, then the column was eluted
with solvent B for 20 min before it was equilibrated 10 min
for another analysis. The total transition time between A and B
was 10 min.
3. Results and discussion
3.1. Spectral characters of DNFB and DNP-amino acids
The color of DNFB appears yellow-green in alkaline solution,
and colorless in neutral or acidic solution. These phenomena are
consistent with its changes of spectral absorbance in solutions
with different pH conditions (Fig. 1). Yellow-green compounds
could be formed when DNFB was reacted with L-theanine and L-
glutamic acid, and the color of DNP derivatives would not fade in
neutral or acidic solution. To get rid of disturbance of DNFB for
analysis of amines, excess DNFB was recommended to be
hydrolyzed by strong alkali, and that the dinitrophenylamine
could be extracted from the alkaline aqueous reaction mixture
into an immiscible solvent such as cyclohexane for spectro-
photometric determination (Kolbezen et al., 1962). Because of
the existence of 1,4-dioxane, which could be well mixed with
DNFB and water, this procedure was not suitable for our
experiment to extract DNP-amino acids into cyclohexane. On
the contrary, 0.5 mL of 1 mol/L HCl was added into a reaction
mixture, thereafter DNFB and the derivatives of amino acids
could be both extracted into ethyl acetate. Because the mixture
became an acidified solution after addition of HCl, no background
 L. Chen et al. / Journal of Food Composition and Analysis 22 (2009) 137–141 139

Table 1
Orthogonal experimental scheme for derivatization reaction and the absorbance of
resulting DNP-amino acids.

Treatment NaHCO3 Temperature Time Absorbance of DNP-amino

(mol/L) (8C) (min) acids (l = 420 nm)

1 0.0 40 20 Not detecteda Not detectedb

2 0.0 50 30 Not detecteda Not detectedb
3 0.0 60 40 Not detecteda Not detectedb
4 0.0 70 50 Not detecteda Not detectedb
5 0.1 40 30 0.966a 0.640b
6 0.1 50 20 0.993a 0.767b
7 0.1 60 50 0.980a 0.833b
8 0.1 70 40 0.978a 0.765b
9 0.2 40 40 0.986a 0.947b
10 0.2 50 50 0.981a 0.926b
11 0.2 60 20 0.999a 0.929b
12 0.2 70 30 0.995a 0.857b
Fig. 1. Spectral absorbance curves of DNFB. (a) 1.0 mL 0.2 mol/L NaHCO3 + 0.1 mL 13 0.4 40 50 0.965a 0.945b
1% DNFB + 0.9 mL H2O; (b) 1.9 mL H2O + 0.1 mL 1% DNFB; (c) 1.0 mL 0.2 mol/L 14 0.4 50 40 1.010a 0.942b
HCl + 0.1 mL 1% DNFB + 0.9 mL H2O and (d) 1.0 mL 0.2 mol/L HCl + 0.1 mL 1,4- 15 0.4 60 30 1.023a 0.953b
dioxane + 0.9 mL H2O (reference). 16 0.4 70 20 0.989a 0.909b
a
DNP-theanine.
b
DNP-glutamic acid.

Fig. 2. Spectral absorbance curves of DNP-amino acids. (a) DNFB & derivatives of L-
theanine extracted by ethyl acetate; (b) DNFB & derivatives of L-glutamic acid
extracted by ethyl acetate; (c) DNFB extracted by ethyl acetate and (d) ethyl acetate
(reference).
effects from DNFB existed in the extractions of ethyl acetate
when the absorbance values of DNP-theanine and DNP-glutamic
acid were measured over 420 nm (Fig. 2).
3.2. Optimization of conditions for derivatization reaction
Both amino acids and amines could be derivatized by DNFB in
weak basic solutions, but the need for time/temperature control
would vary according to the characters of compounds. In a typical
experiment reported by Lockhart to derivatize apliphatic amines,
DNFB (0.15 mL) in 0.2 mL of ethanol, equal volume of 0.2 mol/L
sodium bicarbonate solution was mixed with 0.1 mL amines
containing 10 mg of the amine or its hydrochloride. After standing
for 15 min at room temperature, the solution was placed at 105 8C
in a sealed tube for 2 h (Lockhart, 1956). Moreover, procedures
presented by Friedman et al. to analyze theanine content of tea
leaves, 0.1 mL of DNFB and 2 mL of 1% sodium bicarbonate solution
were added to the residues containing theanine, and the mixtures
were allowed to stand in the dark for 3 h at 40 8C (Friedman et al.,
2007). These time-consuming methods with no definite tempera-
ture control made us design an orthogonal experimental scheme
with three factors and four levels to investigate the effects of
concentrations of sodium bicarbonate solution, temperature and
time for reactions on the absorbance values of DNP-amino acids in
extractions of ethyl acetate (Table 1).
A variance analysis was carried out and its result showed that
concentration of sodium bicarbonate was of notable effects on
absorbance values of DNP-amino acids in ethyl acetate at 420 nm
(P < 0.01), but not the other two factors. It could be seen that DNP-
amino acids in extractions of ethyl acetate would not be detected
from treatment 1 to treatment 4 when equal volume of water was
added into the reaction system instead of sodium bicarbonate
solution. In addition, the absorbance values of extractions tended
to be increased by adding higher concentrations of sodium
bicarbonate solutions. Although the average absorbance values of
DNP-theanine (0.997 mg/mL) and DNP-glutamic acid (0.937 mg/
mL) were obtained with 1 mL of 0.4 mol/L sodium bicarbonate
solution added into reaction systems, background effects from
DNFB in ethyl acetate could be also found with the absorbance
values less than 0.012 at 420 nm. Therefore, the 11th treatment
would be the optimum conditions for derivatization reaction, by
which the highest absorbance values of DNP-theanine (0.999 mg/
mL) and DNP-glutamic acid (0.929 mg/mL) could be measured
with no interference from DNFB. Given the possible effects from
reaction time on the derivatization of the other amino acids in tea
infusions, the treatment by incubating reaction systems at 60 8C
for 40 min with addition of 1 mL of 0.2 mol/L sodium bicarbonate
solution described in the previous method was applied in the
latter analyses.
3.3. Comparison of total free amino acids content estimated by DNFB
method and ninhydrin method
L-theanine and L-glutamic acid standard solutions were
prepared over seven different concentrations, which were
0.02 mg/mL, 0.10 mg/mL, 0.20 mg/mL, 0.40 mg/mL, 0.60 mg/mL,
0.80 mg/mL and 1.00 mg/mL. The studies indicated that gradient
changes of absorbance values of DNP-amino acids could be found
between 420 nm and 480 nm, and the absorbance values of DNP-
derivatives were linear with the concentrations of amino acids in
the range of 0.02–1.00 mg/mL at 420 nm (Figs. 3 and 4).
With these different concentrations of amino acids, the
standard calibration curves for ninhydrin method and HPLC (L-
theanine) were also established according to corresponding
manipulations described in Sections 2.4 and 2.5. HPLC analysis
was of the same linear range with the peak area (Y0 ) against the
concentration of L-theanine (X0 ) at 200 nm as derivative reaction,
but the color reaction with ninhydrin was in the range of 0.10–
140 L. Chen et al. / Journal of Food Composition and Analysis 22 (2009) 137–141
Fig. 3. Spectral absorbance curves of derivatives with different concentrations of L-
theanine (mg/mL).
Fig. 4. Spectral absorbance curves of derivatives with different concentrations of L-
glutamic acid (mg/mL).
1.00 mg/mL at 567 nm (maximum absorbance values could be
reached at this wavelength) (Figs. 5 and 6).
The estimated contents of free amino acids and contents of L-
theanine in various tea infusions were listed in Table 2. It was
shown that no significant differences between two methods to
predict the free amino acids content with L-theanine or L-
glutamic acid, except that some higher content could be found in
black tea, and free amino acids could be also detected in dark
tea with DNFB method. Although the correlation coefficient
Fig. 5. Spectrum of compounds formed by ninhydrin reaction with different
concentrations of L-theanine (mg/mL).
Fig. 6. Spectrum of compounds formed by ninhydrin reaction with different
concentrations of L-glutamic acid (mg/mL).
between L-theanine content in each tea infusions determined by
HPLC and the free amino acids content estimated with L-theanine
reached 0.953 by DNFB method and 0.971 by ninhydrin method,
the mean contents of L-theanine only accounted for 0.069 mg/mL.
Therefore, it could be concluded that most of yellow-green
derivatives (DNFB method) or blue-purple compounds (ninhy-
drin method) were produced from the other amino acids.
Theoretically, other compounds with amine groups in tea
infusions would contribute to higher estimation of free amino
acids content by ninhydrin reaction, but it would be less
interference for DNFB method, because DNP-amino acids were
determined by extraction into ethyl acetate, and derivatives of
short peptides, soluble proteins and their complexes were left in
the lower phase with 1,4-dioxane. According to these similarities
and differences between two methods, it would be more
sensitive and accurate for estimation of free amino acids content
in tea infusions by the derivative method.
3.4. Validation of DNFB method
A series of sample analyses were performed to validate the
performance of the DNFB method. Precision was evaluated from
replicate determinations (n = 5) performed on the same day for
each tea infusions. The mean contents of free amino acids in
various tea infusions were shown in Table 3. The known quantities
of L-theanine or L-glutamic acid were added to various tea
infusions, and the recoveries were also calculated by comparing
the found amount of standards to those of added. The results
indicated that relative standard deviation (R.S.D.) of free amino
acids content in various tea infusions were from 0.507% to 1.299%
estimated by L-theanine and from 0.500% to 1.271% estimated by L-
glutamic acid. Average recoveries of tea infusions added with
standards were from 99.194% to 101.250% for L-theanine and from
98.947% to 101.840% for L-glutamic acid. As a routine method to
analyze free amino acids content in tea infusions, reproducible
results would be obtained by the described ninhydrin method, but
the colorimetric determination should be finished not more than
2 h because of the instability of blue-purple products. However, a
continuous study indicated that the spectral curves of DNP-amino
acids (standards or free amino acids in various tea infusions)
extracted into ethyl acetate stored at dark place with room
temperature could be completely overlapped in the range of 420–
480 nm with their corresponding primitive curves in a week.
Therefore, free amino acids content in large numbers of tea
samples would rather be determined by DNFB method for its good
reproducibility and stability.
 L. Chen et al. / Journal of Food Composition and Analysis 22 (2009) 137–141 141

Table 2
Content of free amino acids and L-theanine in various tea infusions (mg/mL).

Analyte Black tea Green tea Oolong tea Yellow tea White tea Dark tea The linear regression equation
a a a a a a
Free amino acids 0.355 0.311 0.184 0.141 0.572 0.138 Y = 0.9920X + 0.0036 (R2 = 0.9998)a
(DNFB method) 0.379b 0.333b 0.199b 0.153b 0.608b 0.150b Y = 0.9403X0.0008 (R2 = 0.9995)b
Free amino acids 0.311a 0.330a 0.199a 0.132a 0.563a Not detecteda,b Y = 3.0286X0.2688 (R2 = 0.9973)a
(Ninhydrin method) 0.291b 0.309b 0.186b 0.123b 0.527b Y = 3.2278X0.2664 (R2 = 0.9980)b
L-theanine 0.074 0.098 0.060 0.007 0.171 0.005 Y0 =6E + 06X0 +13055 (R2 = 0.999)
a
Contents of free amino acids estimated by L-theanine.
b
Contents of free amino acids estimated by L-glutamic acid.

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