Chapter 57

Development of HPLC Method

for Determination of the Content
of Tyramine in Rice Wine

Xuewu Guo, Lina Li, Yefu Chen and Dongguang Xiao

Abstract The method of detecting the contents of tyramine in rice wine by HPLC
was developed. The chromatographic separation was performed in an analytical
column, C18 (4.6 9 150 mm ID, 5 lm) and a UV detector was used with
wavelengths at 278 nm. The mobile phase was 0.02 mol/L disodium hydrogen-
acetonitrile (v: v 85:15), 10 % phosphoric acid adjusted pH = 8.4, with a flow rate
was 0.8 ml/min, the column temperature was 35 °C. The results showed that the
correlation coefficients were greater than 99.9 %, the limit of detection was
0.7 mg/L, the repeatability (RSD = 1.1 %), reproducibility (RSD = 2.91 %), and
accuracy were of satisfying quality. The recovery was more than 97 %. This
method has been successfully applied for the content of tyramine in rice wine.

Keywords Tyramine
HPLC
Rice wine

57.1 Introduction

Biogenic amines are low molecular weight and formed by the descarboxylation
reaction from corresponding free amino acids by microbial activity in many foods
and especially in fermented food products (Fig. 57.1) [1, 2].
Tyramine, 4-(2-aminoethyl)phenol, is one of the hazards and highest content in
biogenic amines [3]. Trace amounts of Tyramine do not usually represent any
health hazard to individuals. For many drugs synthesis, Tyramine is the necessary
initial substance [4], and is also one of the important components of active cells
and has obvious dilation and shrinkage effect to muscle and vascular. Large

X. Guo
L. Li
Y. Chen
D. Xiao (&)

Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education Tianjin
Industrial Microbiology Key Lab, College of Biotechnology, Tianjin University of Science
and Technology, Tianjin 300457, People’s Republic of China
e-mail: xiao99@tust.edu.cn

T.-C. Zhang et al. (eds.), Proceedings of the 2012 International Conference on Applied 557
Biotechnology (ICAB 2012), Lecture Notes in Electrical Engineering 249,
DOI: 10.1007/978-3-642-37916-1_57, Ó Springer-Verlag Berlin Heidelberg 2014
558 X. Guo et al.

Fig. 57.1 The main

production way of biogenic
amines in fermented food.
The catalyst of this reaction is
tyrosine decarboxylation
enzymes

amounts of tyramine can make the person elevated blood pressure and even
poisoning [5–8].
Many methods have been developed for the determination of biogenic amines in
fermented foods, for example, gas chromatography, capillary electrophoretic
method (CE), thin-layer chromatography (TLC), and high performance liquid
chromatography (HPLC) [9]. At present, HPLC method is the most widely used and
convenient method. But, most of the biogenic amines need be derived by HPLC for
lacking fluorescence or ultraviolet absorption [10–13]. Angela used dansyl chloride
as derived reagent before chromatographic detection by HPLC to determine the
content of eight biogenic amines in Chilean young varietal wines [14]. Mardiana
developed a method to determine of the biogenic amines (tryptamine, putrescine,
cadaverine, histamine, tyramine, spermidine) in food samples using dansyl chloride
as derived reagent by HPLC [15]. These methods have high cost and time-con-
suming, and the repeatability and reproducibility are not satisfying.
In this paper, a rapid and accurate method of determination without derivative
action of tyramine content in rice wine was developed. The method was based on
the benzene structure with the ultraviolet absorption characteristics in tyramine,
and it was necessary to ensure the quality of rice wine.

57.2 Materials and Methods

57.2.1 Materials

Tyramine, HPLC grade, purity is greater than 99.9 %, was purchased from Sigma
(St. Louis, MO, USA). Acetonitrile, HPLC grade, was obtained from KaiTong
(Tianjin, China). Phosphoric acid, 85 %, analytically pure (Tianjin, China),
Sodium phosphate dibasic dodecahydrte, analytically pure, (Tianjin, China);
Distilled water.

57.2.2 Solution Preparation

Tyramine standard fluid (mother solution): take 10 mg tyramine (HPLC

grade, purity is greater than 99.9 %) with the capacity of mobile phase to 10 mL
57 Development of HPLC Method 559

as 1 mg/mL tyramine standard fluid; The mobile phase: 0.02 mol/L disodium
hydrogen-acetonitrile (v: v 85:15), 10 % phosphoric acid adjusted pH = 8.4; 10 %
Phosphoric acid: take 85 % Phosphoric acid 10 mL to the capacity of 85 mL with
distilled water; 0.02 mol/L disodium hydrogen: take sodium phosphate dibasic
dodecahydrte 0.7160 g and add water to dissolve to 100 mL; rice wine fermented
liquid, centrifugal, 0.2 lm filter.

57.2.3 Instruments and Equipment

High performance liquid chromatography, Agilent, Germany; Ultraviolet detector,

Agilent, Germany; One over ten thousand balance, Mettler Toledo (Shanghai,
China); Numerical control ultrasonic cleaning device, Hengao, Tianjin, China;
Diaphragm vacuum pump, Tianjin, China; Centrifugal machine, Eppendorf,
Germany; pH meter, Mettler Toledo, Shanghai, China.

57.2.4 Chromatographic Condition

Chromatographic column: C18 (4.6 9 150 mm ID, 5 lm); column temperature:

35 °C; flow rate: 0.8 ml/min; UV detector: 278 nm.

57.2.5 The Preparation of the Standard Solution

Take different volumes of the mother liquor tyramine (1000 ll, 900 ll, 800 ll,
600 ll, 500 ll, 300 ll, 200 ll, 100 ll), add mobile phase to the capacity of 1 mL
to get tyramine standard fluids with different density.

57.3 Results and Discussion

57.3.1 HPLC Chromatogram of Tyramine Standard Fluid

Under the condition of column temperature: 35 °C; flow rate: 0.8 ml/min; UV
detector: 278 nm, the retention time of tyramine was 4.445 min. This result proves
tyramine can be directly tested without derivative reaction (Fig. 57.2).

57.3.2 The Establish of Standard Curve

The standard curve was constructed using tyramine standard solutions. The cor-
responding regression equation and other characteristic parameters are shown in
560 X. Guo et al.

Fig. 57.2 HPLC chromatograms of Tyramine standard fluid

Compound #1, VWD1 A

Area = 11095.4497*Amt +106.59199
Area Rel. Res%(8): 4.789

1
10000
2
8000
5
6000

4000 6
7
2000 8

0 Correlation: 0.99927

0 0.5 Amount[mg/ml]

Fig. 57.3 The standard curve of Tyramine standard solutions

Fig. 57.3. LOD [16], defined as three times the basis of signal-to-noise ratio, for
tyramine was 0.7 mg/L with coefficients of determination greater than 0.999.
The results showed that the analytical curves exhibited excellent linear behavior
over the examined concentration range.

57.3.3 The Repeatability and Reproducibility

The repeatability and reproducibility of the method were evaluated. The Tyramine
standard fluid was performed seven times by duplicate under optimum conditions
during a working day to determine repeatability and three replicate analyses of the
same sample were made on three different days to determine reproducibility.
Tables 57.1 and 57.2 showed the results of the test for repeatability and repro-
ducibility. The relative standard deviations (RSD) were less than 3 % for both
reproducibility and repeatability. Considering these RSD values, the repeatability
and reproducibility are satisfactory. These results indicate that this method can be
applied as quantitative analyses of tyramine.
57 Development of HPLC Method 561

Table 57.1 Precision of the method for determination the repeatability

Compound Repeatability (n = 6 mg/L)
1 2 3 4 5 6 RSD (%)
Tyramine Standard fluid 28.8 28.8 27.7 28.5 28.9 28.4 1.56

Table 57.2 Precision of the method for determination the reproducibility

Compound Reproducibility (n = 3) Date
Tyramine standard fluid 2012.6.6 2012.6.7 2012.6.8 RSD (%)
28.8 29.2 27.6 2.91

57.3.4 The Determination of Tyramine in Rice Wine

The chromatogram of the rice wine sample without tyramine addition was showed
in Fig. 57.4, the results prove the presence of tyramine in rice wine, and the
retention time was 4.399 min.
Figure 57.5 represents the chromatogram of the rice wine sample with tyramine
standard fluid addition (v: v 1:1) and the amount of tyramine addition was 0.5 mg.
The retention time of tyramine was 4.292 min, and the amount of detected
tyramine was higher than the amount in Fig. 57.4 obviously. The results
demonstrate that the recovery of tyramine can be obtained according to the
different amounts between Figs. 57.4 and 57.5.

57.3.4.1 The Recovery of Tyramine

The accuracy was estimated by means of recovery assays. Different amount of

tyramine addition in rice wine samples to determine the recovery of tyramine was
performed (Table 57.3), the concentrations of tyramine were 500 mg/L, 400 mg/L,
300 mg/L, respectively. The accuracy was satisfying for the recovery higher than
98 %.

Fig. 57.4 HPLC chromatograms of sample rice wine

562 X. Guo et al.

Fig. 57.5 HPLC chromatograms of sample rice wine with tyramine standard fluid addition

Table 57.3 The recovery of tyramine in rice wine with tyramine standard fluid addition by
HPLC
Rice No Amount Amount Amount Average RSD
wine addition addition addition addition recovery (%)
(mg/L) (500 mg/L) (400 mg/L) (300 mg/L)
Amount 28.8 525.6 420.1 325.7 98.7 % 0.8
found
Percent recovery = (amount found-amount no addition)/(amount addition) 9 100 [13]

57.4 Conclusions

The amount of tyramine detected by our method was similar to the reported in
previous works in rice wine by derivative action. This HPLC method makes
possible the identification of tyramine in rice wine without derivative and also
promotes a sensitive, precise, and accurate procedure for their quantification.
The method would be very useful for the analysis of other foods and by-products
fermented like measure of theirs sanitary and nutritional quality.

Acknowledgments This work was financially supported by the program of National High
Technology Research and Development Program of China (863 Program) (Grant No.
SS2012AA023408), the Cheung Kong Scholars and Innovative Research Team Program in
University of Ministry of Education, China (Grant No. IRT1166), and the National Science and
Technology Support Program under contract NO. 2012BAK17B11-05.

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