OPTIMIZATION OF COMPLEX-FORMING METHODS FOR DETERMINATION OF TAURINE IN REAL SAMPLES USING
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH UV DETECTION
Lukáš NEJDL1, Jiri SOCHOR1,2, Ondřej ZÍTKA1,2, Vojtěch ADAM1,2, Rene KIZEK1,2*
1Department
of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, 613 00 Brno, Czech Republic
2Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00 Brno, Czech Republic
*[email protected]
This study aims at analysing of complex-forming reactions with respect to use them for easy-to-use and sensitive determination of taurine in biological samples including tissues extracts and body
fluids. Particularly, we focused on studying of taurine complexes with phenol and sodium hypochlorite, and/or of taurine with sodium hypobromite. The formed complexes were studied under various
temperatures (10 °C, 20 °C, 30 °C, 40 °C, 50 °C and 60 °C) and in various time intervals (0, 5, 10, 15, 20, 25 and 30 min.). Separation of individual complexes of taurine with hypochlorite and
hypobromite were analyzed by HPLC-UV-VIS and spectrophotometrically in the range λ = 220 – 730 nm.
MATERIAL AND METHODS
0,2
220
270
320
370
Wavelenght(nm)
420
2
4
6
40
20
00
0
8
0,09
0,06
2
400
200
0
2
4
Time (minutes)
6
C
1
0
4
6
0.8
0.4
0
2
4
450
520
590
660
6
8
F
Taurine
4
3
2
1
0
8
0
2
Time (minutes)
0,03
E
Time (minutes)
2
2
8
1.2
0
3
0
6
Sodium hypobromite
1.6
8
Sodium hypochlorite
4
4
Time (minutes)
Rt: 4.01
600
Absorbance (mAU)
B
Phenol
D
Rt: 1.61
Rt: 4.03
0
Rt: 2.21
Absorbance (mAU)
B
4
6
8
Time (minutes)
Fig. 3: Spectrum of taurine with sodium hypobromite. Measured after 30 min. long
interactions at 60 °C. (A) Spectrum record of sodium hypobromite and taurine. (B) Complex
formed at 288 nm.
0
0
Purple complex
Complex taurin + NaOBr
60
Absorbance (mAU)
a = chlornan 3,7 mM
b = fenol 1,7 mM
0,4
1,2
Absorbance (mAU)
b
0,6
A
Absorbance (AU)
Absorbance (AU)
730
Wavelength (nm)
0,6
0
220
320
420
520
Wavelenght (nm)
620
720
Absorbance (AU)
0,4
0,3
0,6
0,3
O
OH
+
O
OCl-
NH 2
320
0,10
0,06
370
420
Wavelength (nm)
420
0,14
270
282
294
306
318
Wavelength (nm)
Wavelength (nm)
520
620
720
Fig. 5: interaction of temperature and time
S
O
NHCl
Taurine-chloramine
Hypochlorite
Taurine
270
B
A
O
S
b
0,0
220
Fig. 4: Dependences of absorbance of taurine with phenol and hypochlorite (4, 2, 1, 0.5, 0.25
and 0.125 mM of taurine) after 0, 5, 10, 15, 20, 25 and 30 min. long interactions and
temperatures 10 °C, 20 °C, 30 °C, 40 °C, 50 °C and 60 °C. The reaction was recorded at
0,1
290 nm.
Fig. 5: Dependences of absorbance of taurine with sodium hypobromite (4, 2, 1, 0.5, 0.25 and
0.125 mM of taurine) after 0, 5, 10, 15, 20, 25 and 30 min. long interactions and temperatures
0,0
10 °C, 20 °C, 30 °C, 40 °C, 50 °C and 60 °C. The reaction was recorded at 630 nm.
220
320
Fig. 6: (A) Three different complexes of taurine with chlorinated phenol as taurine
chloramine, 4chloroiminocyclohexa-2,5-dien-1one and indophenole. (B) Complex
Fig. 4: interaction of temperature and time
formed mixing taurine with sodium hypobromite.
O
a
0,18
Absorbance (AU)
a = NaOBr 8 mM
A
b = taurine 100 mM
0,9
0,6
Absorbance (AU)
Absorbance (AU)
1,8
a
1
0
0,12
0,8
2
80
Time (minutes)
Fig. 2.: Spectrum of taurine with phenol and sodium hypochlorite. Measured after 30 min. long
interactions at 60 °C. (A) Spectrum record of sodium hypochlorite (a) and phenol (b). (B) Complex
formed at 530 nm (a, b) and 630 nm (c - g).
2,4
A
3
0
RESULTS AND DISCUSSION
1
Blue complex
4
Rt: 3.42
Absorbance (mAU)
USA), Gemini NX 3u column (110A, Phenomenex USA) and a UV-VIS detector (ESA, USA).
Mobile phase consisted from water:methanol , 55:45 v/v of the following flow rate 1.0 ml/min. The
sample (30 μl) was injected using autosampler (Model 540 Microtiter HPLC, ESA, USA).
Chromatograms were registered at 288, 530 and 630 nm.
Spectra were measured within the range from 220 to 800 nm using SPECORD 210 (Analytik Jena,
Germany) in quartz cuvettes with 1 cm diameter (Hellma, UK).
Preparation of taurine
To study taurine complexes, taurine of the following concentrations were prepared: a) 100, b) 50, c)
25, d) 12.5, e) 6.25 and f) 3.13 mM. To study the influence of time and temperature on the measured
absorbance, taurine of the following concentrations were prepared: a) 4, b) 2, c) 1, d) 0.5, e) 0.25 and
f) 0.125 mM. Milli Q water was used for solutions preparation.
Absorbance (mAU)
Fig. 1. A: First study complex taurine with phenol and sodium hypochlorite. B:
Measurements
Reagents phenol. C: Reagents sodium hypochlorite. D: Second study complex taurine
HPLC-UV system consisted of two solvent delivery pumps (Model 582 ESA Inc., Chelmsford, MA, with sodium hypobromite. E: Reagents sodium hypobromite. E: Pure taurine (99%).
O
+ O
O
O
S
NHCl
O
4-Chloroiminocyclohexa-2,5-dien-1-one
Phenol
O
NCl
NCl
+
O
O
O
N
Blue
O
OH
N
Yellow
Purple
Indophenol dye
O
O
O S
OH
Taurine
+
OBr-
NH2
O S
O
Hypobromite
B
NHBr
Taurine-bromamine
CONCLUSION
It was optimized complex-forming method for studying of taurine interactions with phenol and hypochlorite, and/or with hypobromite. Taurine (200 mM and
higher) forms with phenol/hypochlorite mixture two complexes in comparison with one taurine-bromine complex. For both complexes studied, taurine and phenol
with hypochlorite and taurine with sodium hypobromite, significant dependence on the concentration of taurine, reaction temperature and time of reaction was
observed
ACKNOWLEDGEMENT
The work has been supported by NANIMEL GA CR 102/08/1546 and NANOSEMED GA AV KAN 208130801 and CEITEC CZ.1.05/1.1.00/02.0068.