INTRODUCTION:
Taurine has many physiological functions as an antioxidant, neurotransmitter, toxinicide, modulation of the levels of intracellular ions (e.g. Ca+, K+, Na+), membrane stabilization, osmosis and in conjugation of bile acids and plays an important role in prenatal development especially in neural development. Taurine is a useful biomarker of some diseases and pathological disorders3,4.
Changes in the taurine levels in physiological fluids and tissues occur in various diseases or pathological conditions such as psychosis (depression, schizophrenia, epilepsy), inflammation, hepatic damage, sepsis, retinitis pigmentosa, and cancer3. Human clinical studies showed that the oral intake of taurine can influence in physiological functions5. For example, taurine (3 or 6 g/day) decreases blood pressure for hypertension patients. A substantial increase in the plasma concentration of growth hormone was reported in some epileptic patients, stimulation the hypothalamus and modification of neuroendocrine function. There is an indication that taurine (2 g/day) has possibly a role in the induction of the psoriatic state5. The combination of taurine with caffeine, amino- acids and guarana in energy drinks increases the risk of side effects. The most risky of them are hypertensive crisis, arrhythmia, dehydration6.
Another studies showed that taurine intake is generally less than 200mg/day, even in individuals on a high-meat diet7. But some energy drinks contain taurine in high levels between [800-4000] ppm and this is greater than the highest estimated intake of 400 mg/day from naturally occurring taurine in diets. Because of the consumption of energy drinks which contain taurine in high levels, and increasing of the cases with undesired side effects1, a simple new analytical method has been developed to determine the taurine in energy drinks. Scientific papers reveal that there is non-reliable spectrophotometric method to assay taurine as a standard and in energy drinks. In this study, extraction method of taurine was developed by using cation exchange resin. The extract was treated with ninhydrin as a derivatizing reagent in basic medium to form a stable colored complex, which could be determined spectrophotometrically. The developed method has been validated. Then we have determined the amount of taurine in two types of energy drinks (Company A=25, company B=25) which commonly consumed in Syria.
MATERIALS AND METHODS:
Materials:
Ninhydrin (2,2-Dihydroxyindane-1,3-dione) (pure 99%, SCP, America).
Taurine (2-aminoethanesulfonic acid) (pure 99%, ROTH, Germany).
Sodium Hydroxide (NaOH) (pure 99%, SCP, America).
Cation exchange resin (T50) (TULSION, India).
Energy drinks [Company A, Company B].
Apparatus:
Spectrophotometer (HITACHI-1800, Japan), Ultrasonic (BIOBASE), Balance (Sartorius), Water bath.
Standard solution and reagent:
· Preparation of standard solution (taurine):
· 250 mg of std. was dissolved in distilled water and was shaken well, then water was added to adjust the volume up to 100 ml in volumetric flask (2500 ppm).
· Preparation of reagent (ninhydrin):
· 0.8905 g of reagent was dissolved in distilled water and was shaken well, then water was added to adjust the volume up to 100 ml in volumetric flask (0.01 M).
Handling of sample:
It is established that determination of taurine is directly impossible without extraction it, so a simple extraction method was developed by using cation exchange resin (T50).
Extraction procedure:
2.5 ml from each sample was extracted using cation exchange resin then filtrated.
Analysis procedure:
2ml from each of filtrated solution was taken and put it into tightly closed tube, then 2 ml of reagent (0.01 M) and 0.5 ml of NaOH (0.05 M) were added to each tube. Then they were heated at 100˚ C in water bath for 20 min. After cooling, tubes were diluted up to 50 ml volumetric flask. The absorbance was measured at 568 nm against blank which contains all materials except taurine. The concentrations were calculated from the corresponding equation of the calibration curve.
RESULTS AND DISCUSSION:
Taurine is in fact an amino sulphonic acid and it doesn’t contain a carboxy group, therefore it doesn’t have the same structure of known amino acids (NH2-R-COOH)8.
Since taurine doesn’t have a chromophore group and it shows maximum absorption at 284 nm using water as a solvent, ninhydrin was used to form a stable colored complex with taurine, which is the most popular reagent to detect aminoacids. Several features associated with Ninhydrin appear to be anomalous in its reaction9, so in this study the stoichiometric ratio of the reaction between ninhydrin and taurine was estimated and the optimal conditions for the previous reaction were studied. The reaction was applied at 100˚C in basic medium to form a blue-colored complex.
Determination the wavelength:
The absorbance spectrum of the constituted complex was scanned between [450-800] nm against the corresponding blank. The maximum absorbance was at 568 nm. The spectrum was shown in figure (1).
Figure (1): Absorbance spectrum of colored complex.
Optimizing conditions with respect to pH, temperature, period of heating, reagent, stability:
The optimum pH:
The Ammonia/ammonium chloride buffer, carbonate/ bicarbonate buffer, hydrochloric acid, potassium hydroxide, and sodium hydroxide were added to test tubes and the reaction was carried out for each of them, but the colored complex didn’t form except in tube which contains NaOH. The optimum pH for this reaction was determined using 0.5 ml (0.05M) NaOH pH=12.7, the results were shown in figure (2).
Figure (2): Determination of the optimum pH.
Sodium hydroxide (0.05 M) was added in increasing volumes to determine the optimum volume of it, and the results were shown in figure (3).
Figure (3): Determination of the optimum volume of NaOH (0.05M).
· The optimum volume and concentration of reagent:
The concentrations between [0.01-0.1M] of the reagent were prepared and then added in the same volume to the solution of taurine (50 ppm). To study the optimum volume, the reagent was added in increasing volumes in other tubes at same concentration of taurine and the reaction was carried out in both cases. The results were shown in figure (4) and (5) respectively.
The optimum temperature and period of heating:
The optimum temperature was determined by preparing test tubes at same concentration and heating them at 50˚C to 100˚C, then needed period of heating was determined. The results were shown in figure (6) and (7) respectively.
Figure (4): Determination of the optimum concentration of Ninhydrin.
Figure (5): Determination of the optimum volume of Ninhydrin.
Figure (6): Determination of the optimum temperature.
Figure (7): Determination of period of heating.
The stability of complex:
The reaction was carried out in test tube at 40 ppm, and the absorption was obtained for several periods after dilution. The results were shown in figure (8).
Figure (8): Study of the derivatized compound stability.
Stoichiometric ratio10:
job’s method of continuous variation was employed to determine the molar ratio between the analyte (taurine) and the reagent (ninhydrin). The solutions of each taurine and reagent were prepared to be the concentration (0.011 M). A series of tubes was prepared in which the total volume of the analyte solution and the reagent was 5 ml. Then the proposed reaction was carried out as above. The absorption of each solution was plotted against the standard mole fraction [Analyte]/[Analyte+reagent]. The molar ratio was 1:1 as shown in figure (9).
Figure (9): Determination of the stoichiometric ratio.
Validation of developed method11:
Linearity:
A series of standard was
prepared in a concentrations range of [5-60] ppm. The response of formed
complex was found to be linear in this range and the linear regression equation
was 
with correlation
coefficient R2= 0.997.
Figure (10): Linearity curve for taurine.
Limit of detection (LOD) and Limit of quantification (LOQ):
LOD and LOQ were calculated according to ICH [Q2(R1)] using the following equations:
Where
σ is the standard deviation of intercept.
S is the slope of calibration curve.
The results were summarized in table (1).
The quantitative parameters of the developed method were summarized in table (1).
Table (1): Quantitative parameters of the proposed method.
|
Parameter |
Value |
|
Temperature |
100˚ C |
|
Period of heating |
20 min. |
|
Stability of color |
3 hrs. |
|
Linearity range |
5-60 ppm |
|
Molar absorptivity |
2640 (l.mol-1.cm-1) |
|
Regression equation Y = ax + b |
y = 0.021x + 0.007 |
|
Correlation equation R2 |
0.997 |
|
Slope (S) |
0.021 |
|
Intercept |
0.007 |
|
Standard deviation of intercept (σ) |
0.005686 |
|
LOD |
0.893 ppm |
|
LOQ |
2.707 ppm |
Accuracy:
Accuracy is estimated by standard addition method at 3 concentration levels and 5 replicate measurements. Standard quantity equivalents to 50-100-125 % as added to a sample. Accuracy was determined as recovery % and relative standard deviation RSD %. The results were shown in table (2). The percent of recoveries of taurine by standard addition method was in the range of [89.45-99.33] %. The concentration of sample was 39.04 ppm.
Precision:
Six solutions at same
concentration (40 ppm) were measured. Standard deviation SD=0.25 and RSD=
0.64 % were obtained based
on absorption values. The results were shown in table (3).
Robustness:
The evaluation of robustness should be considered during the development. Variation of wavelength was chosen for six samples, and the results have shown that the small shift of wavelength didn’t affect on assay as present in table (4).
Specificity:
The proposed method was applied on each compound present in energy drinks, and the results was negative for each one. But when the reaction has applied on samples, the detected amount of taurine was insignificant. Therefore, extraction procedure has been developed to improve the recovery and obtain precise results which express about the correct concentration in samples.
Samples data:
25 samples of each energy drink were analyzed to determine their content of taurine. The results for each samples (Company A, Company B) were summarized in table (5) and (6) respectively.
