Study of Vitamin-B 1 Interaction with Dihydroxyanthraquinone Dye and its Thermodynamic Elucidation

Interaction of Alizarin Red-S dye with thiamin (vitaminB1) was studied using spectrophotometric method at room temperature and neutral pH. The yellowish color appeared due to the Alizarin Red-S interaction with vitamin B1. According to the Benesi-Hildebrand equation, the related equilibrium constant, Kb, for the interaction has been determined and the Gibbs free energy of interaction has been calculated. Also the Benesi-Hildebrand equation has been used for assay of vitamin B1 in tablets. Results show that the method has good sensitivity for vitamin B1 and is in good agreement with HPLC method.


Thiamin
(2-[3-[(4-amino-2-methyl-pyrimidin-5-yl) methyl]-4-methyl-thiazol-5-yl] ethanol), is a water soluble vitamin also called vitamin B1 (Figure 1).It is a natural essential nutrient which is found in rice bran, nuts, banana, soybean, green peas and etc.Also it is added to some kind of foods and drinks to produce enriched products for special purposes.Thiamin was first discovered in 1910 by Umetaro Suzuki in Japan when researching how rice bran cured patients of beriberi [1].It was first crystallized by Jansen and Donath in 1926 and its chemical composition and synthesis was finally reported by Robert R. Williams in 1935 [2,3].Phosphate derivatives of thiamin play great roles as co-factor in enzymatic processes inside human body including carbohydrate metabolism and nerve transduction [4].It is also used in the metabolism of branched chain amino acids and may have non coenzyme roles in excitable cells [5].Thiamin level in blood is an important diagnostic factor of sudden infant death syndrome [6].Also, beriberi is a chronic deficiency disease that results from inadequate dietary *Address correspondence to this author at the Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran; Tel/Fax: +98 21 77886326; E-mail: biochem.kh@gmail.comintake or impaired absorption of thiamin, as in chronic alcoholism, so quantification of thiamin has vital importance and due to its use in pharmaceutical formulations such as tablets, syrups and ampoules, accurate analytical methods for quality control of thiamin in dosage forms are also needed.
There are various methods for analysis of thiamin in the literature.
Study of the interaction of small molecules (such as drugs) with dyes, polymers, surfactants, biological macromolecules and etc., is a good way in understanding of the molecules structure and functions [15][16][17].In this study, we used spectrophotometric method for the thermodynamic study of interaction between thiamin and Alizarin Red-S (Figure 2) and its application in assay of thiamin in tablets.

Chemicals and Reagents
Thiamin (vitamin B1), Alizarin Red-S and all the salts were purchased from E. Merck (Darmstadt, Germany).The chemicals are analytical grade and all of them used without any purification.All solutions were prepared with double distilled water (conductivity 3 S).Stock solutions of 1 10 -4 M Alizarin Red-S and 1 10 -2 M of thiamin was prepared by dissolving appropriate amounts of them in distilled water.

Sample Solutions Preparation
An accurately weighed amount of 10 powdered Vitamin B1 tablets was dissolved in water.The excipients were separated by filtration and filter paper was washed three times with water.The filtrate and washing solutions of the tablet samples were transferred into 100 ml calibrated flask and diluted to the mark with water

Procedure
All pH measurements were made at 25˚C, using Metrohm 744 pH meter (Metrohm, Switzerland).Absorption spectra were recorded on a Perkin-Elmer Lambda 25, double-beam UV-Vis spectrophotometer with 1.0 cm matched quartz cells and thermostat cell holder for adjusting the temperature.An 1100 series Agilent HPLC apparatus (Agilent technologies, USA) equipped with quaternary pump, degasser and diode array detector was used.Separations carried out on a ZORBAX-ODS column (150 4.6 mm I.D., 5μm particle size).Chromatographic method used as below: solvent A: methanol, solvent B: Water, gradient elution: 0-1 min 5% A and 95% B, 1-2 min A reaches to 13% with a linear gradient, 2-5 reaches to 35%, flow rate =1 ml.min -1 , =288nm.
1.5 mL of dye solution (1 10 -4 M) placed in the cell and different volumes of thiamin stock solution were added.We give the concentration of the stock solution of thiamin higher to avoiding increasing in sample solution volume and consequent dilution in dye solution (thiamin adding solution is less than 20 micro liter) also we add the similar volume of water to the reference cell for considering any dye concentration changes constant.According to the fact that Alizarin Red-S is a pH indicator and due to effect of pH on absorption spectra of dye, pH of solutions adjusted at the neutral pH range (6.5-7.5) using the dilute solutions of sodium hydroxide and hydrochloric acid.
15 minutes after every addition, absorption changes in 422 nm were recorded at 25˚C.Concentration of thiamin in sample solution determined by the Benesi-Hildebrand equation (1/ A= f (1/C T )) [19].It should be noted that, concentration of dye were kept constant by adding very low volumes of thiamin stock solution.

Study of Interaction Between Thiamin and Alizarin Red-S
The electronic spectrum of Alizarin Red-S was recorded and variation in maximum absorbance of dye during addition of thiamin was followed spectrophotometrically (Figure 3).The binding constant was determined from the effect observed in the absorbance of Alizarin Red-S at 422 nm upon addition of the thiamin according with the Benesi-Hildebrand treatment [20].As shown in Figure 4, there is a linear relation between 1/ A and 1/C, which indicates a certain interaction between dye and thiamin.

Determination of Binding Constant
The value of the binding constant, K b , was obtained according to Benesi-Hildebrand equation and the method described previously [18][19][20][21][22][23].By assuming that there is only one type of interaction between thiamin and dye in aqueous solution, so the Eqs, (1) and ( 2) can be established:

Alizarin Red-S (ARS) +Thiamin (T) ARS-T
(1) Where K b is binding constant, by assuming [ARS-T] =C b : Where C ARS and C T are the analytical concentrations of Alizarin Red-S and Thiamin in solution, respectively.According to the Beer's law: and Where A 0 and A are the absorbance of Alizarin Red-S in the absence and presence of Thiamin, respectively.E ARS and b are the molar extinction coefficients of dye and the complex, respectively.l is the light path of the cell (1 cm).
By displacing C ARS and C b in Eq, ( 3) by Eqs, ( 4) and ( 5), Eq, ( 6) can be deduced: Plot of (1/ (A-A 0 )) versus (1/C T ) is linear and the binding constant (K b ) can be estimated from the ratio of the intercept to the slope [19,20].Figure 4 shows the plot of (1/ (A-A 0 )) versus (1/C T ), at specified experimental conditions.The Gibbs free energy of interaction of Alizarin Red-S with Thiamin can be obtained by the following equation: The obtained amount for the Gibbs energy of the interaction between ARS-T is -19.81 kJ/mol.

Study of Dye Concentration
For optimization of dye concentration, differences of absorbance ( A) during addition of constant amount of thiamin, plotted versus concentration of dye.As shown in Figure 5 in concentrations above 1 10 -4 M, A reaches to a limiting value and remain constant.So, 1 10 -4 M selected as optimum concentration of Alizarin Red-S.

Effect of Time
The absorbance of solution was read after 1, 5, 8, 12 and 20 min of thiamin addition.As shown in Figure 6, in the range of 5 to 15 minutes no variation in absorbance was observed.So, 10 minutes selected as optimum time.

ANALYTICAL APPLICATION
We used Benesi-Hildebrand equation for determination of thiamin in tablets.Wide linear concentration range of 1.31 10 -4 -6.60 10 -6 M and detection limit of 4.36 10 -7 M made this method suitable for this purpose.Some of the analytical characteristics of this method were summarized in Table 1.
Further experiments have been performed to assess the reproducibility of the method.Thus, five replicate determinations have been carried out and the relative standard deviation was calculated.RSD% of the method was 1.54 which indicates the proposed method is reproducible.In order to evaluation of the method we used a high performance liquid chromatographic method for determination of thiamin in tablets at 25˚C, too.Typical chromatograms of standard thiamin and thiamin sample were shown in Figure 7. Comparative results of two methods were presented in Table 2. Results clearly show a good agreement between the methods.

CONCLUSION
According to the obtained results for binding studies of interaction between Alizarin Red-S and vitamin B1 (Thiamin), it can be concluded that there is a strong interaction in this system.Benesi-Hildebrand equation was used for determination of thiamin analytical concentrations in the solution in the presence of Alizarin Red-S.The obtained results were in good agreement with the results obtained from HPLC method.

Figure 3 :
Figure 3: UV-Vis spectra of dye during thiamin addition (thiamin concentration was increased as titration method and it is changed from 10 -7 up to10 -3 molar).

Figure 5 :
Figure 5: Plot of A vs. CT at the specified conditions.

Figure 6 :
Figure 6: Effect of time on variation of A at constant dye concentration.

Table 1 : Quantitative Characteristics of the Proposed Method
*Limit of detection (mol/L).† Linear dynamic range (mol/L).