Spectrophotometric Determination of Vitamin B6 by Coupling with Diazotized p-Nitroaniline

A spectrophotometric method has been described for the determination of pyridoxine hydrochloride (vitamin B6) in pure and pharmaceutical preparations. The method is based on coupling reaction of vitamin B6 with diazotized p-nitroaniline in alkaline solution in the presence of CTAB to form an azo dye which has maximum absorption at 480 nm. Beer's law is obeyed over a range of 5-500 μg/25ml with a molar absorpitivity of 2.7 × 10 l. mol cm and Sandell sensitivity index of 0.0076 μg.cm, a relative error +0.22 to + 0.47% and a relative standard deviation of ± 0.11 to ± 0.98%, depending on the concentration level. The proposed method has been applied successfully to the determination of vitamin B6 in pharmactical preparations.


INTRODUCTION
Vitamins are biologically active organic compounds with a diverse chemical nature. They enter in the human organism with food, in small amounts, and play a major role as biocatalysts in metabolism. Both a lack and an excess of certain vitamins in an organism may cause significant disturbances of various functions of the organism, resulting in serious diseases (Melentyeva and Antonova, 1988). The vitamins of the B 6 group are compounds that contain the pyridine ring in their molecules and they are water soluble. There are six forms of vitamin B 6 : pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM), and their phosphate derivatives : pyridoxal 5-phosphate (PLP), pyridoxine 5-phosphate (PNP), and pyridoxamine 5-phosphate (PMP). Pyridoxine was the first isolated vitamin B 6 (Kaplan and Pesce, 1989).
Pyridoxine hydrochloride (5-hydroxy-6-methylpyridine-3, 4-diyl) dimethanol hydrochloride is a white or almost white, crystalline powder, freely soluble in water, slightly soluble in alcohol. It melts at about 205 o C, with decomposition, the molecular structure C 8 H 11 NO 3 .HCl,M.Wt = 205.6 g/mol (British Pharmacopeia, 2005) is as follows: Pyridoxine functions as a coenzyme in the metabolism of amino acids, protein and the maintenance of body cells (Lehne, 2001).
There are various analytical procedures for the assay of pyridoxine, the most important included flow injection systems (Pons et al., 2000;Portela et al., 2004), high performance liquid chromatography methods for the determination of pyridoxine and isoniazid in pharmaceutical formulations (Kompantseva et al., 2005) and in flavored milk mixes (Agostini -Costa et al., 2007), high performance thin layer chromatography (Argeka and Sawant, 1999), other methods such as a capillary electrophoresis method has been developed that allows the separation and estimations of melatonin and pyridoxine in pharmaceutical preparation using electrochemical detection (Chen et al., 2000).A voltammetry (Solange, 2009) and cyclic voltammetry (Teixeira et al., 2003) have also been proposed.. Many spectrophotometric methods have been described for the determination of pyridoxine hydrochloride; these methods included first derivative spectrophotometry to estimate pyridoxine HCl combined with other drugs in tablet (Pathak and Rajput ,2008), colorimetric method based on the oxidation-reduction reaction of vitamin B 6 with cerium (IV) ion has also been described to determine vitamin B 6 in serum and it's pharmaceutical preparations (Qadir and Mosa, 2008). The diazotized sulphanilic acid (Pons et al., 1999) reagent has also used for determine B 6 vitamins in N-cetylpyridinium chloride medium.
The oxidative coupling reaction has been used to determine vitamin B 6 by coupling of pyridoxine with 4-aminoantipyrine in the presence of ammonium persulphate; the product shows maximum absorption at 420 nm (Nirmalchandar et al., 1987).
In the present work, diazotized p-nitroaniline (D-PNA), along with cetyltrimethyl ammonium bromide (CTAB), is used as a reagent for the determination of pyridoxine hydrochloride (vitamin B 6 ) in alkaline medium. The method offers the advantages of sensitivity, simplicity and rapidity without the need for extraction or heating.

Instruments :
Spectrophotometric measurements are performed using Shimadzu UV-visible Recording Spectrophotometer UV-160, with 1-cm matched glass cells.
The pH measurements are performed on pH meter type HANNA 211 pH -Ion meter.

Reagents :
Chemicals used are of analytical reagent grade.
Working pyridoxine hydrochloride (Vitamin B 6 ) solution, (100 µg/ml). A 0.01 g of pyridoxine hydrochloride (NDI-Iraq) is dissolved in distilled water and the volume is completed to 100 ml in a volumetric flask. The solution is kept in a brown bottle, where it is stable for at least one week.
Sodium carbonate solution, (0.1 N). This solution is prepared by dissolving 1.325 g of sodium carbonate (BDH) in 250 ml of distilled water.
Diazotized p-nitroaniline reagent solution (5mM). A 0.1727g of p-nitroaniline (Fluka) is dissolved in about 50 ml distilled water. Then 20 ml of 1M HCl is added and the solution is heated, the clear mixture is then transferred to 250-ml volumetric flask and is cooled to 0-5 o C in an ice-bath. A 8.65 ml of 1% NaNO 2 (BDH) is added and the mixture is stirred vigorously. After 5 min., the solution is made up to volume in 250 ml volumetric flask with cold water. The solution is kept in a brown bottle in a refrigerator and is stable for at least one month ( Othman , 2001) .

Cetyltrimethylammonium bromide (CTAB) solution,(1×10 -3 M).
This solution is prepared by dissolving 0.911 g of CTAB in distilled water then the volume is completed to 250 ml with distilled water .

Vitamin B 6 Tablets solution.
Five tablets were powdered accurately and weighed and a weight of powder equivalent to one tablet was dissolved and transferred into a 100-ml calibrated flask and made up to volume with distilled water. The solution was filtered and the clear filtrate used for the determination. An appropriate volume of the sample solution was diluted further with water so that the concentration of vitamin B 6 in the final solution was within the working range. Ampoule of vitamin B 6 solution, (100 µg/ml). The 100 µg/ml B 6 solution is prepared by diluting 0.2 ml of ampoule (100 mg B 6 /2ml-Troge Medical GMBH, Germany), content to 100 ml with distilled water in a volumetric flask.

Procedure and calibration curve
To series of 25-ml volumetric flasks, aliquots of pyridoxine hydrochloride (B 6 ) solution are transferred to cover the range of 5-600 µg/25ml B 6 , i.e., 0.2-24 ppm. Then 1.5 ml of diazotized p-nitroaniline (5 mM) reagent solution and 3 ml of (1×10 -3 m) CTAB is added . The mixture are shaken , then 3 ml of (0.1 N) sodium carbonate solution is added and the volumes are made up to the mark with distilled water . The mixtures are shaken and the absorbances are measured at 480 nm against the corresponding reagent blank , using 1cm glass cells. Fig.1 shows the calibration curve obeyed Beer's law over the concentration range 5-500 µg/25ml , i.e., 0.2-20 ppm and a concentration above 500µg/25 ml gives a negative deviation. The molar absorptivity is 2.7×10 4 1.mol -1 .cm -1

RESULTS AND DISCUSSION
For the following experiments, 100 µg of vitamin B 6 is taken in 25 ml final volumes and absorbance measurements are performed at 480nm.

Principle of the method
The method involves the coupling of the determinand vitamin B 6 with diazotized pnitroaniline to form, in basic medium and in the presence of CTAB,an intense coloured dye:

Optimum reaction conditions
The effect of various parameters on the absorption intensity of the coloured dye is investigated and the reaction conditions are optimized.

Effect of Base
The preliminary experiments have shown that the coloured dye can be developed only in alkaline medium. Many (strong and weak) bases with different amounts are examined and the results indicated that 3 ml of 0.1 N Na 2 CO 3 produces the highest intensity of the dye and better colour contrast ( ∆ λ ). So, it has been selected in the subsequent experiments (Table 1).

Effect of Diazotized p-Nitroaniline Reagent Amount
The effect of the diazotized p-nitroaniline reagent amount on the color intensity of the dye has been studied. The results are shown in Table 2. From the values of r 2 and absorbance, 1.5 ml of 5 mM diazotized reagent has been recommended for the subsequent experiments .

Effect of Surfactant
The effect of surfactant on the intensity and stability of azo dye was studied by adding 3 ml of various types of surfactant to the medium of reaction. The result in Table 3 showed that cationic surfactant addition causes the highest stability of azo dye with no effect on the intensity of the dye. Therefore, 3 ml of 10 -3 M CTAB solution is used in the subsequent experiments. The selected surfactants are : Cetyltrimethylammonium bromide (CTAB) (cationic ). Cetylpyridinium chloride monohydrate (CPC) (cationic). Sodium dodecyl sulphate (SDS) (anionic). Tween 80 (neutral) .

Order of Surfactant Addition
The three orders of CTAB addition are tried and results are given in Table 4  (S) + Diazotized p-nitoaniline ( R) + CTAB(C) + Base (B) II = S + C + R + B III = S + R + B + C From the results above, order I has been recommended for the following experiments.

Effect of time and amount of vitamin B 6 on absorbance
The effect of time on the development and stability of the dye obtained from three different amounts of vitamin B 6 has been investigated under the optimum experimental conditions described above. The formation of coloured dye being complete immediately, and the absorbance of the coloured species remained constant for at least 1 hour.The results is shown in Table 5.

Final absorption spectra
The absorption spectra of the coloured dye formed from coupling vitamin B 6 with diazotized p-nitroaniline in alkaline medium, in the presence of CTAB, against its corresponding reagent blank show maximum absorption at 480 nm in contrast to reagent blank which shows no absorbance at the wave length of maximum absorption ( Fig 2) .

Accuracy and precision
The accuracy and precision of the calibration curve are checked by determining vitamin B 6 at three different concentrations. The results shown in Table 6 indicate that the method is quite satisfactory.

Nature of the dye
To estimate the composition of the coloured dye species formed between vitamin B 6 and diazotized p-nitroaniline reagent under the optimum conditions. Job's method of continuous variations is applied. The obtained results (Fig. 3) showed that the combining ratio of vitamin B 6 with diazotized p-nitroaniline reagent is found to be 1:1 .

Study of interferences
The effect of some excipients which often accompany pharmaceutical preparations were studied by adding different amounts to 100 µg vitamin B 6 in final volume 25 ml (Table  7). The above data show that the method is very selective to determine vitamin B 6 in pharmaceutical preparation .

Application of the method
To test the applicability of the present method, it has been applied to the determination of vitamin B 6 in pharmaceutical preparation. On applying the proposed procedure, good recovery is obtained as shown in Table 8.

Comparison of the methods and t-test
A comparison between the present method and British pharmacopia standard method (British Pharmcopia, 2000) for the determination of vitamin B 6 in tables, is based on the ttest to show the ability of using the present method in the determination of vitamin B 6 in pharmaceutical preparation. The results shown in Table 9. The comparison between some of analytical variables obtained from present method and another spectrophotometric method is shown in Table 10. The results in Table 10 show that the suggested method for the determination of vitamin B 6 have a good stability and range comparing with the other method.

CONCLUSION
A simple, rapid, accurate and precise spectrophotometric method is evaluated for determination of pyridoxine hydrochloride (vitamin B 6 ) in pure and pharmaceutical formulation. The short analysis time and low cost are the main advantage of this method for routine analysis in quality control.