Determination of Vitamin B 6 (pyridoxine hydrochloride) in Pharmaceutical Preparations Using High Performance Liquid Chromatography

Determination of vitamin B 6 (pyridoxine hydrochloride) was described using high performance liquid chromatographic method. The analysis was achieved by cosmos IL 5C 18 -MS-II column (250 mm x 4.6 mm i. d., 5µm particle size) at room temperature. The mobile phase used was Acetonitrile, buffer solution (Citric acid, Na 2 HPO 4 pH4) buffer solution in the ratio (70:30) (V: V). the flow rate was set to 1.25 mL.min -1 and the retention time 1.82 min with UV-detection at 282 nm. Beer's law was obeyed over the concentration range 10-1250 µg.mL -1 . The method was accurate (relative error % less than 0.05%), precise (RSD better than ±1.05%), average recovery 100.05%, with a limit of detection and quantification of 2.2μg.ml −1 , and 7.34μg.mL −1 respectively. The proposed method was successfully applied to determine the pyridoxine hydrochloride in pharmaceutical preparations in both forms of tablet and injection.


Introduction
Pyridoxine hydrochloride (vitamin B 6 ) is a 5-hydroxy-6-methyl-3, 4-pyridinedimethanol hydrochloride with the chemical formula C 8 H 11 NO 3 .HCl [1,2]. It is a white powder, soluble in water, slightly soluble in acetone, and insoluble in chloroform and ether [3]. The determination of vitamin content in food and in nutritional supplements is important in quality, labeling, and marketing [4].

Mahmood and Ahmad
Iraqi Journal of Science, 2019, Vol. 60, No. 5, pp: 943-951 944 Pyridoxine is used to prevent low levels of vitamin B 6 which plays an important role in the health of nerves, skin and red blood cells [5].

Exprerimental Apparatus
A (shimadzue, LC-2010A, Japan) HPLC instrument was used with a pump (model LC-2010, high flow rate mode) and auto injector sampler. The detector was UV LC-2010 with D 2 lamp (800mv minimum energy). Separation was achieved using a cosmosil 5C 18 -MS-II column (4.6 mm I.D. x 250 mm, 5 µm particle size, Japan). A shimadzue UV-1650 PC double beam spectrophotometer with 1cm quartz cells have been used for scanning spectra. pH measurement has beam done by pH-meter (HANNA pH 211, microprocessor pH meter, Mauritius), balance (Sartorius BL 20 S, Germany) has been used for weight measurements.

Materials
All chemicals and reagents are of analytical grade. Water was double distilled and filtered. Acetonitril (ACN), dichloromethane (DCM), methanol, and ethanol (Loba. Chemie) are of HPLC. Pyridoxine hydrochloride (2000 µg.mL -1 ): this solution was prepared by dissolving 0.10 gm of pure pyridoxine hydrochloride (from BDH) in double distilled water and filtered using 0.45 µm filter, the volume was completed to 50 ml in a volumetric flask, further dilution was followed to prepare 500 µg.mL -1 working solution. Citric acid (0.1 M) solution: this solution was prepared by dissolving 1.92 g of citric acid (C 6 H 8 O 7 ) (from BDH) in 100 mL of double distilled water and filtered using 0.45 µm filter. Disodium hydrogen phosphate (0.2 M) solution: this solution was prepared by dissolving 1.42 g of (Na 2 HPO 4 ) (from Sigma) in 50 mL of double distilled water and filtered using 0.45 µm filter. Buffer (pH 4) solution: this solution was prepared by mixing 61.9 mL of citric acid (0.1 M) with 38.1 mL of (0.2 M) Na 2 HPO 4 . Pharmaceutical preparations Pyridoxine hydrochloride (Smamvit-B6 tablet) 1000 µg.mL -1 solution: this solution was prepared by weighing 5 tablets (40 mg SDI), grinding to fine particles and mixing; a weight of this pharmaceutical powder (0.0457 g) equivalent to 0.01 g of pure pyridoxine hydrochloric acid was dissolved in solvent mixture (CAN: citric acid, Na 2 HPO 4 ) (70: 30), then filtered using 0.45 µm filter, the volume was completed to 10 mL with the same solvent in a volumetric flask. Pyridoxine hydrochloride (Meho B6 tablet) 1000 µg.mL -1 solution: this solution was prepared by weighing 5 tablets (40 mg Bijing company), grinding to fine particles and mixing; a weight of this pharmaceutical powder (0.042 g) equivalent to 0.01 g of pure pyridoxine hydrochloric acid was dissolved in solvent mixture (CAN: citric acid, Na 2 HPO 4 ) (70: 30), then filtered using 0.45 µm filter, the volume was completed to 10 mL with the same solvent in a volumetric flask. Pyridoxine hydrochloride (Mason natural-B6 tablet) 1000 µg.mL -1 solution: this solution was prepared by weighing 5 tablets (50 mg mason natural company), grinding to fine particles and mixing; a weight of this pharmaceutical powder (0.070 g) equivalent to 0.01 g of pure pyridoxine hydrochloric acid was dissolved in solvent mixture (CAN: citric acid, Na 2 HPO 4 ) (70: 30), then filtered using 0.45 µm filter, the volume was completed to 10 ml with the same solvent in a volumetric flask. Pyridoxine hydrochloride (Mefron injection) 1000 µg.mL -1 solution: this solution was prepared by taking 0.5 mL of injection solution (100mg pyridoxine hydrochloric acid /2ml) (Shanohai) and diluted to 25 mL using solvent mixture (CAN: citric acid, Na 2 HPO 4 ) (70: 30), then filtered using 0.45 µm filter.

Recommended Procedure and Calibration Graph
20 μL of pyridoxine hydrochloride in the concentration range between (10 to 1250 μg.mL -1 ) was injected under the optimum condition of analysis acetonitrile, buffer solution (citric acid, Na 2 HPO 4 pH4) in the ratio (70:30)(V:V). The flow rate was set to 1.25 mL. min -1 , which exhibits a retention

Selection of mobile phase
Many solvents in many compositions and ratios were used as a mobile phase for is ocratically elution of sample after injection of 20 µl of 500 µg.ml -1 of the standard solution. The measurements were done at 282 nm at room temperature and using 1 ml min -1 as a flow rate. From Table-1 ACN: water (70:30)(V:V) was selected as a mobile phase of pyridoxine hydrochloride because of the symmetric chromatogram and the ideal capacity factor (k`); therefore, it was selected in subsequent experiments.

Selection of the analysis media
Many different weak acidic and weak basic media have been used to enhance the symmetry and to get a specific chromatogram of 20 µl of 500 µg.mL -1 of standard solutions; the measurement was done at 282 nm at room temperature and using 1 mL min -1 as a flow rate.   Figure-3.

Effect of flow rate
Between 0.5 to 1.5 mL.min -1 a flow rate for the elution of pyridoxine hydrochloride has been followed. Table-3 shows that 1.25 mL.min. -1 gave a good result in which it reduces the time of analysis and gave the best symmetry; therefore, it was used in subsequent experiments.

Effect of temperature
15, room temperature, 30, and 35 ºC as a temperature of the column has been adjusted to follow its effect on the performance of analysis. Table-4 indicates good results at all temperatures, the room temperature was selected. Figure-4 shows the chromatogram at room temperature.   Table-5 shows the accuracy and precision of three different concentrations of pyridoxine hydrochloride (50, 250, and 500 µg.mL -1 ) in the form of recovery %, relative error RE %, and relative standard deviation RSD %.  Table-5 shows that the proposed method provides a good accuracy (average R.E. % is 0.05) and a good precision (average of RSD% is better than ±1.05).

Effect of ingredients
The recovery % of 10 µg.ml -1 of pyridoxine hydrochloride in the presence of the same amount of expected ingredient has been followed under the optimum analysis conditions.  Table-6 shows that only Arabic gum is seriously and positively interfered in the analysis of pyridoxine hydrochloride.

Application of the method
Using the proposed chromatographic method, assay of pyridoxine hydrochloride in its pharmaceutical preparations, (Samavit B 6 (Tablet) 40mg SDI-Iraq), (Mason natural B 6 (Tablet) 50mg USA),(Meheco B 6 (Tablet) 40mg Beijing-China), and(MefronB 6 (Injection) 100mg/2ml Shanohai-China) has been followed under the optimum analysis conditions. The results are listed in Table-7.  Table-7 shows a good applicability of pyridoxine hydrochloride in its pharmaceutical preparations in the both forms tablets and injections.

Experimental t-test
The table of t-test (at 95% confidence and for four degrees of freedom [18]) shows good trustability, according to Table (8) there is no significant difference between the proposed method and the literature method [19].  [15,16] ( Table-9) shows that the three methods are applicable. Moreover, the present method is more rapid, simple, and precise.  [16] Litruturemethod [15] Column Cosmosil 5C 18 -MS-II (250 mm x 4.6 mm i.d., 5µm particle size) Spherisort OSD C 18

Conclusion
A simple, precise, and accurate HPLC method for the determination of vitamin B6 (pyridoxine hydrochloride)in different pharmaceutical preparations using the proposed procedure without pre separation steps and/or adjustment of temperature has been reported in this paper.