Stability-Indicating HPLC Methods for the Quantification of Cyproheptadine Hydrochloride, Vitamins B 1 , B 5 And B 6 in Tablets using RP-HPLC with UV Detection

The availability of reliable and fast analytical method is crucial for the quality control of multi-API in a single dosage form. Herein, two methods have been developed and validated for the determination of Cyproheptadine hydrochloride (CH), Thiamine mononitrate (Vit. B 1 ), Calcium pantothenate (Vit. B 5 ) and Pyridoxine hydrochloride (Vit. B 6 ) in uncoated tablets. To assay the vitamins, a mobile phase combination of Phosphate buffer pH 3.5 and Methanol in the ratio 93:7, using Eurospher ODS (150 x 4.5mm) as the stationary phase with a flow rate set at 1.0 mL/min at ambient temperature conditions was adopted. Wavelength of detection was 270 nm with a run time of 0.00 to 5.50 minutes for Thiamine mononitrate and Pyridoxine hydrochloride, and 205 nm from 5.50 to 11.00 minutes for Calcium pantothenate. A good resolution and a short run time of 11 minutes were achieved with the validated conditions. The retention times of Thiamine mononitrate, Pyridoxine hydrochloride and Calcium pantothenate were 2.823±0.020, 4.184±0.007 and 10.025±0.015 respectively. The mobile phase for Cyproheptadine HCl was methanol and an ion-pairing solution (70:30), set at a flow rate of 1mL/min, a runtime of 8 minutes, an injection volume of 20 µL and a wavelength of 285 nm using Eurospher ODS (150 x 4.5 mm) as the stationary phase. The retention time for Cyproheptadine HCl was 4.961±0.006. Both methods were found to be specific, robust, accurate and precise over the concentration ranges of 0.0192 mg/mL– 0.0288 mg/mL for Thiamine mononitrate, 0.0128–0.0192 mg/mL for Pyridoxine hydrochloride, 0.032–0.048 mg/mL for Calcium pantothenate and 0.032-0.048 mg/mL for Cyproheptadine hydrochloride. The Correlation Coefficient (r 2 ) for Cyproheptadine hydrochloride, Thiamine mononitrate, Pyridoxine hydrochloride and Calcium pantothenate were greater than 0.999. The purpose of this study was to develop and validate simple HPLC methods for the estimation of Cyproheptadine hydrochloride, Vitamins B 1 , B 5 and B 6 in combined dosage forms. The proposed methods were found to be precise, specific, accurate and robust for the estimation of Cyproheptadine hydrochloride, Vitamins B 1 , B 5 and B 6. Thiamine mononitrate, Pyridoxine hydrochloride and Calcium pantothenate in fixed dosage forms over the concentration ranges of 0.0192 mg/mL–0.0288 mg/mL for Thiamine mononitrate, 0.0128–0.0192 mg/mL for Pyridoxine hydrochloride, 0.0320–0.0480 mg/mL for Calcium pantothenate and 0.0320-0.0480 mg/mL for Cyproheptadine hydrochloride.


Introduction
Cyproheptadine hydrochloride is the hydrochloride salt of a synthetic methyl-piperidine derivative with antihistaminic and anti-serotoninergic properties. Cyproheptadine competes with free histamine (HA) for binding at HA-receptor sites, thereby competitively antagonizing histamine stimulation of HA-receptors in the gastrointestinal tract, large blood vessels, and bronchial smooth muscle. This agent also competes with free serotonin for binding at serotonin receptor sites. Cyproheptadine exhibits anticholinergic and sedative properties and has been shown to stimulate appetite and weight gain. Cyproheptadine has a molecular formula of C21H22CIN (National Center for Biotechnology Information 2020a). Thiamine mononitrate has a molecular formula of C12H17N5O4S. It plays a key role in intracellular glucose metabolism and it is thought that thiamine inhibits the effect of glucose and insulin on arterial smooth muscle cell proliferation. Thiamine plays an important role in Chemistry and Materials Research www.iiste.org ISSN 2224-3224 (Print) ISSN 2225-0956 (Online) Vol.12 No.6, 2020 36 helping the body convert carbohydrates and fat into energy. It is essential for normal growth and development and helps to maintain proper functioning of the heart and the nervous and digestive systems (Wishart et al. 2018). Pyridoxine hydrochloride has a molecular formula of C8H12ClNO3. It is the hydrochloride salt form of pyridoxine, a water-soluble vitamin B. Pyridoxine hydrochloride is converted into the active form pyridoxal 5 1 -phosphate (PLP), an essential cofactor in many enzymatic activities including synthesis of amino acids, neurotransmitters, and sphingolipids. This vitamin is essential to red blood cell, nervous system, and immune systems functions and helps maintain normal blood glucose levels (National Center for Biotechnology Information 2020b). Calcium pantothenate has a molecular formula of C18H32CaN2O10. It is the calcium salt of the water-soluble vitamin B5, ubiquitously found in plants and animal tissues with antioxidant property. Pantothenate is a component of coenzyme A (CoA) and a part of the vitamin B2 complex. Vitamin B5 is a growth factor and is essential for various metabolic functions, including the metabolism of carbohydrates, proteins, and fatty acids. This vitamin is also involved in the synthesis of cholesterol, lipids, neurotransmitters, steroid hormones, and hemoglobin (National Center for Biotechnology Information 2020c). Several chromatographic methods for the determination of Cyproheptadine hydrochloride, Thiamine mononitrate, Pyridoxine hydrochloride and Calcium pantothenate have been reported (Shino et al. 2008;Hasimoglu and Ghodke 2018;Yantih et al. 2011;Staroverov et al. 2004). The aim of this study is to develop simple RP-HPLC methods. This study was done in accordance with the International Conference on Harmonization (ICH) guidelines (Q2(R1) 2005).

Instrumentation and Chromatographic conditions
A High Performance Liquid Chromatographic (HPLC) system, controlled by Knauer Advanced Scientific Instrument data handling system (Knauer 2000) and coupled with an autosampler was used for the analysis. The data was recorded using Euro Chrome software. For the analysis of the Vitamins, a stainless steel column 150 mm long, 4.5 mm internal diameter filled with Octadecyl silane chemically bonded to porous silica particles of 5 µm diameter (Make: Knauer Advanced Scientific, Germany) was used with the mobile phase composed of Phosphate buffer pH 3.5 and Methanol (93:7) at ambient temperature. The mobile phase was pumped through the column at a flow rate of 1 mL/minute. The sample injection volume was 20 µL. The UV detector was set at a wavelength of 270 nm from 0.00 to 5.50 minutes for the detection of Thiamine mononitrate and Pyridoxine hydrochloride, and 205 nm from 5.50 to 11.00 minutes for the detection of Calcium pantothenate. The run time was 11 minutes. For Cyproheptadine, same column as for the vitamins was used. The mobile phase consisted of an ion paring reagent prepared separately and mixed with methanol in the ratio (30:70). The wavelength was 285 nm, injection volume of 20 µL, flow rate of 1mL/min and Runtime of 8 minutes.
Model tablets containing 1.5 mg Thiamine mononitrate, 1 mg Pyridoxine hydrochloride and 2.5 mg Calcium pantothenate were prepared with the following excipients; Lactose, Dicalcium phosphate granules, Microcrystalline cellulose Avicel 12, Sodium starch glycolate, Talc, Magnesium stearate and Cab-O-sil. Samples were analysed using the developed RP-HPLC method. The results are reported as means ± SEM (standard error of the mean). Results were statistically analysed for significant differences.

Preparation of Solution A
Approximately 700 mg of 1-Hexanesulphonic acid sodium salt was weighed and transferred into a 500 mL volumetric flask containing 250 mL of distilled water, dissolved, added 5 mL of Glacial acetic acid and made to volume with distilled water.

Preparation of Mobile phase
Into a 500 mL volumetric flask, about 350 mL of methanol (HPLC grade) was transferred and mixed with 150 mL of Solution A.

Preparation of Standard solution
This was prepared by accurately weighing and transferring about 40 mg of Cyproheptadine HCl working standard into a 100 mL volumetric flask and dissolved to the mark with the diluent with intermittent shaking for 30 minutes. 5 mL of the resultant solution was dissolved into a 50 mL volumetric flask and diluted to the mark with the diluent.

Preparation of Sample Solution
About 240 mg of the synthetic blend was weighed into a 100 mL volumetric flask and dissolved with the diluent for 30 minutes to the mark.

Preparation of Analytical
Solutions for Analysis of Vitamins B1, B5 and B6.

Preparation of Buffer solution
About 500 mg of monobasic potassium phosphate was dissolved in 1000 mL of water and adjusted with phosphoric acid to a pH of 3.5.

Preparation of Mobile phase
Into a 1000 mL volumetric flask, 900 mL of the phosphate buffer was transferred, and 100 mL of Methanol HPLC grade was added with thorough mixing.

Preparation of Standard solution
About 24 mg of Vit. B1 working standard, 40 mg of Vit. B5 working standard and 16 mg of Vit. B6 working standard were accurately weighed and transferred into a 100 mL volumetric flask. They were dissolved and diluted to the mark with distilled water. 5 mL was transferred into a 50 mL volumetric flask and diluted to the mark with distilled water.

Preparation of Sample solution
About 960 mg of the synthetic blend was weighed and transferred into a 250 mL volumetric flask. 10 mL of methanol was added and swirled to disperse the specimen. Distilled water was added to volume, mixed and filtered.

Method Development
In order to develop a relatively cheap chromatographic system for Vitamins B1, B5 and B6, Methanol (HPLC grade) was chosen as the only organic solvent during the developmental stages. In order to obtain the best resolution possible, various mobile phase ratios were utilized. A mobile phase ratio of Phosphate buffer pH 3.5 and Methanol (93:7) was found appropriate at a flow rate of 1 mL/min with wavelength of 270 nm from 0.00 to 5.50 minutes for the detection of Thiamine mononitrate and Pyridoxine hydrochloride, and 205 nm from 5.50 to 11.00 minutes for the detection of Calcium pantothenate. Eurospher ODS (150 x 4.5mm) was the column used at an injection volume of 20 µL and Run time of 11 minutes. Column temperature was ambient.
The method for Cyproheptadine hydrochloride consisted of an Ion pairing reagent (1-Hexanesulphonic acid sodium salt) used in the mobile phase to enhance the separation of the vitamins and Cyproheptadine hydrochloride on the reversed phase column. A mobile phase composition of Methanol and ion paring solution (70:30) was used with a flow rate of 1 mL/min, runtime of 8 minutes, injection volume of 20 µL and a wavelength of 285 nm.

Specificity and specificity by forced degradation
In evaluating specificity, there were no interferences. Injections of the placebo and the mobile phase (diluent) gave no peaks. The order of elution is as follows; Thiamine mononitrate followed by Pyridoxine hydrochloride and then Calcium pantothenate.    The system was found suitable as per predetermined acceptance criterion prior to the generation of all other data from the execution of other method performance characteristics.

Repeatability
Six sample preparations of the synthetic blend were analysed as per the protocol. The percentage contents and the % RSD were determined. RSD for the sample preparation of Cyproheptadine was 1.39% That of Vit. B1,Vit. B5 and Vit. B6 were 0.90%, 1.01% and 1.51% respectively.

Inter-day Precision
Analysis conducted on Day 1 and Day 2 were compared. For Cyproheptadine, RSD for Day 1 was 1.39% and that for Day 2 was 1.80%. For the Vit. B1, RSD for Day 1 was 0.90% and Day 2 was 1.53%. For Vit. B6, Day 1 was 1.51% and Day 2 was 1.47%. For Vit. B5, Day 1 was 1.01% and Day 2 was 1.05%. Results obtained from Day 1 and Day 2 were statistically analyzed to determine any significant differences. For acceptance of the null hypothesis, F-calculated is not more than F-critical value of 5.05 at 95% confidence level (Olaniyi 2005 Vol.12 No.6, 2020 between 80.0 % to 120.0 % level. The correlation coefficient is greater than 0.998. In addition, the analysis of residuals shows that, the values are randomly scattered around zero, which fits, and well within the linear model. To evaluate whether the y-intercepts are significantly different from zero, the P-value was determined. If P value is > 0.05, then intercept is statistically equal to zero. For Cyproheptadine, P value is 0.843937. Hence, it is statistically equal to zero. In addition, the origin is within the lower and the upper limit of the 95 % confidence level that gives a high degree of confidence to the values obtained for intercept. Moreover, the value of the intercept is less than 5 % of the area response at 100 % level.
From the statistical treatment of the linearity data of Thiamine mononitrate, Pyridoxine hydrochloride and Calcium pantothenate, the responses of Thiamine mononitrate, Pyridoxine hydrochloride and Calcium pantothenate were linear between 80.0 % to 120.0 % level. The correlation coefficients are greater than 0.998. In addition, the analysis of residuals shows that, the values are randomly scattered around zero, which fits, and well within the linear model. To evaluate whether the y-intercepts are significantly different from zero, the P-values were determined. If P value is > 0.05 then intercept is statistically equal to zero. For Thiamine mononitrate, Pyridoxine hydrochloride and Calcium pantothenate, P values were 0.946327, 0.641731 and 0.756602. Hence, it is statistically equal to zero. In addition, the origin is within the lower and the upper limit of the 95 % confidence level that gives a high degree of confidence to the values obtained for intercept. Moreover, the value of the intercept is less than 5 % of the area response at 100 % level.
The Limit of Detection (LOD) and Limit of Quantitation (LOQ) were determined. For Cyproheptadine HCl, LOD was 0.0019 mg/mL and LOQ was 0.0058 mg/mL. Vitamin B1 had LOD of 0.0027 mg/mL and LOQ of 0.0083 mg/mL. Vitamin B5 had LOD of 0.0014 mg/mL and LOQ of 0.0042 mg/mL. Vitamin B6 had LOD of 0.0014 mg/mL and LOQ of 0.0043 mg/mL.

Robustness
Changes in flow rate (± 0.2 mL/min) and wavelength (±2) were effected to determine the robustness of the method. Irrespective of the changes in chromatographic conditions, all the system suitability parameters remained unaffected and results conformed to acceptance criteria indicating robustness of the methods.

Stability in Analytical Solution
The standard and sample solutions were injected within a period of 24 hours and was concluded that, standard and sample solutions of Thiamine mononitrate, Pyridoxine hydrochloride and Calcium pantothenate can be assayed within 24 hours after preparation.

Conclusion
The validation of the proposed isocratic RP-HPLC methods for the estimation of Cyproheptadine hydrochloride, Thiamine mononitrate, Pyridoxine hydrochloride and Calcium pantothenate in model tablets were carried out as per ICH guidelines. The methods were found to be specific, robust, accurate and precise for the estimation of Cyproheptadine hydrochloride, Thiamine mononitrate, Pyridoxine hydrochloride and Calcium pantothenate in fixed dosage forms over the concentration ranges of 0.0192 mg/mL-0.0288 mg/mL for Thiamine mononitrate, 0.0128-0.0192 mg/mL for Pyridoxine hydrochloride, 0.0320-0.0480 mg/mL for Calcium pantothenate and 0.0320-0.0480 mg/mL for Cyproheptadine hydrochloride.