High-Throughput UPLC Method for Accurate Quantification of Betahistine in Pharmaceutical Tablets

The Betahistine is an Orally active, some H₁ selective histamine analogue, which is used to control vertigo in patients of  Meniere’s disease. It possibly acts by causing vasodilatation in the internal ear. It is contraindicated in asthamatics and ulcer patients. Chemically it is N-Methyl-2-pyridine-2-ylethanamine[1,2]. Its Molecular formula is C₈H₁₂N₂ and Molecular weight is 139.19 g/mol. The present work reports simple, rapid, sensitive and economical rapid UHPLC method with UV detection, useful for the routine analysis of Betahistine in bulk and pharmaceutical formulations. The method parameters such as linearity, accuracy, precision, robustness, stability and system suitability were validated as per International Conference on Harmonization (ICH) guidelines.

Figure1: Chemical structure of Betahistine

UPLC

Ultra-Performance Liquid Chromatography (UPLC) [6,7] is specially designed to withstand higher system pressures during chromatographic analysis so that it enables significant decreases in separation time and solvent consumption. The UPLC columns packed with 1.7 µm sized particles provides not only increased efficiency but also the ability to work at increased linear velocity without loss of efficiency but also the ability to work at increased linear velocity without loss of efficiency, providing both resolution and speed. Using advantages of UPLC, a number of applications in different fields including pharmacy, clinical analysis, pesticide analysis and tetracycline inhuman urine have been reported. The UPLC is based on the principle of use of stationary phase consisting of particles less than 2µm, while UPLC columns are typically filled with particles of 3 to 5µm. The underlying principles of this evolution are governed by the Van Deemter equation, which is an empirical formula that describes the relationship between linear velocity (flow rate and plate height (HETP or column efficiency). The Van Deemter curve, governed by an equation with three components shows that the usable flow range for a good efficiency with a small diameter particle in much greater than for larger diameters.

2. MATERIALS AND METHODS:

INSTRUMENTATION: UHPLC– SOFTWEAR: Open Lab - CHEM STATION (Make - Agilent)

Chromatographic conditions

• Mobile phase : Dissolve 2.76g of Sodium dihydrogen phosphate monohydrate and 1.6g of Sodium dodecyl-sulphate in 600ml of water add 0.4g of Hexylamine and 400ml of Acetonitrile adjusted to pH 3.5 with Orthophosphoric acid.
• Flow rate : 1 ml/min
• Column: C18; 4.6mm X 250 mm; 5 microns
• Detector wave length : 254 nm
• Injection volume : 5 µl
• Run time : 5 min
• Results : Retention time and peak shape is good.

All analysis was carried out at a temperature of 30°C under isocratic conditions.

METHOD DEVELOPMENT

The RP-UPLC method was developed by conducting number of trails in which the values of chromatographic parameters like wavelength, mobile phase composition and ratio, flow rate, stationary phase etc. were altered to determine the effect  of them on separation and identification of selected drug, finally the chromatographic parameters were optimized as follows.

Preparation of mobile phase:

Dissolve 2.76g of Sodium dihydrogen phosphate monohydrate and 1.6g of Sodium dodecyl-sulphate in 600ml of water add 0.4g of Hexylamine and 400ml of Acetonitrile adjusted to pH 3.5 with Orthophosphoric acid, degassed in the mixture by ultrasonication, and was filtered through the 0.45μm Membrane filter using vacuum filtration assembly

Preparation of standard stock solution:

Weigh accurately and transfer about 32 mg of Betahistine standard into 100 ml volumetric standard flask. Dissolve with Mobile phase and makeup to the volume with Mobile phase.

Preparation of Standard solution:

Transfer, 5ml from standard stock solution into 25ml volumetric flask makeup to the volume with Mobile phase.

Preparation of Sample solution:

Weigh and transfer 136.52 of Betahistine sample into 25 ml volumetric flask. Dissolve with Mobile phase and makeup to the volume with Mobile phase and filter through 0.45 µm membrane filter. Further dilute 5ml into 25ml volumetric Flask dilute and makeup with Mobile phase.

Method Validation:

Validation of the optimized method parameters includes linearity, system suitability, accuracy, precision, ruggedness, robustness, limit of detection and limit of quantification according to ICH guidelines.

System suitability parameters:

The system suitability parameters were determined by preparing standard solution of Betahistine injected Five times and the parameters like peak tailing and USP plate count were determined.

The % RSD for the area of five standard injections results should not be more than 2%.

Specificity:

Checking of the interference in the optimized method.

Precision:

Precision of an analytical procedure expresses the closeness of agreement between series of measurements obtained from multiple sampling of the same homogenous sample under the prescribed conditions. The precision of ananalytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements. ICH has defined precision to contain three components: repeatability, intermediate precision, and reproducibility.

Linearity:

The linearity of the developed method was determined by preparing the standard drug solutions with concentrations of 32-95 µg/ml of Betahistine from the standard stock solution, the linearity parameters like regression value (r²), slope, y-intercept, were evaluated by plotting the calibration curve taking concentration in µg/ml on x-axis, and peak area on y-axis for linearity concentrations. The correlation coefficient (r²) should be not less than 0.9999, % RSD of peak areas for replicated individual linearity concentrations should be not more than 2.0% to accept the linearity of the method.

Accuracy:

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. This is also termed as trueness. It was done by recovery study. Sample solutions were prepared with 100% in triplicate. To the formulation, the working standards of the drug were added at the level of 80%, 100%, 120%. Prepare each level in triplicate injection and the average is take to calculate the percentage relative standard deviation.

Acceptance Criteria: The % Recovery for each level should be between 98.0% - 102.0%

Robustness:

The robustness of the proposed method was determined by triplicate injection of working stock solution from homogenous lots by differing physical parameter like flow rate and mobile phase composition, temperature variation which may differ but the response was still within the specified limits of the assay.

Acceptance criteria: % Relative standard deviation of peak areas and R_t should not be more than 2.0%.

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The limit of detection and limit of quantification was calculated by the average value of standard deviation and slope. The LOD and LOQ were determined by the linearity studies.

LOD = 3.3 x (SD / SLOPE)

LOQ = 10 x (SD / SLOPE)

Where,

SD = standard deviation of y- intercepts

S = slope

3. RESULTS AND DISCUSSION:

Method development and optimization:

The current research work enumerates the validated UHPLC method development  for  Betahistine in tablet formulation. The effective separation and good peak symmetry was achieved by using mobile phase Buffer : ACN having pH3.5 [600:400, v/v] as mobile phase, on water C18 (4.6 x 250mm, 5 microns), analytical column under isocratic conditions.

System suitability:

The retention time (R_t) for Betahistine were found as 1.951 min respectively with consistent reproducibility represented by % RSD shown in Figure 2. The chromatographic parameter like USP plate count, tailing factor, resolution were found to be within the limits and given in Table1 which indicates the system suitability of the developed method.

Figure 2: Optimized Chromatogram of Betahistine

Table 1: System suitability parameters of Betahistine

Specificity:

Retention time of Betahistine was eluted at 1.951min. We did not find and interfering peaks in blank and placebo at retention times of these drugs in this method. So, this method was said to be specific.

Linearity:

The developed method showed the proportional relationship between peak area and concentration at different level of standard drug in the range 32-95μg/ml with regression coefficients (r²) 0.9999 for Betahistine. Respectively the linearity result were given in the Table 2. The linearity plots were shown in Figure 3.

Figure 3: Calibiration curve of Betahistine

Table-2. Linearity result of Betahistine

Precision:

Repeatability:

Six working sample solution of 64 ppm are injected and the percentage amount was calculated and %RSD was found to be 0.37 (Table 3)

Table 3: Repeatability data of Betahistine

Six working sample solution of 64 ppm are injected on the next day of the preparation of sample and the % amount found was calculated and %RSD was found to be 0.97. (Table 4)

Table 4: Intermediate precision of Betahistine