SPECTROPHOTOMETRIC METHOD DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR DETERMINATION OF GRANISETRON HYDROCHLORIDE IN BULK AND INJECTIONS

SPECTROPHOTOMETRIC METHOD DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR DETERMINATION OF GRANISETRON HYDROCHLORIDE IN BULK AND INJECTIONS

P. B. Badhan ^{* 1}, S. S. Sonawane ², A. V. Shelke ¹ and A. G. Jadhav ¹

Department of Pharmaceutical Chemistry ¹, Sandip Institute of Pharmaceutical Sciences, Mahiravani, Nashik - 422213, Maharashtra, India.

Department of Pharmaceutical Chemistry ², Met’s Institute of Pharmacy, Adgaon, Nashik - 422003, Maharashtra, India.

ABSTRACT: The present UV Spectrophotometric and RP-HPLC method was simple, accurate, precise, specific, and sensitive. Spectrophotometric method was developed in water and very useful for analysis of bulk drug and injection formulation. RP-HPLC method was developed in Phosphate buffer (pH 3.0 adjusted with orthophosporic acid) and acetonitrile (70:30) using column   HiQsil C18 column (250 × 4.6 mm; 5 μm). Flow Rate: 1.0 ml / min. The method was validated for the determination of granisetron hydrochloride in bulk and parenteral dosage form. The standard solution of granisetron hydrochloride in water showed maximum absorption at 301 nm with correlation, slope, and intercept 0.9998, 0.04198, and 0.01255, respectively and the percentage recovery of the formulation was 99.965% by UV spectrophotometry. By RP-HPLC, the % RSD of intraday and interday precision was   0.82% and 1.40 %, respectively. The % means recovery amount of Granisetron hydrochloride in 80 %, 100%, 120% was 94.3%, 94.9%, 95.2%, respectively, which reflect that the method was free from the interference of the impurities and other additives during the estimation of drug in the formulation. The proposed method can be successfully used for analysis of granisetron hydrochloride in marketed preparations. The results of analysis have been validated statistically and by recovery studies. This method was found suitable for quantitative and qualitative estimation of granisetron hydrochloride in bulk and parenteral dosage form.

Granisetron hydrochloride, UV Spectrophotometry, RP-HPLC, Validation, Small volume parenteral

INTRODUCTION: Granisetron hydrochloride, endo - 1 - methyl - N - (9 - methyl - 9 - azabicyclo 3.3.1 non - 3 - yl) H-indazole-3-carboxamide Fig. 1, is a selective 5-HT3 receptor antagonist ¹. Granisetron hydrochloride is an effective and potent antiemetic drug which is used in the postoperative treatment of vomiting and nausea resulting from cancer chemotherapy and radio-therapy in adults and children ^2-6.

Granisetron hydrochloride is also effective in the management of postoperative nausea and vomiting due to anesthetics ⁷. Granisetron hydrochloride exhibits its antiemetic effects through central and peripheral 5-HT3 receptors ¹⁸. Granisetron, as Granisetron hydrochloride, is used both in oral tablets and injection ¹.

Literature survey reveals that the drug has been estimated by HPLC ^8-13, HPTL C ²⁰, tandem LC-MS ^15, and Bioanalytical ^16-17 methods in biological fluids like plasma and pharmaceutical dosage forms but no simple UV Spectro-photometric and RP-HPLC method in pharmaceutical formulations of injection has been reported so far. A thorough literature survey reveals that only few analytical methods have been reported for the determination of granisetron hydrochloride in bulk and pharmaceutical formulations, including RP-HPLC stability-indicating and bioanalytical method. The present work deals with the estimation of granisetron hydrochloride in injection by UV spectrophotometry and by RP-HPLC. Experimentation was carried out in MET’s institute of pharmacy Nashik.

FIG. 1: CHEMICAL STRUCTURE OF GRANISETRON

MATERIALS AND METHODS:

Instruments: A Jasco UV-Visible Spectrophotometer (UV-1700) with a matched pair cell of 10 mm quartz was used for experimental purpose.  The HPLC system used consisted of a pump PU- 2080 plus (JASCO Corporation, Japan) fitted with 20 μL Rheodyne loop injector (7725i). Detection was carried out on UV -2075 detector (JASCO Corporation, Japan). The data acquisition was done on BORWIN chromatography software (version 1.50).

Materials: Pharmaceutical grade Granisetron hydrochloride was kindly provided as gift sample from Sunpharma Ltd., and it was used without further purification.  All analytical grade reagents were purchased from S.D Fine chemicals, Mumbai. Granisetron hydrochloride injection (KYTRIL) was purchased from market. Each 1 mL contains 1.12 mg granisetron hydrochloride equivalent to 1mg of granisetron. Double distilled water was freshly prepared by all double distillation glass Assembly (Borosil, Mumbai, India) for the preparation of solutions and further used in analysis after filtering through 0.45 × 47 mm membrane filter papers purchased from PCI Analytics Ltd. Was used for degassing the prepared mobile phase.

Preparation of Standard Stock Solution and Calibration Curve: The standard stock solution was prepared by dissolving granisetron hydrochloride in distilled water to make the final concentration of 100 μg/ml. Different aliquots were taken from the stock solution and diluted with distilled water separately to prepare a series of concentrations from 5-30 μg/ml.

The λ_max was found by the UV spectrum of granisetron hydrochloride in distilled water in the range of 200-400 nm, and it was found to be 301 nm. Absorbance was measured at 301 nm against distilled water as blank. The calibration curve was prepared by plotting absorbance versus concentrations of granisetron hydrochloride.

Analytical Method Validation by UV and RP-HPLC: The developed method was validated in terms of specificity and selectivity, linearity and range, precision, accuracy, the limit of detection, the limit of quantitation, and robustness as per USP and ICH guidelines ^21-23.

Linearity and Range: The calibration graph of the absorbance versus concentration was found to be linear over the range of 5-30 μg/ml. The calibration graph was constructed after an analysis of 6 different concentrations. Each point of the calibration graph corresponded to the mean value obtained from three independent measurements. The regression equation was y = 0.04198 × -0.01255, where y is the absorbance, and x is the concentration in μg/ml. (r² = 0.9998).

Precision and Accuracy: Precision is the degree of repeatability of an analytical method under normal operational conditions. The precision and accuracy were determined with standard quality control samples (in addition to calibration standards) prepared in triplicate at different concentration levels covering the entire linearity range. The precision of the assay was determined by repeatability (intraday) and intermediate precision (inter-day) and reported as %RSD for a statistically significant number of replicate measurements. The intermediate precision was studied by comparing the assays on three different days, and the results are documented as the standard deviation and % RSD. Accuracy is the percent of analyte recovered by assay from a known added amount. Data from nine determinations over three concentration levels covering the specified range were obtained.

Sensitivity: The limit of quantification (LOQ) is the lowest concentration of Granisetron hydro-chloride on the calibration curve that can be quantified with acceptable precision and accuracy. The LOQ was found as 1.35 μg/ml for the proposed method. The limit of detection (LOD) was found to be 0.4465 μg/ml. The results indicate that the proposed method is sensitive to detect and quantify 0.4465 μg/ml and 1.35 μg/ml respectively.

Specificity: The spectra obtained from the test solution were identical with that obtained from a standard solution containing an equivalent concentration of Granisetron hydrochloride indicating that the wavelength of maximum absorbance of Granisetron hydrochloride did not change.

It was concluded that the excipients did not interfere with the quantification of Granisetron hydrochloride in pharmaceutical formulations by the proposed method. Thus, the proposed method can be used for the determination of Granisetron hydrochloride in the presence of excipients.

Robustness: The terms robustness refer to the ability of an analytical method to remain unaffected by small variations in method para-meters (mobile phase composition, column age, column temperature, change in wavelength, etc.) and influential environmental factors (room temperature, air humidity, etc.) and characterize its reliability during normal usage. It was making small deliberate changes in the wavelength of detection used for testing the robustness of method.

Analysis of Marketed Formulation: Granisetron hydrochloride injection (KYTRIL) (containing Granisetron equivalent to 1 mg per ml) was purchased from the market. 0.15 ml of this solution was diluted with 10 ml distilled water (15 μg/ml) and subjected to UV and RP-HPLC analysis. The amount of Granisetron hydrochloride was obtained from the regression equation of calibration curve.

Specificity: Complete separation of Granisetron hydrochloride was noticed in the presence of excipients of the tablet formulation. In addition there was no any interference at the retention time of in the chromatogram of placebo solution, which shows that the peaks of analyte were pure and excipients does not interfere the analyte. Hence, Identification and Specificity of Granisetron is established.

System Suitability: The chromatographic systems used for analysis must pass the system suitability criteria before sample analysis can commence. Set up the chromatographic system; allow the HPLC system to stabilize for 30 min. Inject blank pre-paration (single injection) and standard preparation (six replicates) and record the chromatograms.

RESULTS AND DISCUSSION:

UV Spectrophotometry Selection of Detection Wavelength: The UV spectrum of diluted solutions for various concentrations of Granisetron hydrochloride in the mobile phase was recorded using UV spectrophotometer. The Wavelength of maximum absorbance was observed at 301 nm.

Linearity: The response for the drug was linear in the concentration range of 5-30 µg/ml. The regression equation was y = 0.04198 × -0.01255, where y is the absorbance, and x is the concentration in μg/ml. (r² = 0.9998). The regression data, values of correlation coefficient (r) and other statistical parameters are listed in Table 1.

TABLE 1: ANALYTICAL CHARACTERISTICS OF GRANISETRON HYDROCHLORIDE BY PROPOSED UV SPECTROPHOTOMETRIC METHOD

TABLE 2: REPEATABILITY

Precision and Accuracy: The results of the repeatability listed in Table 2. The developed method was found to be precise as the % RSD values for repeatability, and intermediate precision studies were found < 2. From accuracy data, as shown in Table 3 the % recovery of the drug was 102.1% to 101.2% found.

Analysis of Marketed Formulation: The % recovery of drug content in the marketed formulation was found to be 99.965% with 0.6227 % RSD; the low RSD value indicated the stability of method for the routine analysis of granisetron hydrochloride in pharmaceutical formulation.

Validation using HPLC:

Specificity: There was no any interference at the retention time of in the chromatogram of the drug in the presence of placebo, which shows that the peaks of analyte were pure, and excipients do not interfere with the analyte.

TABLE 3: ACCURACY AND PRECISION TABLE

FIG. 4: CHROMATOGRAMS OF GRANISETRON HYDROCHLORIDE STANDARD

System Suitability: System is suitable if the tailing factor should NMT 1.5, theoretical plate count should NLT 2000 and % RSD for peak area of six replicate injections of Granisetron hydrochloride standard should NMT 2.0. Results of system suitability are presented in Table 1, which shows that all results were within acceptance criteria, which proves the reproducibility of the method.

Precision: The intraday and inter-day precision results were shown in Table. The percent relative standard deviation (% RSD) was calculated, which is within the acceptable criteria of not more than 2.0, which proves the repeatability of the method.

TABLE 4: SYSTEM SUITABILITY BY HPLC

TABLE 5: PRECISION BY HPLC

TABLE 6: ACCURACY BY HPLC

CONCLUSION: The proposed methods can be successfully applied for granisetron hydrochloride assay by UV Spectrophotometry and RP- HPLC in small volume parenteral dosage forms without any interference in quality control. Analysis of the small volume parenteral by this method was reproducible, reliable, and in good agreement with the labeled claim of the drug.

ACKNOWLEDGEMENT: Authors are thankful to the management and trustees of Mumbai Educational Trust’s Bhujbal Knowledge City, Adgaon, Nashik, for providing necessary Analytical requirements and Sunpharma Ltd. for providing gift sample of Granisetron hydrochloride.

CONFLICTS OF INTEREST: Nil

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    How to cite this article:

    Badhan PB, Sonawane SS, Shelke AV and Jadhav AG: Spectrophotometric method development and validation of RP-HPLC method for determination of granisetron hydrochloride in bulk and injections. Int J Pharm Sci & Res 2021; 12(2): 1184-89. doi: 10.13040/IJPSR. 0975-8232.12(2).1184-89.

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