Validated Analytical Method for Multicomponent Analysis of Famotidine and Ofloxacin in Bulk drug and Tablet Formulation by using UV-Visible Spectrophotometer and RP-HPLC

 

Pooja Kaushal¹, Shiv Kumar Kushawaha¹*, Manish Majumder², Mahendra Singh Ashawat¹

¹Laureate Institute of Pharmacy, Kathog, Kangra, H.P. 176031.

²Adichunchanagiri University, Karnataka.

 

ABSTRACT:

A simple, sensitive, accurate, precise, and reproducible UV- spectrophotometric method and RP-HPLC methods were developed and validated for the estimation of Famotidine and Ofloxacin in bulk drug and pharmaceutical formulation. The Linearity regression was detected and shows a good linear relationship; in the concentration range of 10-50µg/mL (R² >0.9908) for famotidine and 10-50µg/mL (r²>0.9913) for ofloxacin. The UV – spectrophotometric estimation was carried out by the first-order derivative spectrophotometric method and absorbance were recorded at 273 and 280nm. Beers range were found to be 5-50µg/mL, respectively for both drugs while, correlation coefficient r² > 0.9988 and 0.9941 for famotidine and ofloxacin. The isoabsorptive point was found to be 274nm in HPLC optimized mobile phase composition, potassium dihydrogen orthophosphate: methanol (60:40). Chromatographic condition consisted of mobile phase potassium dihydrogen orthophosphate buffer pH 2.3, methanol (60:40v/v), run time 30 min, C-18 column (ODS Hypersil) and flow rate 0.8mL/minute. The retention time for famotidine and ofloxacin were found to be 2.44 min, 7.99 min. respectively, and detection at λ_max 274nm for both drugs (overlain spectra). The UV methods and RP-HPLC showed good reproducibility and recovery with the percent relative standard deviation (RSD) less than 5%. As per ICH guidelines, the developed method was validated for linearity, accuracy, precision, Sandell's sensitivity, and repeatability proving its utility in the estimation of famotidine and ofloxacin in house tablet formulation.

 

 

 

1. INTRODUCTION:

Peptic ulcer disease (PUD), poses a global health crisis up to 10% as a chronic disease. It is characterized by disrupting the integrity in the inner lining area of the gastrointestinal (GI) tract established with gastric acid secretion or pepsin factor.

 

Unfortunately, available combat zone therapies have demonstrated adverse effects, relapse due to gastrointestinal infection.

 

Famotidine (Fig-1A) is a potent, highly selective histamine H₂-receptor antagonist and chemically known as (1Z)-3-[({2-[(diaminomethylidene) amino]-1, 3-thiazol-4-yl} methyl) sulfanyl]-N' sulfamoyl propanimidamide. Nowadays, it is used for gastro-intestinal ailments such as gastroesophageal reflux disease (GERD), duodenal peptic ulcer in place of older analogues, cimetidine and ranitidine due to its efficacy and patient compliance². Noteworthy, it acts by decrease the basal and nocturnal gastric acid by blocks the site for histamine located on the basolateral membrane of the parietal cell^3-4.

 

Ofloxacin (Fig-1B) is a fluoroquinolone derivative and chemically known as 9-fluro-2, 3-dihydro-3-methyl-10-(4-methyl-1-piperizinyl)-7-oxo-7H-pyrido [1, 2, 3-de]-1, 4benzoxaine-6-carboxylicacid⁵. It is used in the treatment of urethritis, cervicitis, atypical pneumonia, M. tuberculosis, and M. leprae as an alternative drug regimen⁶.

 

(A)                                                      (B)

Fig 1:  Structure of (A) Famotidine and (B) Ofloxacin

 

The combined regimen will help to heal the ulcers as well as combat microbial infection. Both drugs are official in Indian pharmacopeia 2007⁷, United States Pharmacopoeia 2000⁸, and British Pharmacopoeia 2005⁹. Notably, several scientists developed the analytical methods for the analysis of single component of Famotidine and Ofloxacin by using RP-HPLC, UV-spectrophotometric methods, HPLC, RP-UPLC, and Liquid Chromatography^10-22. Till date, there is no efficient analytical method reported for the multicomponent analysis of Famotidine and Ofloxacin in bulk drug as well as formulation. Hence, an attempt has been made to develop novel, simple, accurate, sensitive, and economical optimized analytical methods for both drugs and validation of optimized methods according to ICH guidelines.

 

2. MATERIALS AND METHOD:

2.1. Reagent and Chemicals:

All the reagents used in this assay were analytical grade (Qualigens and Rankem, CDH) and HPLC grade water was procured from sanjay biological museum, Amritsar.  Famotidine (API) was purchased from Sri Sai Lab Technology, Hoshiarpur, Panjab and while Ofloxacin (API) was procured from Aarti drugs Ltd. Palghar.

 

2.2 Instrumentation:

UV-Visible Spectrophotometer (UV-VIS Spectrophotometer Lab India UV 3000⁺, Software win-chrome 5.0) and RP-HPLC: High-performance liquid chromatography (Water 600) software Winchrome, and detector (Water 486). Column C-18 (ODS Hypersil) 150 × 4.6mm i.d. particle size 5µm±0.30 were used for analytical method development and validation.

 

3. UV SPECTROPHOTOMETRIC METHOD:

3.1. Determination of λ_max of FAM and OFL:  Based on scanning the solution of FAM and OFL (10µg/mL) was determined the wavelength of maximum absorption λ_max by UV–Visible double spectrophotometer from 200-400nm using methanol and distilled water (50:50 v/v) as blank.

 

3.2. Preparation of stock solutions and test solutions:  

The stock solution of 100µg/mL was prepared by dissolving FAM and OFL (10mg each) that were weighed accurately and separately transferred into 100 mL volumetric flasks. Test dilutions were prepared by taking aliquot amounts of stock solution to get concentration in Beer’s range of 5-50µg/mL for FAM and OFL respectively.

 

3.3. UV Spectrophotometric methods:  

In this part, FAM and OFL in pure drug and in-house tablet formulation were determined successfully applied in first-order derivative methods.

 

3.4. Assay method validation:

Optimized in house formulation is shown in table1.

 

Table 1. (A)Tablet in house formulation

 

Table 1. (B) Tablet Composition and

 

Table 1. (C) Placebo Composition

 

3.5. First order method: 

The wavelengths of both drug concentrations the same 5 – 50µg/mL in solvent methanol: water (50:50 v/v) were selected for an individual determination as FAM 273nm and OFL 280nm respectively.

 

3.6. Methods for validation: 

UV spectroscopy methods were validated as per International Conference on Harmonization (ICH) guidelines for developed validation analytical procedures.

 

3.6.1. Linearity:

As per ICH Q2B guidelines, the linearity methods were validated^23-25. For validation of analytical procedure, for each analyte were performed linearity curve at λmax FAM 273nm and OFL 280nm, both drugs same concentration 5 – 50µg/mL respectively in both UV methods. The least-square regression method was applied to perform the linearity.

 

3.6.2 Accuracy:  

This was determined by the recovery studies using the standard addition method. The pre-analyzed samples prepared were spiked with standard drugs FAM and OFL at different concentration level 80, 100, and 120 %, and these triplicates method proposed by the re-analyzed method^26,27. The percentage recovery was calculated based on observed data. In both UV– spectrophotometric methods, a known amount of the standard solution of FAM (8, 10, and 12µg/mL) and OFL (8, 10, and 12 µg/mL) were added to the sample solution containing 10 µg/mL FAM and OFL respectively.

 

3.6.3. Precision:

The precision method was determined by repeatability, and intermediate precision (Intraday and Interday), and reproducibility^28-30. The observed data was calculated as % RSD for both drugs FAM and OFL with a number of replicate measurements. The obtained result %RSD deviation was found to be less than 2% that shows the was reproducible and accurate.

 

3.6.4. LOD and LOQ: 

The LOD is the method, the smallest concentration of analyte that can be detected (signal to noise ratio3:1) but cannot be quantified respectively. Whereas the LOQ is the method, the smallest concentration of the analyte which gives response (signal to noise ratio 10:1) that can be quantified accurately. Based on the standard deviation of response and slope were generated equation, is used to calculate both LOD and LOQ. Hence the parameters of LOD and LOQ were calculated as: 

LOD = 3.3×σ/s

LOQ = 10×σ/s

Where, σ = Standard deviation of the lowest standard concentration

s    =    Slope of the standard curve

 

3.6.5. Optimization of chromatographic conditions:  

To achieve the best separation of the peak, we have changed different conditions of chromatography parameters. Suitable conditions were selected for the method validation. In the above-optimized parameters individually or combination have been done by using various trials.  The proper separation was done by using various conditions, which include mobile phase composition, i.e. 0.1M potassium dihydrogen orthophosphate buffer: methanol (60:40 v/v) at pH 2.3 and flow rate 0.8mL/min for both drugs. The work was done in an air-conditioned room maintained at temperature 25±2^oc and the total run time was 30min.

 

3.6.6. Chromatographic conditions:

The composition of potassium dihydrogen orthophosphate buffer pH 2.3, methanol (60: 40 v/v) has been found to be satisfactory for the complete separation of individual compounds. Before use, the mobile phase was filtered through a nylon 0.45µm membrane filter and degassed for 30min. The standard stock solutions (100µg/mL) of both drugs are prepared separately. The concentration of both stock solution FAM and OFL (10 mg) each were weighed accurately and separately transferred into individual 100mL volumetric flasks. The mobile phase added up to 75mL in both volumetric flask and solutions were sonicated to get the maximum dissolution of the drugs. The volume was made up to the mark 100mL with the mobile phase. Further dilutions were made in the concentration range 10 – 50µg/mL for both drugs FAM and OFL from stock solutions (100 µg/mL) respectively. A volume 20µL of each sample was used for injection.

 

3.6.7. Selection of wavelength: 

The standard stock solution 100µg/mL of FAM and OFL were prepared separately by weighing 10mg drug samples and transferred to 100mL volumetric flask separately and volume made up to mark with the mobile phase. From stock solution pipette out 1mL was transferred to 10mL volumetric flask and made up the volume up to the mark with the mobile phase for both drugs separately. The resulting solutions were scanned over the UV range (200 - 400nm), maximum absorbance was found at λ_max 274nm for both the drugs (overlain spectra).

 

3.6.8. Preparation of buffer: 

The 0.1M potassium dihydrogen orthophosphate buffer (pH 2.3) was used for method development. Buffer was prepared by dissolving 13.6gm of potassium dihydrogen orthophosphate with Mili – Q water to 1000mL. The pH was adjusted with dilute orthophosphoric acid to 1000 mL. The prepared buffer was filtered through a 0.45µm membrane filter.  

 

3.6.9. Preparation of mobile phase:  

Mobile phase was prepared by mixing 0.1M potassium dihydrogen orthophosphate buffer (pH 2.3) and methanol (HPLC grade) in 60:40v/v proportions. The mixture solution was sonicated for 30min prior to use.

 

3.6.10. Preparation of stock solutions and test solutions: 

The stock solution was prepared by dissolving FAM and OFL (10mg each) that were weighed accurately and separately transferred into 100mL volumetric flasks. Similarly, both drugs mixture of FAM and OFL was prepared as that of the stock solution by dissolving 10 mg of both drugs in 70mL of the mobile phase, then sonicated for 30min and diluted up to 100mL. A series of solutions were prepared in the concentration range of 10 – 50µg/mL both drugs FAM and OFL respectively.

 

3.6.11. Preparation of calibration curve:  

The calibration curve was prepared by injecting a concentration of 10 – 50µg/mL of FAM and OFL, both drugs mixture solutions manually in duplicate to the HPLC system at a detection wavelength of 274nm. The mean of n = 3 determinations was plotted as the standard curve. The calibration curve was tested and validated with inter and intraday, reproducibility, specificity, and ruggedness.

 

4. RP–HPLC METHOD:  

For tablet formulation, an assay was performed to check the percentage purity of each drug and also calculated. The 20µL of standard and sample solution was injected by knowing the peak area of FAM and OFL, the amount of both drugs in the sample were calculated.

 

4.1. Method validation:

The simultaneous spectrophotometry and RP–HPLC methods were validated as recommended by the International Conference on Harmonization for all validation parameters including specificity, linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ).

 

4.1.6. Assay method validation:

For in-house formulation, the label claim contains FAM 40 mg and OFL 200mg. Its dissolution time was found to be 3 min and hardness was found to be 2.5kg/cm². An assay was performed to check the purity of each drug in the formulation and also calculated the percentage purity of the drugs.

 

 

(a)

 

(b)

Fig. 2 (a) First order spectra of Famotidine 5-50μg/ml in methanol: water (50:50), and (b) First-order spectra of Ofloxacin 5-50μg/ml in methanol: water (50:50).

5. RESULT AND DISCUSSION:

5.1. UV – spectrophotometric method:

5.1.1 Method validation:

5.1.1.1. Linearity:

In zero-order, method (Vierodt’s method) was established for the linearity range of both drugs in the concentration range 5 – 50µg/mL at λ_max 273nm FAM and 280nm OFL, respectively. The spectra of drugs are shown individually in Fig. 2 (a) and (b). Similarly, calibration curves were as plotted shown in Fig. 3 (a) and (b). These calibration curves slope and intercept with its correlation coefficient were obtained and data shown in Table 2. Thus, regression equations were obtained for both drugs.

 

In the first-order derivative method, the wavelengths were selected for determination of individual 273nm FAM and 280nm OFL with the same concentration range of 5 - 50µg/mL respectively. The linearity curves of FAM and OFL were developed by the first-order derivative method was shown in Table1. All the parameters of first-order derivative spectroscopy are mentioned in Table 2.

 

Fig. 3 (a) calibration curve at 273 nm for Ofloxacin in methanol: water (50:50) by first-order derivative spectroscopy, (b) Calibration curve for Ofloxacin at 280 nm in methanol: water (50:50) by first-order derivative spectroscopy.

Table 2. Statistical data of Famotidine and Ofloxacin at 273 nm and 280 nm respectively by first-order derivative spectroscopy.

 

Table 2. Determination of Accuracy of Famotidine and Ofloxacin by first order derivative spectroscopy.

 

5.1.1.2. Accuracy as a recovery method:  

The accuracy of zero and first-order derivative methods was determined by the recovery studies. Recovery study results were found to be within the acceptance criteria 100±10%, indicating a good degree of sensitivity of the method towards the detection of analytes in house formulation sample. The recovery study results of FAM and OFL are mentioned in Table 2 for both UV spectrophotometric methods.

 

5.1.1.3. Precision: 

Standard dilutions were prepared and three replicates of each dilution were analyzed interday and intraday for repeatability. The values of precision studies for UV – spectrophotometric methods are shown in Table 3 and 4 (for the first derivative method). 

Table 3.  Determination of Precision for Famotidine at 273 nm by first order derivative spectroscopy.                      n=3

 

Table 4. Determination of Precision for Ofloxacin at 280 nm by first order derivative spectroscopy.

 

 

5.1.1.5. Repeatability:

Standard solutions of FAM and OFL (5-50μg/ml) were prepared and absorbance was measured for both drugs wavelength at 273nm and 280nm respectively. The absorbance of the same concentration solutions was measured three time and the standard deviation was calculated. Result was shown in Table 5 and 6.

 

5.1.1.6. LOD and LOQ:  

In the first order derivative method, LOD and LOQ were found to be the same concentration 5 - 50 µg/mL for both the drugs FAM and OFL in Table 8

 

Assay of in house formulation: 

For in house formulation, an assay was performed to check the purity of each drug in the formulation and also calculated the percentage purity of the drugs. Percentage purity estimation for FAM and OFL was found to be first order method results are shown in Table 7.

 

Table 5. Repeatability data for Famotidine at 273 nm by first order derivative spectroscopy.                    n=3

 

 

 

Table 6. Repeatability data for Ofloxacin at 280 nm by first order derivative spectroscopy

 

 

Table 7. Assay results of Marketed Formulation by first order derivative spectroscopy.

 

 

Table: 8. Summary of validation parameters of spectrophotometry by first order derivative spectroscopy

 

 

Figure 4. (a) Overlain spectra of FAM and OFL at 274 nm for RP- HPLC method.

 

 

Figure 4. (b) Chromatogram representing retention time of a standard solution containing 50µg/mL FAM.

 

 

Figure 4. (c) Chromatogram representing retention time of a standard solution containing 50 µg/mL OFL.

 

 

Figure 4. (d) Chromatogram representing retention time of a mixed standard solution containing 40µg/mL for FAM and 200µg/mL OFL.

 

5.1.2. RP – HPLC method:

5.1.2.1. Validation of the proposed method specificity:  

The retention time for FAM and OFL was 2.44min and 7.99 min, respectively. The wavelength and chromatograms have been shown in Fig. 4 (a) (b) (c) and (d).

 

 

(a)

 

(b)

Figure 5. (a) Calibration Curve for FAM by RP-HPLC (b) Calibration Curve for OFL by RP-HPLC.

 

5.1.2.2. Linearity: 

Linear correlations were obtained for both drugs FAM and OFL in the same concentration range 5 – 50µg/mL with the correlation coefficient of 0.9913 and 0.9908 respectively. The calibration curve was plotted using the area under the curve versus concentration of a standard solution as shown in Fig. 5 (a) and (b). Peak areas were recorded for all the peaks. The linearity data values were shown in Table 9. A six-replicate calibration curve was obtained with working standards and was found linear (r²>0.991) for each of the analyte over their calibration ranges. The slopes were calculated using the plot of drug concentration versus the area of the chromatogram. Linearity was found to be quite satisfactory and reproducible as shown in Figure 5.

 

 

Table 9. Statistical data of Famotidine and Ofloxacin by RP-HPLC

 

 

5.1.2.3. Accuracy as recovery: 

The accuracy was determined by recovery studies. The recovery study results were found to be within the acceptance criteria 100 ±10% indicating the good degree of sensitivity of the method towards the detection of analytes in the sample. The accuracy values studies for the RP – HPLC methods are shown in Table 10.

 

 

Table 10. Determination of Accuracy of Famotidine and Ofloxacin by RP-HPLC

 

5.1.2.4. Precision:

Six replicates of each dilution were analyzed on intermediate intraday and interday for repeatability. The values of the RP-HPLC precision studies are shown in Table11 and 12.

 

Table 11. Determination of Precision for Famotidine by RP-HPLC

 

Table 12. Determination of Precision for Ofloxacin by RP-HPLC

 

5.1.2.5. LOD and LOQ:

For the RP-HPLC method LOD were found to be 7.0240 and 9.006 µg/mL, whereas LOQ were found to be 12.951 and 16.898 µg/mL for both drugs FAM and OFL respectively. The values of the LOD and LOQ in the RP-HPLC method are shown in Table 9.

 

5.1.2.6. Assay of in house formulation:

For in house formulation, an assay was performed to check the purity of each drug in the formulation and the percentage purity of the drugs was calculated. Percentage estimation of FAM and OFL was found to be 97.35 and 97.70%, respectively. The results are shown in Table 13.

 

 

Table 13. Assay results of in-house Formulation by RP-HPLC

 

 

Table 14. Summaries of Validation Parameters by RP-HPLC

 

6. CONCLUSION:

Pharmaceutical analysis occupies a pivotal role in statuary certification of marketed drugs and their formulations either by the industry or by the regulatory authorities. Noteworthy, the purpose of new analytical methods development for the determination of drugs in pharmaceutical dosage can make, it feasible to study pharmacokinetics, toxicological and biological. The proposed method has been developed and it was novel, simple, accurate, precise, reproducible, and economical, which can be used to estimate famotidine and Ofloxacin in their combined dosage form without any interference from the excipients. The developed method was successfully validated as per ICH guidelines. In the absence of an official monograph, these validated methods can be used for routine quality control analysis.

 

7. ACKNOWLEDGEMENTS:

We are thankful to our Managing Director Dr. Ran Singh, Laureate Institute of Pharmacy, Kathog, H.P. for providing the proper resources and infrastructure to carry out the research work. The author would like to acknowledge Dr. Amar Deep Ankalgi for helping in writing the manuscript.

 

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Received on 20.07.2022       Modified on 03.10.2022

Accepted on 07.12.2022   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2023; 13(3):162-170.