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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2023 The   Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access  Full Text Article                                                                                                                                       Research Article

Comparative UV Spectroscopic Method Analysis and Validation for Estimation of Rifaximin in Pharmaceutical Preparation          

Dr. Aney Joice¹* and Farheen Mohammed Zubair Sange²                                                               

¹ Assistant Professor Department of Pharmaceutics, M.C.E Society’s Allana College of Pharmacy, Pune-411001, Maharashtra, India.

² Postgraduate Student M. Pharm Department of Pharmaceutical Quality Assurance, M.C.E Society’s Allana College of Pharmacy, Pune-411001, Maharashtra, India

INTRODUCTION

Rifaximin

Rifaximin is an oral antibiotic with broad spectrum of action that acts locally in the gastrointestinal tract with minimal systemic adverse effects. It is used for the treatment of traveler’s diarrhea caused by non-invasive strains of E. coli. It is benzimidazole derivative. Rifaximin is a product of synthesis of Rifamycin, an antibiotic with low gastro- intestinal absorption and good anti-bacterial activity. It acts on the ß-subunit of the deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase enzyme of the microorganism to inhibit RNA synthesis. Rifaximin (C₄₃H₅₁N₃O₁₁ and molecular weight 785.9 g mol⁻¹) is a derivative of rifamycin and is a structural analogue of rifampicin. ^1-5

Figure 1: Chemical Structure of Rifaximin

UV Spectroscopy

A UV-visible spectrophotometer records a UV or visible spectrum as a plot of wavelengths of absorbed radiations versus the intensity of absorption in terms of absorbance (optical density) A or molar absorptivity (molar extinction coefficient) e as defined by the Lambert-Beer law. According to Lambert's law, the fraction of incident. Monochromatic radiation absorbed by a homogeneous medium is independent of the intensity of the incident radiation while Beer's law states that the absorption of a monochromatic radiation by a homogeneous medium is proportional to the number of absorbing molecules ^6,7.

The absorption of electromagnetic radiations in the UV and visible regions induces the excitation of an electron from a lower to higher molecular orbital (electronic energy Level). UV Visible spectroscopy is also called as electronic spectroscopy in which the light is absorbed at each wavelength of UV and Visible region of electromagnetic spectrum. Organic chemists use ultraviolet and visible spectroscopy mainly for detecting the presence and elucidating the nature of the conjugated multiple bonds or aromatic rings ⁷.

UV-Visible spectrophotometry is one of the most important technique used in analytical chemistry in the pharmaceutical analysis which is being used in the quantitative analysis of a specific analyte. It involves measuring the amount of ultraviolet or visible radiation absorbed by a substance in solution. Instrument which measures the ratio, or function of ratio, of the intensity of two beams of light in the U.V. visible region is called Ultraviolet-Visible Spectrophotometer ⁸.

In qualitative analysis, the analysis of conjugated organic compounds and transition metal ions can be identified by use of spectrophotometer, if any recorded data is available, and quantitative spectrophotometric analysis is used to ascertain the quantity of molecular species absorbing the radiation. Spectrophotometric technique is simple, rapid, moderately specific and applicable to small quantities of compounds ⁹.

Ultraviolet and visible (UV) spectroscopy records the absorption of radiations in ultraviolet and visible regions of the electromagnetic spectrum. The ultraviolet radiation extends from 10nm to 400nm and the visible radiation extends from 400nm to 800nm. 

MATERIAL AND METHODS 

Shimadzu UV - 1700 UV/VISIBLE spectrophotometer with UV probe 2.10 software and 1 cm matched quartz cells were used for absorbance measurements. Make- Mettler Toledo, Model- X was used as analytical balance for weighing standard and sample.

2.1. Preparation of Standard Stock Solution

Standard Stock Solution was prepared by dissolving 10mg of the drug in 10ml of 0.1N HCL pH 1.2, Phosphate buffer pH 6.8 & Phosphate buffer pH 7.4 to get concentration of 1000 µg/ml. From the above Standard Stock Solution, working standard solution was prepared containing 100 µg/ml of Rifaximin.

2.2. Selection of Wavelength for Analysis

From the Standard Stock Solution (1000ug/ml) further dilutions were made using 0.1N HCL pH 1.2, Phosphate buffer pH 6.8 & Phosphate buffer pH 7.4 and scanned over the range of 200-800 nm and the spectra was obtained using 0.1N HCL pH 1.2, Phosphate buffer pH 6.8 & Phosphate buffer pH 7.4 as a blank.

2.3. Preparation for Calibration Curve

Aliquots of standard stock solution were further diluted with 0.1N HCL pH 1.2, Phosphate buffer pH 6.8 & Phosphate buffer pH 7.4 to get the solutions of concentration within range 2, 4, 6, 8, 10 µg/mL. The absorbance was measured using 0.1N HCL pH 1.2, Phosphate buffer pH 6.8 & Phosphate buffer pH 7.4 as blank. All measurements were repeated three times for each concentration.

2.4. Assay of Rifaximin in Tablet 

Twenty tablets were weighed; their average weight was determined and finely powdered. Powder equivalent to 50mg Rifaximin of was accurately weighed and dissolved in small amount of methanol in 50 mL volumetric flask and then the volume was adjusted with methanol to obtain the final concentration 1000 µg/mL. From this, 10 mL solution was taken and diluted up to 100 mL with the same solvent in a volumetric flask to obtain the solution of concentration 100 µg/mL. From this solution, aliquot of 2 mL was diluted to 10 mL using methanol. The drug content was measured using UV spectrophotometer.

2.5. METHOD VALIDATION ^10,11,12

The analytical method was validated as per ICH guidelines for following parameters

Aliquots of standard stock solution were further diluted with 0.1N HCL pH 1.2, Phosphate buffer pH 6.8 & Phosphate buffer pH 7.4 to get the solutions of concentration within range from 2, 4, 6, 8, 10 µg/mL. The absorbance was measured at wavelength 440 nm, 439 nm & 440nm. Linear calibration graph was obtained by plotting the absorbance value versus concentration of Rifaximin.

To ensure accuracy of the method, recovery studies were performed by standard addition method at 80%, 100% and 120% level to pre-analyzed samples and subsequent solutions were reanalyzed. At each level, three determinations were performed. Accuracy is reported as % recovery.

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision of the method was determined in terms of repeatability and intra-day and inter-day precisions.

In UV method development LOD & LOQ was determined by utilizing the following equation 

LOD = 3.3 X SD/S

LOQ = 10 X SD/S

Where, S= Slope, SD= Standard deviation of Y-intercepts.

Robustness of the method was determined by carrying out the analysis under additions during which scanning wavelength was altered. Time was also changed from spotting to development to scanning and the effect on the area were noted.

3.1 Selection of Wavelength for Analysis 

The UV spectrum of Rifaximin showed the maximum absorbance at the wavelength 439 nm,440 nm & 433 nm respectively for 0.1N HCL pH 1.2, Phosphate buffer pH 6.8 & Phosphate buffer pH 7.4 [ Figure 2-4]. It was selected for the analysis of Rifaximin in bulk and tablet formulation.

Figure 2: UV Spectrum of Rifaximin in 0.1N HCL (pH 1.2)

Figure 3: UV Spectrum of Rifaximin in Phosphate buffer (pH 6.8)

Figure 4: UV Spectrum of Rifaximin in Phosphate buffer (pH 7.4)

3.2 Preparation of the Calibration Curve 

The calibration curve was constructed by plotting absorbance against corresponding concentration as shown in [Figure 5, 6 & 7] The calibration curve for Rifaximin. The drug obeyed Beer–Lambert’s law in the concentration range of 2, 4, 6, 8, 10 µg/mL with coefficient of correlation (r²) of 0.998. [Table 1]

Figure 5: Calibration Curve of Rifaximin in 0.1N HCL (pH 1.2)

Figure 6: Calibration Curve of Rifaximin in Phosphate buffer (pH 6.8)

Figure 7: Calibration Curve of Rifaximin in Phosphate buffer (pH 7.4)

Table 1: Linearity data of Rifaximin in 0.1N HCL of 0.1N HCL pH 1.2, Phosphate buffer pH 6.8 & Phosphate buffer pH 7.4.

3.3 Assay of Rifaximin in Tablet

The amount of Rifaximin present in formulation was calculated by comparing the absorbance of sample with standard absorbance. Content of Rifaximin in tablet formulation determined by developed method was in good agreement with the label claim. [ Table 2]

Table 2: Assay of Tablet Formulation by UV method

3.4.1. Accuracy

The responses were reanalyzed using the suggested method, and the accuracy results are shown in [Table 3-5], which demonstrate that the percentage amount recovered was between 98.60%-99.96%, 95.12% - 95.59% & 98.17%-98.87% with % RSD less than 2.

Table 3: Results of Accuracy for Rifaximin in 0.1N HCL (pH 1.2) 

Table 4: Results of Accuracy for Rifaximin in Phosphate buffer (pH 6.8)

Table 5: Results of Accuracy for Rifaximin in Phosphate buffer (pH 7.4)

3.4.2. Precision

The developed method's precision was reported as a % RSD. These findings demonstrate the assay's repeatability. % RSD values less than 2 shows that the method for determining rifaximin is precise. [Table 6-8]

Table 6: Results of Precision for Rifaximin in 0.1N HCL (pH 1.2) 

Table 7: Results of Precision for Rifaximin in Phosphate buffer (pH 6.8)

Table 8: Results of Precision for Rifaximin in Phosphate buffer (pH 7.4) 

3.4.3. LOD & LOQ

By using the given formula, the LOD & LOQ were calculated for rifaximin in 0.1N HCL pH 1.2, Phosphate buffer pH 6.8 & Phosphate buffer pH 7.4 respectively in [ Table 9]

Table 9: Results of LOD & LOQ                                                               

3.4.4. Robustness

This method's robustness was tested using variations in wavelength change. The experimental results demonstrated that the suggested UV technique is robust, with the change since% RSD being less than 0.9%. [Table 10-12]

Table 10: Results of Robustness for Rifaximin in 0.1N HCL (pH 1.2)

Table 11: Results of Robustness for Rifaximin in Phosphate buffer (pH 6.8) 

Table 12: Results of Robustness for Rifaximin in Phosphate buffer (pH 7.4) 

3.5 THE SUMMARY OF VALIDATION PARAMETERS BY UV METHOD

Table 13: Summary of Results of Validation Parameters by UV Method

The present study reports a comparative validations data of UV spectrophotometric analysis for qualitative determination of Rifaximin in bulk drug and formulation. Validation of UV method was conducted at different pH (1.2, 6.8 & 7.4) solutions with the wavelengths of 439nm, 440nm & 433 nm respectively. The results of the study suggest that the analytical approach described is relatively simple, accurate, precise & reproducible. Hence, the UV method is suitable for routine determination of Rifaximin in pharmaceutical formulations.

REFERENCES

1. https://link.springer.com/chapter/10.1007/978-1-4020-2575-4_2

2. Rivkin A., Gim S. Rifaximin: new therapeutic indication and future directions. Clinical Therapeutics. 2011; 33(7):812-827. https://doi.org/10.1016/j.clinthera.2011.06.007 PMid:21741091

3. Sudha T., Anandakumar K., Hemalatha P. V., Ravikumar V. R., Radhakrishnan Spectrophotometric estimation methods for Rifaximin in tablet dosage form. International Journal of Pharmacy and Pharmaceutical Sciences. 2010; 2(1):43-46.

4. Mullen K. D., Sanyal A. J., Bass N. M., et al. Rifaximin is safe and well tolerated for long-term maintenance of remission from overt hepatic encephalopathy. Clinical Gastroenterology and Hepatology. 2014; 12(8):1390-1397. https://doi.org/10.1016/j.cgh.2013.12.021 PMid:24365449

5. Yoshida, T. Bioorg & Med Chem., 2012; 20:5705-5719.

6. Scott, A.I., Interpretation of Ultraviolet Spectra of Natural Products, Pergamon Press, New York, 1964.

7. Skoog, D.A. Holler, F.J., Nieman, D.A., Introduction to UV Spectroscopy in, principle of instrumental analysis, 5thed., Cole publication, 2004.

8. Chatwal G. R, Anand S. K, "Instrumental Methods of Chemical Analysis", 5 th Edn., Himalaya Publishing House, New Delhi, 2002; 566-587, 624-639. 3. Beckett. A. H., Stenlake J. B. Practical Pharmaceutical chemistry CBS Publishers and distributors, New Delhi., 1997, Ultraviolet visible absorption spectrophotometric. 2002; 275-278.

9. Rivkin A., Gim S. Rifaximin: new therapeutic indication and future directions. ClinicalTherapeutics. 2011; 33(7):812-827. https://doi.org/10.1016/j.clinthera.2011.06.007 PMid:21741091

10. International Conference on Harmonization. ICH. (2005). ICH - Q2 (R1): Guideline on Validation of Analytical Procedure: Text and Methodology.

11. International Conference on Harmonization, ICH (2003). Q2 (R1): Validation of analytical procedures: test and methodology.

12. Martindale. The Extra Pharmacopoeia. Published by direction of the Council of Royal Pharmaceutical Society of Great Britain. 34th ed, Vol. 11. London Royal Pharmaceutical Society, 2005; 653-656.