Development and Validation of Simultaneous Spectroscopic Method for Tetracycline Hydrochloride and Clotrimazole

Abstract

A simple and accurate simultaneous method was developed for tetracycline hydrochloride (TH) and clotrimazole with a mixture of solvents. the combination is used to Validate the developed UV-spectrophotometric method as per ICH guidelines, The concentration of Tetracycline hydrochloride & clotrimazole in the sample solution was determined by solving the respective simultaneous equations generated by using absorptivity coefficients and absorbance values of TH & Clotrimazole at these wavelengths 361 nm and 259 nm respectively. and both satisfy Beer\'s law in concentration ranges of 4-20 µg/ml for TH and 10-30 µg/ml for Clotrimazole. The method was effectively used to estimate TH and clotrimazole in Buccal film without the interference of common excipients.

Introduction

I. INTRODUCTION

A combination of tetracycline hydrochloride and clotrimazole has been devised to treat Staphylococcus aureus-induced infective endocarditis. Antibiotic resistance has developed in Staphylococcus aureus. The S. aureus major facilitator superfamily (MFS) efflux pump Tet causes tetracycline resistance (K). Whereas biofilm hinders the diffusion of antibiotics. None of the compounds that are currently known have had efflux pump inhibitors (EPIs) licensed for use in clinical settings.  We discovered clotrimazole, which is inhibitor of fungus-produced ergosterol production, with possible EPI-like potential and fractional inhibitory concentrations, indicating synergism. The literature search revealed that, although a promising combination, there is no method available for simultaneous estimation of TH & Clotrimazole. Therefore, developing a UV spectrophotometric method for the simultaneous estimation of TH and clotrimazole is challenging. Therefore, the aim of the present work was to develop & validate a UV spectroscopic method for simultaneous estimation of TH and Clotrimazole.

Tetracycline hydrochloride, is chemically (4S,4aS,5aS,6S,12aS)-4- Dimethylamino- 3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo- 1,4,4a,5,5a, 6,11,

12a-octahydrotetracene-2- carboxamide monohydrochloride, as shown in fig.1[1]

Over the range of 200-400 nm, a UV-spectrophotometer UV-1800 (Shimadzu, Japan) with a spectral bandwidth of 2 nm and 10 mm matched quartz cells was utilised to create an analytical procedure.

II. MATERIAL AND METHOD

A. Chemical and Instrument

The reference standard Clotrimazole (99.4%) was received as a gift sample from halcyon sewri west, Mumbai-400015, and tetracycline hydrochloride (99%) was received as a gift sample from Medley, Andheri, Mumbai-400069. All solvents used for analysis were of analytical grade and were procured from Vishal Chem. India.

Over the range of 200-400 nm, a UV-spectrophotometer UV-1800 (Shimadzu, Japan) with a spectral bandwidth of 2 nm and 10 mm matched quartz cells was utilised to create an analytical procedure.

B. Development of the Method

During the development of the method, the spectrum of each drug was measured in different solvents. This was done to enable the selection of the solvent system and concentration range. Based on the spectrum, the solvent selected was methanol and water for both drugs. The method was developed and validated as per the procedures mentioned.

C. Preparation of Standard Stock Solution

Accurately weighed clotrimazole (50 mg) and tetracycline hydrochloride (TH) (50 mg) were transferred to a 50 mL volumetric flask, dissolved, and diluted to the mark with methanol to obtain a standard solution having a concentration of tetracycline and clotrimazole (1000 ppm). Once more, 10 ml of TH and 10 ml of clotrimazole were withdrawn respectively, and diluted to 100 ml each with methanol in a volumetric flask of 100 ml (100 ppm).

D. Calibration curve of Tetracycline Hydrochloride and Clotrimazole

A series of calibrated 10mL volumetric flasks were taken and appropriate aliquots of stock solution of TH and clotrimazole were withdrawn and diluted up to 10mL with water[2], Different dilutions were made from this stock solution, ranging from 4 µg/ml to 20 µg/ml for TH. The absorbance was measured at absorption maxima 361 nm for TH against the reagent blank prepared in a similar manner without TH i.e., water. The same procedure was applied for clotrimazole stock solution solutions having concentrations of 10, 15,20,25,30 μg/mL) and absorbance was measured at 259nm, against a reagent blank prepared in a similar manner without clotrimazole.

Conclusion

The quantitative determination of tetracycline hydrochloride and clotrimazole using UV spectroscopy was done quickly, cheaply, linearly, repeatedly, specifically, and economically. After the technique was validated, it was determined that all the method validation parameters were satisfactory. Regular analysis of samples of marketed tetracycline hydrochloride and clotrimazole as well as in-process quality control can be done using the described method.

References

[1] Khaleel, Roaa & Mohammed, Dawooh. (2020). Spectrophotometric Determination of tetracycline hydrochloride Using 2,4 –dinitrophenyl hydrazine as Coupling Reagent. Journal of Physics: Conference Series. 1664. 012084. 10.1088/1742-6596/1664/1/012084. [2] Aleem H, Zhao Y, Lord S, McCarthy T and Sharratt P. Pharmaceutical process validation: an overview, Journal of Process Mechanical Engineering,2003; 2(217) :P 141-151. [3] Nash R. A, Wachter A,Marcel Dekkar H. Pharmaceutical Process Validation An International Third Edition. Revised and Expanded, New York, 2003; 129:P760-792. [4] Shah VP, Midha KK, Dighe S, McGilveray IJ, Skelly JP, Yacobi A, Layloff T, Viswanathan CT, Cook CE, McDowall RD, Pittman KA, Spector S. Analytical methods validation: bioavailability, bioequivalence, and pharmacokinetic studies. Conference report Pharma Research. 1992; 9:P 588-592. [5] US FDA Technical Review Guide: Validation of Chromatographic Methods.Center for Drug Evaluation and Research (CDER). Rockville, MD. 1993: P 112- 119. [6] Hartmann C, Smeyers-Verbeke J, Massart DL, McDowall RD. Validation of bioanalytical chromatographic methods. Journal of Pharmaceutical and Biomedical Analysis, 1998; 17:P 193-218. [7] Dadgar D, Burnett PE, Choc MG, Gallicano K, Hooper JW. Application issues in bioanalytical method validation, sample analysis and data reporting, Journal of Pharmaceutical and .Biomedical Analysis.1995; 13: P 89-97. [8] ravichandran v, 1 shalini s,1 sundram k. and harishrajak. validation of analytical methods – importance and strategies. International Journal of Pharmacy and Pharmaceutical Sciences.2010; 5:P112-115. [9] Bressolle F, Bromet PM, Audran M. Validation of liquid chromatographic and gas chromatographic methods Applications to pharmacokinetics. Journal of Chromatography.1996; B 686:P3-10. [10] Causon R. Validation of chromatographic methods in biomedical analysis viewpoint and discussion. Journal of Chromatography. 1997; B 689 :175-180. [11] Hartmann C, Smeyers-Verbeke J,Massart DL, McDowall RD. Validation of bioanalytical chromatographic methods. Journal of Pharmaceutical and Biomedical Analysis. 1998; 17:P 193-218. [12] Shah VP, Midha KK, Findlay JW, Hill HM, Hulse JD, McGilveray IJ, McKay G, Miller KJ, Patnaik RN, Powell ML, Tonelli A, Viswanathan CT, Yacobi A.Bioanalytical method validation--a revisit with a decade of progress.journal of Pharmaresearch. 2000; 17:P 1551-1557.

Copyright

Copyright © 2023 Janhavi Mishra, Jaya Agnihotri. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.