Development and Validation of UV–Spectrophotometric method for the Determination of Enrofloxacin in Synthetic form and Veterinary Injectible Dosage forms

 

Tasneem O. ElmahadiI², Shaza W. A Shantier¹ and Mohamed E. Adam^1*

¹Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Khartoum, Sudan

²Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Omdurman Islamic University Omdurman, Sudan

 

ABSTRACT:

Simple, sensitive and accurate spectrophotometric method was developed for the estimation of Enrofloxacin (EFC) in bulk and injection dosage form. The method was based on the measurement of EFC solution in 0.1M potassium hydroxide at 271nm. Validation was conducted in accordance to ICH guidelines. The developed method was found to be linear in the concentration range 2-10 µg/ml (r= 0.999), sensitive (LOD was found 0.127 µg/ml) and precise (RSD% values are less than 2%). Freedom from interference by the formulation excipients was proved by the good recovery percentages’ results (100.06% ± 1.12, n=3). The proposed method was successfully used to estimate EFC in laboratory mixture and in the injection dosage forms.

 

 

 

INTRODUCTION:

Enrofloxacin (EFC) (Figure 1), a zwitterionic molecule, is a synthetic chemotherapeutic agent of the fluoroquinolone carboxylic acid derivatives¹.  It is active against a wide spectrum of Gram-negative and Gram-positive bacteria. It is believed that this drug inhibits bacterial DNA gyrase (a type-II topoisomerase), DNA supercoiling and DNA synthesis². Animals with impaired kidney or liver function may need monitoring and dose adjustments to prevent excess drug accumulation, because enrofloxacin is eliminated by both renal and hepatic metabolism³.

 

The literature reveals many methods for analysis of enrofloxacin in formulations and biological fluids alone or combined with another drugs. These methods include: HPLC methods^4-8, FPT and ELISA methods⁹, electrophoresis–electrochemiluminescent method¹⁰. No previous UV spectrophotometric method has been reported for the estimation of EFC in pharmaceutical formulations. Thus, the present study describes a direct, simple and accurate UV spectrophotometric method for the estimation of EFC laboratory mixture and injection dosage forms.  

 

MATERIALS AND METHODS:

Materials:

Standard and sample:

Enrofloxacin working standard (Purity 99.0%, Expiry date: 27.11.2019) was kindly provided by Bach Pharma CO. Ltd, Sudan. Injections dosage form (10%w/v; 100ml) were purchased from the local market.

 

Diluent:

Potassium hydroxide solution (0.01M) was used as diluent in all experiments.

 

Instrument:

UV-Visible spectrophotometer, Shimadzu, Kyoto, Japan.

 

Preparation of stock solutions:

EFC standard solution:

EFC standard (10mg) was accurately weighed and dissolved in about 60ml of 0.01M potassium hydroxide. The resultant solution was transferred to 100ml volumetric flask and the volume was completed to mark with the diluent (solution A; 100µg/ml).

 

EFC sample solution:

One ml of enrofloxacin injection (10%w/v) was withdrawn, transferred into 100 ml volumetric flask and the volume was completed to 100ml with the diluents. 1 ml of the resultant solution was further diluted to 100ml (solution B; 100µg/ml). 

 

Synthetic mixture:

EFC standard (0.1g) was accurately weighed and transferred into 100 ml volumetric flask; 50 ml of placebo was added, shaken and then completed to mark with the diluent (Solution C; 100µg/ml).

 

Selection of wavelength of maximum absorbance (λ_max):

Solution A was scanned in the range 200-400 nm against the diluent as blank. The wavelength of maximum absorbance was then selected.

 

Method validation:

Specificity:

One ml of placebo was transferred into 100 ml of volumetric flask and volume was completed to mark.  1ml of solution was further diluted to 100ml then the solution was scanned between 200-400nm.

Linearity:

Aliquot volumes of solution A (1- 5ml) each were transferred into set of 50 ml volumetric flasks. Each solution was completed to 50 ml with the diluent to obtain concentrations of 2-10 µg /ml. The absorbance values of these solutions were measured at 271 nm against the diluent as blank. Calibration curve was then constructed by plotting absorbance vs. concentration.

 

Precision:

In order to assess the precision of the developed method in terms of inter-day and intra-day precision, Absorbance values of six replicates of solution A (8µg/ml) were measured in the same day and in three different days. RSD% values were then calculated.

 

Accuracy:

Three volumes of solution B (2ml each) were transferred into 100ml volumetric flasks. 2ml, 4ml and 6ml of solution A were added respectively to the above solutions. Volumes were then completed to mark with diluent and absorbance was recorded at 271nm. The recovery% was calculated using the formula¹¹:

 

                Absorbance of mixture –Absorbance of sample

Recovery% =-------------------------------------------- X 100

                                 Absorbance of standard

 

Determination of content %:

Serial dilutions of solution B and C were treated as under linearity. The injection and synthetic mixture content were determined by the direct sample/ standard comparison.

                              Absorbance of sample

 Content % =-------------------------------------------- X 100

                                 Absorbance of standard

 

RESULTS AND DISCUSSION:

Despite the fact that UV spectrophotometry lacks selectivity, it is the most commonly used method for quantitative analysis of many drugs in their raw materials and pharmaceutical formulations. This is due to its simplicity and accuracy.  Scanning of the placebo showed no absorption at 271 nm (Figure 2) where solution of EFC in 0.01M potassium hydroxide has maximum absorption (Figure 3).

 

 

 

Figure 2: UV Spectrum of the Placebo

 

 

Figure 3: UV Spectrum of EFC standard solution (10µg/ml)

 

Linearity:

Linearity was checked by measuring the absorbance of serial dilutions of solution A and plotting the absorbance values vs. concentrations (Figure 4). The obtained regression analysis data was summarized in Table 1.

 

Table 1: Linearity data of the developed method

*Standard error of slope calculated at 95% confidence level for n-2 degrees of freedom

** Standard error of intercept calculated at 95% confidence level for n-2 degrees of freedom

 

 

Figure 4: Calibration curve of EFC solution (271nm)

 

 

 

Precision:

Method precision was assessed in term of repeatability and reproducibility. The RSD% values were 1.024% and 1.46%, respectively which reflects the precision of the developed method (RSD% ˂ 2).

 

Accuracy:

The accuracy of the method was determined by recovery experiments. The recovery studies were carried out for test concentration at spiking levels of 50%, 100% and 150%. Obtained results are summarized in Table 2 which are satisfactory and reflecting the accuracy of the method and its freedom from interferences by excipients.

 

Table 2. Accuracy of the developed method

 

Assay of Enrofloxacin in laboratory mixture and injection dosage form:

The developed method was applied for determination of EFC laboratory mixture   and injection formulation. The mean content percent of three independent analyses gave good results (99.2 ±0.14% ± and 101.00 ± 0.64%, respectively).

 

CONCLUSION:

An accurate and precise UV spectrophotometric method has been developed and validated for the analysis of Enrofloxacin in laboratory mixture and in injection formulation. The percentage recovery and concentration of active ingredient in pharmaceutical formulations showed that the amount of drug present is consistent with the label claim. Hence, it is a very useful simple and accurate method; it can be used for the routine analysis of Enrofloxacin in pharmaceutical dosage form.

 

CONFLICT OF INTEREST:

Authors declare no conflict of interest.

 

REFERENCES:

1.     Barbosa J, Barrón D, Jiménez-Lozano E and Sanz-Nebot V. Comparison between Capillary Electrophoresis, Liquid Chromatography, Potentiometric and Spectrophotometric Techniques for Evaluation of pKa Values of Zwitterionic Drugs in Acetonitrile-Water Mixtures. Analytica Chimica Acta. 2001; 437: 309-321

2.     Boothe DM. Enrofloxacin revisited. Vet. Med., 1994; 8: 744- 753.

3.     Gardiner H. Administration seeks to restrict antibiotics in livestock. The New York Times, 2008, P A18.

4.     Pavani P, Rajeswari TR, Ramana RG, and Satyanarayana. Development and Validation of Stability Indicating RP-LC Method for Estimation of Related Substances of Enrofloxacin in Bulk and Its Pharmaceutical Formulations. Research Journal of Pharmaceutical, Biological and Chemical Sciences, RJPBCS. 2016; 7(1): 1440-1450.

5.     Violeta T, Elena P and Viorica C. Development and validation of an HPLC method for the determination of oxytetracycline and enrofloxacin in veterinary formulations. Medicamentul Veterinar / Veterinary Drug. 2015; 9(2): 65-69.

6.     Sujittra P and Naraya T. Validation of a confirmatory method for the detection of Enrofloxacin and Ciprofloxacin in egg by liquid chromatography-tandem mass spectrometry. BQCLP E-Journal. 2016: 100-103.

7.     Baena-Nogueras RM, Ortori C, Barrett DA and Gomes RL. Analysis of veterinary antibiotics in dairy environments by liquid chromatography – mass spectrometry. 15th International Conference on Environmental Science and Technology Rhodes, Greece, 31 August to 2 September 2017 CEST2017_00889.

8.     Nidal B, Shorouq W and Al-Rimawi F. A Validated Stability-Indicating HPLC Method for Simultaneous Determination of Amoxicillin and Enrofloxacin Combination in an Injectable Suspension, Sci. Pharm. 2017; 85(6): 2-8.      

9.     Razzagh M, Reza N. Determination of enrofloxacin residue in chicken eggs using FPT and ELISA methods Journal of Research & Health Social Development & Health Promotion Research Center. 2015; 5(2): 159-164.

10.   Xiaoming Z, Da X, Debin Z, Yabing T, Li J. Development and application of a capillary electrophoresis–electrochemiluminescent method for the analysis of enrofloxacin and its metabolite ciprofloxacin in milk. Talanta. 2008; 75: 1300–1306.  

11.   Adam MA, Shantier SW, Hussien MA, Garalnabi EA and  Gadkariem EA. Development of Spectrophotometric Method for the Determination of Amikacin Sulphate in Pure and Pharmaceutical Formulations using Ascorbic Acid. European Journal of Pharmaceutical and Medical Research. 2017; 4 (2): 235-239.

 

 

Received on 26.12.2018       Accepted on 12.02.2019     

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Asian J. Pharm. Ana. 2019; 9(1):11-14.