INTRODUCTION:

Cefquinome is a fourth generation cephalosporin with pharmacological and antibacterial properties valuable in the treatment of coliform mastitis and other infections¹. It is only used in veterinary applications. The chemical name² of Cefquinome Sulphate is Quinolinium, 1-[[(6R,7R)-7-[[(2Z)-(2-amino-4-thiazolyl)(methoxyimino)acetyl]amino]-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl]-5,6,7,8-tetrahydro-,sulfate; and the molecular formula is C₂₃H₂₄N₆O₅S₂^.H₂SO₄ with a molecular weight of 626.68. Literature survey^3-7 reveals no chromatographic methods for the estimation of Cefquinome Sulphate from pharmaceutical dosage forms. The availability of an HPLC method with high sensitivity and selectivity will be very useful for the determination of Cefquinome Sulphate in pharmaceutical formulations. The aim of the study was to develop a simple, precise and accurate reversed-phase HPLC method for the estimation of Cefquinome Sulphate in bulk drug samples and in pharmaceutical dosage form.

Fig 1: Structure of Cefquinome Sulphate

MATERIALS AND METHODS:

Cefquinome Sulphate was obtained as a gift sample from M/s. Vishnu Chemicals Ltd, Hyderabad. Acetonitrile, Ammonium acetate and water used were of HPLC grade (Qualigens). Commercially available Cefquinome Sulphate suspension containing 25mg/mL cefquinome (Cobactan^® 2.5%, MSD Animal Health) were procured from local market.

Instrument:

Quantitative HPLC was performed on liquid Chromatograph, Shimadzu LC 2010 dual λ detector equipped with automatic injector with injection volume 20 µl. The HPLC system was equipped with empower software.

HPLC Conditions:

The contents of the mobile phase were mixture of buffer 0.02M Ammonium acetate in water and acetonitrile in the gradient program (shown in table-IV). They were filtered before use through a 0.45 μm membrane filter, and pumped from the respective solvent reservoirs to the column at a flow rate of 1.0 mL/min. The run time was set at 30.0 min and the column temperature was ambient. Prior to the injection of the drug solution, the column was equilibrated for at least 30 min with the mobile phase flowing through the system. The eluents were monitored at 238 nm.

Preparation of Standard Stock solution:

A standard stock solution of the drug was prepared by dissolving 10 mg of Cefquinome Sulphate in 10 mL volumetric flask and dissolved in diluent (Acetonitrile and Water:50:50), sonicated for about 15 min and then made up to 10 mL with diluent get 1000 mcg/mL standard stock solution.

Working Standard solution:

1mL of the above stock solution was taken in 10 mL volumetric flask and thereafter made up to 25 mL with diluent (Acetonitrile and Water: 50:50) to get a concentration of 40 µg/mL.

Preparation of Sample solution:

Cobactan® Suspension 1 mL was taken from dosage form was transferred to 25 mL volumetric flask containing diluent, which is equivalent to 1000 ppm of Cefquinome. The resultant sample solution was further diluted to get working sample solution of 40 mcg/mL. The mixture was allowed to stand for 15 min with intermittent sonication to ensure complete solubility of the drug, and then filtered through a 0.45 μm membrane filter to get clear solution.

Linearity:

Aliquots of standard Cefquinome Sulphate stock solution were taken in different 10 mL volumetric flasks and diluted up to the mark with the mobile phase such that the final concentrations of Cefquinome Sulphate are in the range of 4-48 mcg/mL. Each of these drug solutions (20 μL) was injected three times into the column, and the peak areas and retention times were recorded. Evaluation was performed with PDA detector at 234 nm and a Calibration graph was obtained by plotting peak area versus concentration of Cefquinome Sulphate (Fig 3).

The plot of peak area of each sample against respective concentration of Cefquinome Sulphate was found to be linear in the range of 4–48 mcg/mL with correlation coefficient of 0.9999. Linear regression least square fit data obtained from the measurements are given in table I. The respective linear regression equation being y= 849408x-849408. The regression characteristics, such as slope, intercept, and %RSD were calculated for this method and given in table I.

Assay:

20 µL of sample solution was injected into the injector of liquid chromatograph. The retention time was found to be 6.06 minutes. The amount of drug present per suspension was calculated by comparing the peak area of the sample solution with that of the standard solution. The data are presented in table II.

Recovery Studies:

Accuracy was determined by recovery studies of Cefquinome Sulphate, known amount of standard was added to the preanalysed sample and subjected to the proposed HPLC analysis. Results of recovery study are shown in table II. The study was done at three different concentration levels.

RESULTS AND DISCUSSION:

The system suitability tests were carried out on freshly prepared standard stock solution of Cefquinome Sulphate. Parameters that were studied to evaluate the suitability of the system are given in table III.

Table I: Linear Regression Data for Calibration curves.

Drug

Cefquinome Sulphate

Concentration range (mcg/mL)

Slope (m)

Intercept (b)

Correlation coefficient

% RSD

4-48

849408

-849408

0.9999

0.83

Table II: Results of HPLC Assay and Recovery studies

Sample

Amount claim

(25mg/mL Suspension)

% found by the proposed method

% Recovery*

25mg/mL

99.49

99.36

99.37

99.36

99.52

99.56

*Average of three different concentration levels.

Table III Validation Summary

Validation Parameter

Results

System Suitability

Theoretical Plates (N)

Tailing factor

Retention time in minutes

% Area

4528

1.20

6.06

98.66

LOD (mcg/mL)

LOQ (mcg/mL)

0.01

0.03

Table IV: Gradient Program in HPLC method

Time in mins	Buffer	Acetonotrile
0.01	90	10
15	10	90
25	10	90
26	90	10
30	90	10

Fig 2: Typical Chromatogram of Cefquinome Sulphate by HPLC

Fig.3:Calibration curve of the Cefquinome Sulphate by RP-HPLC.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The limit of detection (LOD) and limit of quantification (LOQ) for Cefquinome Sulphate were found to be 0.01µg/mL and 0.03µg/mL respectively. The signal to noise ratio is 3 for LOD and 10 for LOQ. From the typical chromatogram of Cefquinome Sulphate as shown in fig 2, it was found that the retention time was 6.06 min. A mixture of buffer 0.02M Ammonium acetate in water and acetonitrile in the gradient program (shown in table-IV) was found to be most suitable to obtain a peak well defined and free from tailing. In the present developed HPLC method, the standard and sample preparation required less time and no tedious extraction were involved. A good linear relationship (r²=0.9999) was observed between the concentration range of 4-48 mcg/mL. Low values of standard deviation are indicative of the high precision of the method. The assay of Cefquinome Sulphate suspension was found to be 99.4%. From the recovery studies it was found that about 99.5% of Cefquinome Sulphate was recovered which indicates high accuracy of the method. The absence of additional peaks in the chromatogram indicates non-interference of the common excipients used in the suspension. This demonstrates that the developed HPLC method is simple, linear, accurate, sensitive and reproducible.

Thus, the developed method can be easily used for the routine quality control of bulk and suspension dosage forms of Cefquinome Sulphate within a short analysis time.

ACKNOWLEDGEMENTS:

The authors are grateful to M/s Vishnu chemicals Limited, Hyderabad for the supply of as a gift sample Cefquinome Sulphate and to the Management, Vishnu Chemicals Limited, Hyderabad, for providing the necessary facilities to carry out the research work.

REFERENCES:

1. The Merck Index, 13, 1960, (2001)

2. Martindale-The Complete Drug Reference, 36, 233.1, (2009).

3. Bahk, J. and Marth, E.H. (1990). Listeriosis and Listeria monocytogenes. In: Cliver, D., ed. Foodborne Diseases. Academic Press, Inc., San Diego, California.

4. Bradford, P.A., Petersen, P.J., Fingerman, I.M., and D.G. White (1999). Characterization of expanded-spectrum cephalosporin resistance in E. coli isolates associated with bovine calf diarrhoeal disease. J. Antimicrob. Chemother.; 44: 607-610.

5. Bryskier, A., Aszodi, J, and Chantot, J.-F. (1994), Parenteral cephalosporin classification. Expert Opin. Invest. Drugs; 3(2): 145-171

6. Bryskier, A. (1997). New concepts in the field of cephalosporins: C-3’ quarternary ammonium cephems (Group IV). Clin. Microbiol. Infect. 3 (Suppl 1):S1-S6.

7. Bywater, R., Deluyker, H., Deroover, E., Jong, A. de, Marion, H., McConville, M., Rowan, T., Shryock, T., Shuster, D., Thomas, V., Vallé, M., and Walters, J. (2004). A European survey of antimicrobial susceptibility among zoonotic and commensal bacteria isolated from food-producing animals. J. Antimicrob. Chemother.; 54: 744-754

Received on 14.05.2012 Accepted on 26.05.2012

Asian J. Pharm. Ana. 2(2): April-June 2012; Page 33-35