METHOD DEVELOPMENT AND ITS VALIDATION FOR QUANTITATIVE SIMULTANEOUS DETERMINATION OF DEXAMETHASONE AND GATIFLOXACIN IN OPHTHALMIC SOLUTION BY RP-HPLC

About author :
Ankit Agarwal*, Subhash Dadhich, Sunil Kumar Tiwari, Kashyap Nagariya
R & D Division, Ahlcon Parenterals (I) Ltd., Bhiwadi, Rajasthan, India
*e-mail: [email protected]

ABSTRACT :
To develop a simple, rapid and accurate HPLC method for simultaneous quantitative determination of Dexamethasone and Gatifloxacin in ophthalmic solution. Chromatographic separation was achieved with PDA detector using Inertsil C18, 250 x 4.6mm, 5µ reverse phase analytical column. The mobile phase consist of buffer: acetonitrile (70: 30 v/v), was passed through the column at flow rate of 1.0 ml/min. The method was performed at wavelength 254. The experiment was carried out at 30^oC. The calibration curves were linear in the concentration range of 25% to 150% of the working concentration (r2 >0.999). The lower limit of quantification was 1.6, 2.8 µg/ml for Dexamethasone and Gatifloxacin respectively. The accuracy of Dexamethasone and Gatifloxacin was also found to be in the limits (ie 98% – 102%). Developed procedure was used for simultaneous quantitative estimation of Dexamethasone and Gatifloxacin in ophthalmic solution and validated as per ICH and most useful for academic as well as industrial scale.
Key words: Dexamethasone, Gatifloxacin, RP-HPLC, Validation.
INTRODUCTION 
Dexamethasone is a glucocorticoid agonist. Unbound dexamethasone crosses cell membranes and binds with high affinity to specific cytoplasmic glucocorticoid receptors. This complex binds to DNA elements (glucocorticoid response elements) which results in a modification of transcription and, hence, protein synthesis in order to achieve inhibition of leukocyte infiltration at the site of inflammation, interference in the function of mediators of inflammatory response, suppression of humoral immune responses, and reduction in edema or scar tissue. The anti-inflammatory actions of dexamethasone are thought to involve phospholipase A2 inhibitory proteins, lipocortins, which control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes. Gatifloxacin is a synthetic broad-spectrum 8-methoxyfluoroquinolone antibacterial agent for oral or intravenous administration. is bactericidal and its mode of action depends on blocking of bacterial DNA replication by binding itself to an enzyme called DNA gyrase, which allows the untwisting required to replicate one DNA double helix into two. Notably the drug has 100 times higher affinity for bacterial DNA gyrase than for mammalian. Gatifloxacin is a broad-spectrum antibiotic that is active against both Gram-positive and Gram-negative bacteria. It should be used only to treat or prevent infections that are proven or strongly suspected to be caused by bacteria. The aim of this study was to develop a RP HPLC method for the quantitative simultaneous determination of Dexamethasone and Gatifloxacin. The method developed was validated as per ICH Q2 (R1).
MATERIALS & METHODS
Chemicals and reagents
HPLC grade acetonitrile, sodium dihydrogen, GAA and TEA were used to prepare the mobile phase and were purchased from Merck Specialties. The working standards of Dexamethasone and Gatifloxacin were purchased from LG Promochem. Deionized and purified water using a Mili-Q system (Millipore) was used for the mobile phase and the standard solutions preparation. All experiment was performed using ‘A’ class volumetric glassware. All other reagents were of analytical grade.
Instrument and Chromatographic Conditions 
Shimadzu LC 2010 C_HT HPLC was used for the chromatographic separation equipped with autosampler and Photo diode array (PDA) detector. The software used was LC Solution. The chromatographic separation of Dexamethasone and Gatifloxacin were carried out using Inertsil C18 250 x 4.6 mm, 5µ reverse phase analytical column. Mobile phase consisted of Acetonitrile: Buffer (0.1 M sodium di hydrogen phosphate and 1ml triethylamine in 1000 ml WFI and pH adjusted to 3.0 with GAA) in the ratio 30: 70. The mobile phase was filtered by passing it through 0.45 µm filter and the filtrate is degassed by using bath sonicator. Mobile phase was used as diluent. Injection volume was 10 μl. Oven temp was set at 30^oC. The mobile phase was pumped at 1 ml/min at room temperature. Detection was carried by using wavelength 254.
CONCLUSION 
The present study shows that the method developed for the determination of Gatifloxacin and Dexamethasone were specific, linear, accurate, precise and robust. The method clearly shows that all the peaks had tailing factor less than 2. The RSD for areas and theoretical plates (> 2500) was also found to be satisfactory. Validation parameters were performed according to ICH Q2 (R1) guidelines. The recoveries achieved were highly significant in the developed method. Hence it can be concluded that the method developed can be effectively used in the industries as well as research purposes.

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