Development and Validation of HPLC Method Using Hydrotropic Mobile Phase for the Estimation of Gatifloxacin

In the present work eco-friendly, precise, accurate RP-HPLC method has been developed for the determination of gatifloxacin using the concept of hydrotropy. Hydrotropy is a concept of increasing solubility of a solute in water by adding an agent termed as hydrotropes. This concept reduces the use of organic solvents for analysis of drugs which makes analysis eco friendly and less expensive. The method was developed using 3% sodium benzoate (pH 6.5) as mobile phase and Shimadzu ODS-C18 column as stationary phase. The flow rate was maintained at 1.4 ml/min and detection was performed at 293 nm using diode array detector. The retention time was 2.5 min and method was found to be linear over a concentration range of 0.5-30 μg/ml with correlation coefficients of 0.999. Developed method was validated as per the ICH Q2 (R1) guidelines and applied to the estimation of gatifloxacin in eye drop formulation.


Introduction
Hydrotropy is a concept of increasing solubility of a solute in water by adding an agent termed as hydrotropes. Hydrotropic solvents can be as solvent in spectrophotometry and chromatography for accurate, rapid and precise analysis. Hydrotropes are amphiphilic substances composed of hydrophilic functional group used as a media for promoting solubilization of sparingly soluble substances in aqueous solution. Hydrotropes are organic salts which present in aqueous solutions can substantially improve the solubility of hydrophobic organic substances in the aqueous phase. Commonly used hydrotropes are hydroxy benzenes, hydroxy benzoates, benzene sulfonates, sodium benzoate and sodium Citrate [1][2][3][4].
A literature survey regarding quantitative analysis revealed that various analytical methods have been reported for the estimation of GATI. Estimation of GATI in Human Plasma and Blood has been reported using methods like RP-HPLC, UV Spectroscopy & HPTLC from bulk and tablet dosage form

Accuracy
The accuracy of the method was determined by calculating recovery of GATI by method of standard additions. Known amounts of GATI (2.5, 5, 7.5 μg/ml) standard stock solutions were taken and added to pre-quantified sample mixture. The solution was injected into HPLC system and analyzed by proposed method. The amount of GATI was estimated by measuring the areas and by fitting these values to the straight-line equations of the calibration curves.

Intermediate precision
Intraday precision was determined by analyzing sample solutions of GATI (0.5, 5 and 30 μg/ml) at three levels cover-ing low, medium and high concentrations of the calibration curve three times on the same day. Interday precision was determined by analyzing sample solutions of GATI (0.5, 5 and 30 μg/ml) at three levels covering low, medium, and high con-centrations over a period of 3 days. The peak areas were ob-tained and RSD values were determined.

Repeatability
Repeatability of sample application was assessed by ana-lyzing GATI (5 μg/ml) six times and peak area was recorded.
The percent relative standard deviation (% RSD) of mean peak areas was obtained.

Specificity
Specificity is the ability to assess unequivocally the analyte in the presence of excipients, degradants, impurities which may be expected to be present. The specificity of method was ascertained by analysing GATI in presence of excipient like benzalkonium chloride (0.02%) and interference was noted.

Sensitivity
The limit of detection (LOD) is defined as the lowest con-centration of an analyte that can reliably be differentiated from background levels. The limit of quantification (LOQ) of an individual analytical procedure is the lowest amount of analyte that can be quantitatively determined with suitable precision and accuracy.
Limit of detection (LOD) and limit of quantitation (LOQ) for HPLC method were calculated by using the equation given in ICH Q2 (R1) guideline. LOD = 3.3 × σ/S, LOQ = 10 × σ/S; Where, σ = Standard deviation of y intercepts of regression line obtained from calibration cure, S = Mean slope of cali-bration curve.

Robustness
Small deliberate changes were introduced in the chro-matographic conditions to observe the effect of such chang-es. Flow rate and mobile phase composition were changed upto ± 10% and the effects on the results were examined.
Robustness of the method was determined at concentration level of 5 μg/ml of GATI. The mean and % RSD values of the peak areas were calculated.

System suitability
System suitability tests were carried out on freshly preare toxic to the environment and increases pollution. In pres-ent study, eco-friendly, cost-effective, sensitive and accurate liquid chromatographic method has been developed for the estimation of GATI from eye drop formulation.

Materials and Methods
Instrument HPLC instrument of Agilent 1220 Infinity make with Diode array Detector (Software: Chem Station, Open LAB Control) and stationary phase Shimadzu ODS-C18 (250 × 4.6 mm × 5 μm particle size) column was used in the study. All drugs and chemicals were weighed on digital balance (Shimadzu Ltd., Japan, Model AUX220) and pH was measured using digital pH meter (Equip tronics, India, Model EQ-610). Mobile phase and samples were sonicated using Ultrasonicator (EIE Instru-ments Pvt.Ltd., India).

Chemicals and reagents
Gatifloxacin was obtained as gift samples from Primal En-terprise Limited, India. Sodium benzoate (HPLC grade) and water of HPLC Grade were purchased from Merck Ltd., India.
Methanol of HPLC grade was purchased from Thermo fisher scientific Pvt. Ltd., India while glacial acetic acid analytical re-agent grade were purchased from S.D. Fine Chem. Ltd., India.

Chromatographic conditions
The HPLC system was equilibrated with the mobile phase 3% sodium benzoate (pH 6.5) before starting analysis for 30 minutes. The flow rate was maintained at 1.4 ml/min and elu-ent were monitored with DAD detector at 293 nm. Total run time was kept at 5 min.
Preparation of working standard solution (100 µg/ml) GATI (10 mg) was accurately weighed and transferred to 10 ml volumetric flask. Methanol was added and swirled to dissolve the drug. Volume was made up to the mark with methanol in order to prepare standard stock solution of 1000 μg/ml solution. 1 Vml of this solution was withdrawn in 10 ml volumetric flask and volume was made to 10 ml with metha-nol to get working standard solution of 100 µg/ml.

Method validation
Validation of the developed RP-HPLC method was carried out according to International Conference on Harmonization (ICH) guidelines Q2 (R1) for specificity, sensitivity, accuracy, precision, repeatability, and robustness [39].

Linearity
Calibration curve was prepared by taking appropriate aliquot of working standard solution in different 10 ml volu-metric flask. The volume was made up to 10 ml using mobile phase as a solvent to obtain final concentration of 0.5-30 μg/ ml. The calibration curve was plotted using mean peak area versus concentration. The regression equation was computed and correlation coefficient was determined.

Analysis of marketed formulation
An aliquot of eye drops equivalent to 10 mg of GATI was  The percentage (%) RSD value was found to be less than 2%.
Repeatability study was performed by analyzing target con-centration six times over a short period of time which gave % RSD value less than 1% which indicates that the method is precise.
The method was found to be specific and no interference of excipients was observed. Under the experimental conditions used, the lowest amounts of drug that could be detected (LOD) was found to be 0.1 μg/ml and the limit of quantification (LOQ) for GATI was found to be 0.5 μg/ml. This indicates that the nano gram quantity of drug can be estimated accurately and precisely which indicate the sensitivity of the method. To study robustness of the method, small but deliberate changes were introduced in flow rate, pH and concentration of Sodium benzoate. In each study peak shape was not affected but there was variability in retention time of the GATI and the % RSD was observed to be less than 2% (Table  3). System suitability parameters are summarized in (Table 4).
The developed method was applied for the analysis of eye drop formulation. The % drug content was found in the range of 99.4%-100.6%. The analysis result of GATI eye drops complied with the specification. Hence, the developed method can be used for routine quality control test of GATI.
ml. Solution was injected in a system equilibrated with optimized chromatographic conditions. Peak area and retention time were obtained and quantification was carried out using regression equation.

Results and Discussion
Selection and optimization of mobile phase was performed based on ability of method to give acceptable value of theoretical plates, asymmetric factor and retention time. Different proportions of hydrotrope sodium benzoate (2-6%) were tried with different pH (6-7.4) to obtain sharp peak of GATI. A mobile phase composition of 3% sodium benzoate (pH 6.5), gave sharp peak with asymmetry of 1.3. The retention time was found to be 2.5 min and theroratical plates were found to be 2302 ± 15. Hence this mobile phase was selected as an optimized mobile phase. The overlay chromatogram of gatifloxacin has been shown in (Figure 2 and Figure 3). UV spectrum of GATI in Methanol showed absorbance maxima at 293 nm, hence it was selected as detection wavelength.
The method was found to be linear in a concentration range of 0.5-30 μg/ml (n = 5) for GATI. The regression equation was found to be y = 48.29 × +17.82 with a correlation coefficient of 0.999 which indicates a good linear relationship over the concentration range studied, demonstrating the suitability of the method for analysis.
Accuracy study was performed by estimating recovery of drug. The % recovery was found to be 98.93 ± 0.43, 99.06 ± 0.15, 99.44 ± 0.54 for 50%, 100% and 150% of target concentration for GATI (Table 1 and Table 2). The values near to 100% demonstrated that the method is accurate. Intra-day and Inter-day precision study was performed by taking 3 different concentrations covering entire range of the method.

Conclusion
Precise, accurate reversed phase liquid chromatographic method has been developed for the estimation of Gatifloxacin from eye drop formulation. The method utilizes an aqueous 3% solution of sodium benzoate as mobile phase which reduces the use of organic solvents. The method is less time consuming having retention time of 2.5 min. The reported analytical methods  use organic solvents which are toxic to the environment and increases pollution. The present method is eco-friendly, cost effective, sensitive and accurate. Method was successfully used for the estimation of GATI in its formulation and it can be applied for analysis of GATI in bulk and quality control samples.

Parameters
Proposed method