Scholarly article on topic 'Simultaneous determination of dexamethasone and moxifloxacin in pharmaceutical formulations using stability indicating HPLC method'

Simultaneous determination of dexamethasone and moxifloxacin in pharmaceutical formulations using stability indicating HPLC method Academic research paper on "Chemical sciences"

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{"Reverse phase liquid chromatography" / Moxifloxacin / Dexamethasone / "Stress induced degradation products" / "ICH guidelines"}

Abstract of research paper on Chemical sciences, author of scientific article — Syed Naeem Razzaq, Muhammad Ashfaq, Islam Ullah Khan, Irfana Mariam, Syed Saleem Razzaq, et al.

Abstract A simple, sensitive, specific, precise and accurate stability indicating reverse phase liquid chromatographic method was developed for simultaneous determination of moxifloxacin hydrochloride and dexamethasone in bulk drugs and pharmaceutical formulations. The developed chromatographic method was optimized for the separations of moxifloxacin hydrochloride, dexamethasone and stress-induced degradation products by the use of BDS Hypersil C8 column (250×4.6mm, 5μm) as stationary phase with mobile phase consisting of a mixture of phosphate buffer (20mM) containing 0.1% (v/v) triethylamine, at pH 2.8 (adjusted with dilute phosphoric acid) and methanol (38.5:61.5 v/v) at a flow rate of 1.5mLmin−1. Chromatographic separations of analytical peaks and degradation products were achieved within 10min. Detection of the analytes and degradation products was performed at 254nm using a diode array detector. Method validation was performed in accordance with ICH guidelines. Linearity of the method was established over the concentration ranges of 50–350μgmL−1 for moxifloxacin hydrochloride and 10–70μgmL−1 for dexamethasone (correlation coefficients greater than 0.999). The method resulted in good separation of both the analytes and degradation products with acceptable tailing and resolution with peak purity index ⩾0.9999 which indicated complete separation of analyte peaks from the degradation products. The method can therefore be considered as stability-indicating and can be used successfully for simultaneous determination of moxifloxacin hydrochloride and dexamethasone in pharmaceutical formulations and stability studies.

Academic research paper on topic "Simultaneous determination of dexamethasone and moxifloxacin in pharmaceutical formulations using stability indicating HPLC method"

Arabian Journal of Chemistry (2014) xxx, xxx-xxx

King Saud University Arabian Journal of Chemistry

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE

Simultaneous determination of dexamethasone and moxifloxacin in pharmaceutical formulations using stability indicating HPLC method

Syed Naeem Razzaq a, Muhammad Ashfaq b'*, Islam Ullah Khan a, Irfana Mariam c, Syed Saleem Razzaq d, Waqar Azeem a

a Department of Chemistry, Government College University, Lahore 54000, Pakistan b Department of Chemistry, University of Gujrat, H.H. Campus, Gujrat 50700, Pakistan c Department of Chemistry, Queen Marry College, Lahore 54000, Pakistan d Medipharm Pharmaceuticals Kot Lakhpat, Lahore 54000, Pakistan

Received 5 November 2012; accepted 3 November 2014

KEYWORDS

Reverse phase liquid chromatography; Moxifloxacin; Dexamethasone; Stress induced degradation products; ICH guidelines

Abstract A simple, sensitive, specific, precise and accurate stability indicating reverse phase liquid chromatographic method was developed for simultaneous determination of moxifloxacin hydrochloride and dexamethasone in bulk drugs and pharmaceutical formulations. The developed chromatographic method was optimized for the separations of moxifloxacin hydrochloride, dexamethasone and stress-induced degradation products by the use of BDS Hypersil C8 column (250 x 4.6 mm, 5 im) as stationary phase with mobile phase consisting of a mixture of phosphate buffer (20 mM) containing 0.1% (v/v) triethylamine, at pH 2.8 (adjusted with dilute phosphoric acid) and methanol (38.5:61.5 v/v) at a flow rate of 1.5 mL min~\ Chromatographic separations of analytical peaks and degradation products were achieved within 10 min. Detection of the analytes and degradation products was performed at 254 nm using a diode array detector. Method validation was performed in accordance with ICH guidelines. Linearity of the method was established over the concentration ranges of 50-350 igmL-1 for moxifloxacin hydrochloride and 10-70 igmL-1 for dexamethasone (correlation coefficients greater than 0.999). The method resulted in good separation of both the analytes and degradation products with acceptable tailing and resolution with peak purity index P0.9999 which indicated complete separation of analyte peaks from the degradation products. The method can therefore be considered as stability-indicating and can be used successfully for

* Corresponding author.

E-mail address: m.ashfaq@uog.edu.pk (M. Ashfaq). Peer review under responsibility of King Saud University.

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simultaneous determination of moxifloxacin hydrochloride and dexamethasone in pharmaceutical formulations and stability studies.

© 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction

Moxifloxacin hydrochloride is chemically designated as 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-[(4aS,7aS)-octa-hydro-6H-pyrrolo[3,4-b]pyridin-6-yl]-4-oxo-3-quinolinecarb-oxylic acid hydrochloride (Fig. 1A). It is a broad-spectrum antibiotic that functions by inhibiting DNA gyrase, a type II topoisomerase, and topoisomerase IV, enzymes necessary to separate bacterial DNA, thereby inhibiting cell replication. It is used for bacterial conjunctivitis, keratitis, pre and post operatively to control infections of the eyes. Dexamethasone (Fig. 1B) chemically designated as (8S,9R,10S,11S,13S,14S, 16R,17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one is a corticosteroid, used principally for steroid-responsive inflammatory ocular conditions for which a corticosteroid is indicated and where bacterial ocular infection or a risk of infection exists (Martindale, 2009).

Both moxifloxacin hydrochloride and dexamethasone have been analysed by various techniques either alone or in combination with other drugs. Analytical methods existed for moxifloxacin hydrochloride included determination by spectro-photometry (Misra et al., 2010; Chaudhary, 2010), HPTLC (Sanjay et al., 2007) and high performance liquid chromatography (Sultana et al., 2010, 2011; Devi and Chandrasekhar, 2009; Razzaq et al., 2012a-d). Analytical methods existing for dexa-methasone included determination by high performance liquid chromatography (Iqbal et al., 2006; Huetos et al., 1999; Mallinson et al., 1995; Chen et al., 2008; Lemus and Arroyo, 2002; Urban et al., 2009; Kwak et al., 1995), gas chromatogra-phy-mass spectrometry (Mallinson et al., 1995) and thin layer chromatography (Huetos et al., 1999).

The combination of moxifloxacin hydrochloride and dexa-methasone has not been adopted by any official pharmacopoeia like USP, BP or EP, etc. A careful review of the literature did not reveal any stability indicating HPLC method (with forced degradation studies) for simultaneous determination of both drugs. The only HPLC method found in the literature for the said combination was by Dabhi et al. (2012). The reported

method although can separate both the components with reasonable run time yet lacks its ability to separate degradation products. ICH guidelines stress to include forced degradation studies in all the analytical methods to be reported. Therefore attempts were made to develop and validate simple, precise, and sensitive, isocratic reverse phase stability indicating high performance liquid chromatographic method for the simultaneous determination of moxifloxacin hydrochloride and dexamethasone and their stress induced degradation products in pharmaceutical formulations. We are currently engaged in binary combination analysis of different classes of drugs in pharmaceutical formulations and in human plasma (Qutab et al., 2009; Qutab et al., 2007a,b; Ashfaq et al., 2007, 2008, 2013; Khan et al., 2008, 2010a,b, 2013, 2014; Sharif et al., 2010; Razzaq et al., 2012a-d, 2013) and this work is a continuation of that have been done already.

2. Materials and methods

2.1. Chemicals and reagents

Reference standards of moxifloxacin hydrochloride and dexa-methasone with stated purity of 99.97% and 99.96%, respectively, were kindly gifted by Schazoo Zaka Laboratories (Lahore, Pakistan). Occumox DM and Moxiblu-D eye drops (containing 5 mg per ml of moxifloxacin and 1 mg per ml of dexamethasone) were used for method applications. Methanol (HPLC grade), potassium dihydrogen phosphate, phosphoric acid, triethylamine, sodium hydroxide, hydrochloric acid and hydrogen peroxide (analytical reagent grade) were from M.S. Traders Lahore, Pakistan (Fluka origin). Double distilled water was used throughout the analysis. Mobile phase was filtered using 0.45 im nylon filters by Millipore (USA).

2.2. Equipment and chromatographic conditions

The HPLC system consisting of Shimadzu LC-20A system (Kyoto, Japan) is equipped with model LC-20AT pump,

(A) (B)

Figure 1 Chemical structures of moxifloxacin HCl (A) and dexamethasone (B).

SPD-M20A Diode array detector (set at 254 nm), DGU-20A5 online degasser, and a Rheodyne injection valve with a 20 iL loop. Peak areas were integrated using a Shimadzu LC solution (version 1.227) software program. Experimental conditions were optimized on a BDS Hypersil C8 column (250 x 4.6 mm, 5 im) at room temperature. The mobile phase was consisting of methanol and 20 mM phosphate buffer (pH 2.8) in the ratio of 61.5:38.5 v/v, respectively. The phosphate buffer was prepared by taking 2.72 g of potassium dihydrogen phosphate in 1000 mL of water. Triethylamine (1 mL) was added to it and the pH was then adjusted to 2.8 using phosphoric acid. Flow rate of the mobile phase was 1.5 mL min-1 and all chromatographic experiments were performed at room temperature (25 0C ± 2 0C).

2.3. Preparation of standard stock and standard solution

Standard stock solutions of moxifloxacin hydrochloride and dexamethasone were prepared by accurately weighing 62.5 mg moxifloxacin and 12.5 mg dexamethasone dissolved in 5 mL methanol in 25 mL volumetric flask and then made up to the mark with mobile phase. The stock solution was used to prepare the working standard solution of moxifloxacin hydrochloride and dexamethasone. Two mL of the standard stock solution was diluted to 25 mL with mobile phase to prepare working standard solution having concentrations equal to 200 ig mL-1 of moxifloxacin and 40 ig mL-1 of dexamethasone. The solution was filtered through a 0.45 im nylon filter before analysis.

2.4. Preparation of sample solution

Two mL commercial eye drops (composition 5mgmL-1 moxifloxacin and 1 mg mL-1 dexamethasone) were diluted to 50 mL with mobile phase to obtain concentrations equal to 200 ig mL-1 of moxifloxacin and 40 ig mL-1 of dexametha-sone. The solution was filtered through 0.45 im nylon filter before the analysis.

2.5. Linearity

Linear calibration plots of the proposed method were obtained over concentration ranges of 50-350 igmL-1 (50, 100, 150, 200, 250, 300 and 350 igmL-1) for moxifloxacin and 10-70 igmL-1 dexamethasone (10, 20, 30, 40, 50, 60 and 70 igmL-1). Each solution was prepared in triplicate.

2.6. Accuracy

Accuracy of the developed method was determined by two ways (i) standard addition method and (ii) analysis of synthetic mixtures of moxifloxacin and dexamethasone eye drops. In the first method known amounts of moxifloxacin hydrochloride and dexamethasone were supplemented to the previously analysed sample solution and then experimental and true values were compared. Three levels were made corresponding to 50%, 100% and 150% of the nominal analytical concentration. In the second method known quantities of moxifloxacin hydro-chloride and dexamethasone of known purity have been spiked to the placebo components (benzalkonium chloride and sodium

chloride in aqueous base). Synthetic mixture (100% nominal analytical concentration) of moxifloxacin (200 igmL-1) and dexamethasone (40 ig mL-1) was prepared by mixing moxifloxacin (20 mg), dexamethasone (4 mg), benzalkonium chloride (0.02 g) as preservative and sodium chloride (0.3 g) in 5 ml methanol and then in 100 ml of purified water for 30 min using magnetic stirrer. Three levels of synthetic mixtures were prepared corresponding to 50%, 100% and 150% of nominal analytical concentration (200 igmL-1 of moxifloxacin and 40 igmL-1 of dexamethasone) and analysed by the developed method.

2.7. Precision

Repeatability was studied by determination of intra-day and inter-day precision. Intra-day precision was determined by injecting five standard solutions of three different concentrations on the same day and inter-day precision was determined by injecting the same solutions for three consecutive days. Relative standard deviation (RSD%) of the peak area was then calculated to represent precision.

2.8. Specificity (Stress Testing)

Stress testing was carried out using different ICH prescribed stress conditions such as acidic, basic, oxidative, thermal and photolytic stresses. All stress studies were performed in 25 mL volumetric flask and in triplicate.

2.8.1. Acid degradation studies

Acid degradation study was performed in a versatile environmental test chamber (Sanyo, Japan) at 40 0C/75% RH using 5 M HCl. For this purpose, 2 mL of the standard stock solution was taken in 25 mL volumetric flask. Two millilitres of 5 M HCl was added in the flask and kept in versatile environmental test chamber at 40 0C/75% RH for 16 h. After completion of the stress the solution was neutralized by using 5 M NaOH and completed up to the mark with mobile phase.

2.8.2. Base degradation studies

Base degradation study was performed at 22 0C/58% RH using 5 M NaOH. For this purpose, 2 mL of the standard stock solution was taken in 25 mL volumetric flasks. Two millilitres of 5 M NaOH was added in the flask and kept at 22 0C/58% RH for forty minutes. After completion of the stress the solution was neutralized by using 5 M HCl and completed up to the mark with mobile phase.

2.8.3. Oxidative degradation studies

Oxidative degradation study was performed in a versatile environmental test chamber (Sanyo, Japan) at 40 0C/75% RH using 6% H2O2. For this purpose, 2 mL of the standard stock solution was taken in 25 mL volumetric flask. Two millilitres of 6% H2O2 was added in the flask and kept in versatile environmental test chamber at 40 0C/75% RH for 16 h. After completion of the stress, the 25 mL flask was completed up to the mark with mobile phase.

2.8.4. Thermal degradation studies

Thermal degradation studies were performed in a versatile environmental test chamber (Sanyo, Japan) at 40 0C/75%

RH. For this purpose, 2 mL of the standard stock solution was taken in three different 25 mL volumetric flasks and kept in a versatile environmental test chamber at 40 0C/75% RH for 144 h, 288 h and 528 h. After completion of the stress, the 25 mL flask was completed up to the mark with mobile phase.

2.8.5. Photolytic degradation studies

For photolytic degradation study 2 mL of the standard stock solution was taken in 25 mL volumetric flask placed in the direct sunlight for 1.0 h. After completion of the stress the 25 mL flask was completed up to the mark with mobile phase.

2.9. Robustness

Premeditate variations were performed in the experimental conditions of the proposed method to assess the method robustness. For this intention, minor changes were made in mobile phase composition, flow rate and pH of buffer solution. The effect of these changes on chromatographic parameters such as retention time, tailing factor and number of theoretical plates was then measured.

2.10. Limit of detection (LOD) and limit of quantitation

Detection and quantitation limits were determined by the signal-to-noise (S/N) approach. In order to examine the limit of quantitation and limit of detection solutions of different concentrations were prepared by spiking known amounts of moxi-floxacin hydrochloride and dexamethasone into excipients (benzalkonium chloride and sodium chloride). Each solution was prepared according to the defined protocol and analysed repeatedly to determine the S/N ratio. The average S/N ratio from all the analyses at each concentration level was used to calculate the limit of quantitation and limit of detection. The concentration level that gives an S/N ratio of 10:1 at which analytes can be readily quantified with accuracy and precision was reported as the limit of quantitation. The concentration level that gives an S/N ratio of 3:1 at which analytes can be readily detected was reported as the limit of detection.

3. Results and discussion

In RP-LC analysis the selection of the stationary phase depends on the chemical structures of the target molecules. Due to high carbon content and hydrophobic character of moxifloxacin hydrochloride and dexamethasone these drugs can be separated through C8 or C18 stationary phases. The method development process was initiated with different ratios of water and methanol at different pH values. Asymmetrical peaks of moxifloxacin hydrochloride and double peak of dexamethasone were obtained on all stationary phases. Polarity of the mobile phase was then increased by using phosphate buffer in the mobile phase. Further chromatographic experiments were performed using methanol: phosphate buffer as mobile phase along with (0.1% v/v) triethylamine (as silanol blocker). Symmetrical and sharp peaks of moxifloxacin hydro-chloride and dexamethasone were obtained with methanol: 0.02 M phosphate buffer containing 0.1% v/v triethylamine (61.5:38.5, v/v) on C8 columns. The variations in the composition of the mobile phase and dissimilar stationary phases had substantial influences on peak shape, tailing factor, retention factor, theoretical plates and resolution.

In order to optimize the appropriate pH of the phosphate buffer solution, chromatographic experiments were performed at four different pH (2.8, 3.5, 4.5 and 6.5) values of the buffer solution. Highly symmetrical and sharp peaks of moxifloxacin hydrochloride and dexamethasone were obtained at pH 2.8 and 3.5 with better resolution, capacity factor, and theoretical plates at pH 2.8 as compared to pH 3.5. Finally, methanol: phosphate buffer 0.02 M, pH 2.8 (61.5:38.5, v/v) was selected which provided symmetrical peaks using Hypersil BDS C8 column. Under the mentioned chromatographic conditions highly symmetrical and sharp peaks of moxifloxacin hydrochloride and dexamethasone were obtained at retention times of 3.372, and 6.503 min, respectively (Fig. 2). System suitability data are given in Table 1.

The developed chromatographic method was validated using ICH guidelines (ICH 1996). Validation parameters include linearity, accuracy, precision, robustness, specificity, limit of detection and quantitation.

Figure 2 Chromatogram of moxifloxacin (200 igmL and dexamethasone (40 igmL in pharmaceutical formulations.

Linear calibration plots for the proposed method were obtained in concentration ranges of 50-350 igmL-1 (50, 100, 150, 200, 250, 300 and 350 igmL-1) for moxifloxacin and 10-70 igmL-1 dexamethasone (10, 20, 30, 40, 50, 60 and 70 ig mL-1). The linear regression equation for dexamethasone was found to be Y = 33,695 X + 8000 with correlation coefficient greater than 0.999. In this equation 33,695 is the slope (95% confidence interval lies between -6913 and 74,303) and 8000 is the Y-intercept (a point of the Y-coordinate where a given line intersects the Y-axis) at which X is equal to zero. It defines the elevation of the line. The Y-intercept provides with an estimate of the variability of the method. For example, the ratio percent of the Y-intercept with variable data at nominal concentration is used to estimate the method variability. The linear regression equation for moxifloxacin was found to be Y = 80,041 X + 11,371 with correlation coefficient greater than 0.999. In this equation 80,041 is the slope (95% confidence interval lies between 27,589 and 132,494) and 11,371 is the intercept.

The limit of detection (LOD) and quantitation (LOQ) were determined by making serial dilutions. LOD was found to be 0.316 igmL-1 and 0.061 igmL-1 for moxifloxacin and dexamethasone, respectively (signal to noise ratio of 3:1). LOQ was found to be 1.014 ig mL-1 and 0.192 ig mL-1 for moxifloxacin and dexamethasone, respectively (signal to noise ratio of 10:1).

Table 4 Robustness study of moxifloxacin.

Chromatographic Assay tR Theoretical Tailing

conditions (%) (min) plates

Methanol:buffer (63.5:36.5) 98.1 3.241 4387 1.36

Methanol:buffer (61.5:38.5) 100.0 3.372 4358 1.36

Methanol:buffer (59.5:40.5) 101.5 3.415 4458 1.34

Flow rate (1.3 mL/min) 100.8 3.825 4498 1.36

Flow rate (1.5 mL/min) 99.4 3.372 4389 1.36

Flow rate (1.7 mL/min) 98.2 2.789 4328 1.36

Buffer (pH 2.6) 102.0 3.365 4318 1.34

Buffer (pH 2.8) 100.9 3.371 4422 1.36

Buffer (pH 3.0) 100.2 3.379 4478 1.34

Accuracy of the developed method was performed by standard addition and synthetic mixture techniques. Three levels of solutions (50%, 100% and 150%) of the nominal analytical concentrations were prepared and analysed by the developed method. Percentage recoveries along with standard deviation and relative standard deviations for each analyte are given in Table 2. Recovery studies showed the method to be highly accurate and suitable for intended use.

Intra-day precision was determined by injecting five standard solutions of three different concentrations on the same

Table 1 System suitability parameters of moxifloxacin and dexamethasone.

Ingredient Retention time (min) Tailing factor Theoretical plates Resolution k

Moxifloxacin 3.371 1.39 4481 - -

Dexamethasone 6.504 1.09 6588 7.21 1.34

Table 2 Accuracy of the proposed HPLC method.

Drugs Spiked concentration (ig mL-1) Standard addition measured concentration (ig mL-1) ± SD; RSD (%) Synthetic mixtures measured concentration (ig mL-1) ± SD; RSD (%)

Moxifloxacin 100.0 200.0 300.0 100.7 ± 0.5; 0.1 202.2 ± 0.8; 1.2 298.2 ± 1.1; 1.4 99.8 ± 0.4; 0.3 201.4 ± 0.7; 0.8 302.4 ± 0.5; 0.7

Dexamethasone 20.0 40.0 60.0 20.1 ± 0.4; 0.7 40.4 ± 0.9; 0.7 60.7 ± 0.7; 1.2 19.8 ± 0.2; 0.1 40.2 ± 0.5; 0.9 60.0 ± 0.1; 0.1

n = average of 5 analyses, chromatographic conditions: mobile phase methanol: 20 mM phosphate buffer 61.5:38.5, v/v, pH 2.8, Column BDS Hypersil C8 (250 x 4.6, 5 im), flow rate 1.5 mL min-1, injection volume 20 iL, wavelength 254 nm.

Table 3 Intra-day and inter-day precision of the proposed HPLC method.

Drugs Actual concentration Intra-day precision Inter-day precision

(ig mL-1) measured concentrations; RSD (%) measured concentrations; RSD (%)

Moxifloxacin 100.0 100.7 ± 0.4; 1.3 99.6 ± 0.3; 1.8

200.0 202.7 ± 0.2; 0.8 198.2 ± 0.1; 1.0

300.0 300.1 ± 0.4; 1.5 304.7 ± 1.2; 1.5

Dexamethasone 20.0 20.0 ± 0.9; 0.3 19.9 ± 1.7; 1.0

40.0 40.5 ± 0.5; 0.9 40.2 ± 0.9; 0.4

60.0 59.7 ± 0.8; 1.4 59.8 ± 0.7; 1.6

n = average of 5 analyses.

day and inter-day precision was determined by injecting the same solutions for three consecutive days. Relative standard deviation (RSD%) of the peak area was calculated to represent

Table 5 Robustness study of dexamethasone.

Chromatographic Assay tR Theoretical Tailing

conditions (%) (min) plates

Methanol:buffer (63.5:36.5) 101.2 6.047 6322 1.09

Methanol:buffer (61.5:38.5) 101.8 6.503 6274 1.08

Methanol:buffer (59.5:40.5) 99.0 7.211 6344 1.09

Flow rate (1.3 mL/min) 99.1 7.433 6354 1.09

Flow rate (1.5 mL/min) 101.5 6.503 6247 1.08

Flow rate (1.7 mL/min) 100.5 5.569 6378 1.09

Buffer (pH 2.6) 100.4 6.697 6398 1.10

Buffer (pH 2.8) 99.9 6.503 6358 1.09

Buffer (pH 3.o) 98.5 6.498 6241 1.10

precision. Results of intra-day and inter-day precision are presented in Table 3.

Robustness of the method was performed by slightly varying chromatographic conditions. The results showed that slight variations in chromatographic conditions had a negligible effect on the chromatographic parameters (Tables 4 and 5).

Specificity of the developed method was evaluated by applying different stress conditions (acid, base, oxidation, thermal and photolytic) to moxifloxacin hydrochloride and dexamethasone in combination form. The chromatograms under basic and photolytic stress conditions are shown in Figs. 3 and 4 along with the peak purity index of both the active ingredients (others not shown). The results of stress studies are given in Table 6.

All the stress conditions applied were enough to degrade both the drugs. Comparison of the two drugs showed that moxifloxacin hydrochloride is more stable as compared to dexamethasone. Under acidic conditions dexamethasone was degraded up to 22.6% and moxifloxacin hydrochloride was

Figure 3 Chromatogram of moxifloxacin (200 igmL ^ and dexamethasone (40 igmL ^ under basic stress.

Figure 4 Chromatogram of moxifloxacin (200 igmL ^ and dexamethasone (40 igmL ^ under photolytic stress.

degraded up to 9.1%. Under basic stress dexamethasone was degraded up to 94.5% and moxifloxacin hydrochloride was found to be stable. Under oxidative stress dexamethasone was degraded up to 5.5% and moxifloxacin hydrochloride was degraded up to 6%. Under thermal stress studies, moxi-floxacin hydrochloride and dexamethasone were degraded up to 4.2% and 3.8%, respectively. Under photolytic stress dexa-methasone and moxifloxacin hydrochloride were degraded up to 6.3% and 10.2%, respectively. From these stress studies it is thus concluded that dexamethasone and moxifloxacin hydro-chloride drugs are not stable in acidic, basic, oxidative, thermal and photolytic stress conditions.

The developed method effectively separated the degradation products or impurities (1 impurity peaks under acidic stress, 6 impurity peaks under basic stress with IMP 4 as major degradation peak, 3 impurity peaks under oxidative stress with IMP 1 as major degradation peak, 4 impurity peaks under photolytic stress with IMP 3 as major degradation peak) from analyte peaks. The resolution of impurity in acidic stress was 2.03. In basic stress the resolution of six impurities were 2.03, 3.12, 3.43, 1.51, 4.32 and 2.76, respectively, from impurities 1-6. Resolution of impurities under oxidative stress was 2.03, 3.98 and 2.32 whereas under photolytic stress the resolution was 2.11, 3.22, 1.59 and 4.98, respectively. Therefore, the developed method is considered to be highly specific and selective for intended use.

Application of the developed method was checked by analysing the moxifloxacin hydrochloride and dexamethasone in commercially available pharmaceutical products. The results are provided in Table 7 which showed high percentage recoveries and low RSD (%) values for both analytes.

4. Conclusion

A simple, sensitive, isocratic and accurate reverse phase HPLC method is described for simultaneous determination of moxi-floxacin hydrochloride and dexamethasone in pharmaceutical formulations. The developed method was validated by testing its linearity, accuracy, precision, specificity, limits of detection and quantitation. The method is simple, fast and is without the use of ion pair or any derivatization reagent. The method is good enough to separate the peaks of active pharmaceutical ingredients (APIs) from the degradation products (produced during forced degradation studies). It is also clear from the chromatograms that both the active ingredient peaks in all the stress conditions were free from any sort of degradation impurities. All these convince us to conclude that the method can be successfully used for any sort of stability and validation studies.

References

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Chem. Soc. 52, 1220-1223. Ashfaq, M., Khan, I.U., Asghar, M.N., 2008. J. Chil. Chem. Soc. 2008 (53), 1617-1619.

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Table 6 Stress testing results of moxifloxacin and dexamethasone.

Nature of Storage Time (h) Amount of moxifloxacin Amount of Dexamethasone Extent of

stress conditions remaining ± RSD (%) remaining ± RSD (%) decomposition

5 M HCl (40 °C/75% RH) 16 90.9 ± 2.8 (PPI = 1.0000) 77.4 ± 2.0 (PPI = 1.0000) Substantial

5 M NaOH (22 °C/58% RH) 0.67 102.0 ± 1.1 (PPI = 1.0000) 5.5 ± 2.3 (PPI = 1.0000) Substantial

6% H2O2 (40 °C/75% RH) 16 94.0 ± 1.9 (PPI = 1.0000) 94.5 ± 1.8 (PPI = 1.0000) Substantial

Thermal (40 °C/75% RH) 144 100.1 ± 1.7 (PPI = 1.0000) 99.0 ± 2.2 (PPI = 1.0000) None

288 97.8 ± 1.5 (PPI = 1.0000) 96.4 ± 1.7 (PPI = 1.0000) Slight

528 95.8 ± 1.5 (PPI = 1.0000) 96.2 ± 1.7 (PPI = 1.0000) Slight

Photolytic Sunlight 1 89.8 ± 1.7 (PPI = 1.0000) 93.7 ± 2.8 (PPI = 1.0000) Substantial

n = average of 3 determinations, PPI = peak purity index, chromatographic conditions: mobile phase methanol: 20 mM phosphate buffer

61.5:38.5, v/v, pH 2.8, Column BDS Hypersil C8 (250 x 4.6, 5 im), flow rate 1.5 mL min , injection volume 20 iL, wavelength 254 nm.

Table 7 Assay results of moxifloxacin and dexamethasone in commercial eye drops.

Products eye Ingredient Label value % Recovery

drops (mg per mL) ± RSD (%)

Occumox DM Moxifloxacin 5 100.3 ± 0.3

Dexamethasone 1 99.0 ± 0.7

Moxiblu-D Moxifloxacin 5 100.7 ± 0.1

Dexamethasone 1 98.9 ± 0.7

n = average of 10 determinations, chromatographic conditions: mobile phase methanol: 20 mM phosphate buffer 61.5:38.5, v/v, pH 2.8, Column BDS Hypersil C8 (250 x 4.6, 5 im), flow rate 1.5 mL min-1, injection volume 20 iL, wavelength 254 nm.

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