Stability Indicating HPLC Method for the Determination of Zonisamide as Bulk Drug and in Pharmaceutical Dosage Form 2006, 64, 261–266

D. V. G. Rao1,&, I. E. Chakravarthy1, S. R. Kumar2 1 2

Department of Chemistry, Sri Krishna Devaraya University, Post Graduate Center, Kurnool 518002, AP, India; E-Mail: [email protected] Analytical Research and Development, Dr. Reddy’s Laboratories, Hyderabad 500072, India

Received: 6 May 2006 / Revised: 1 July 2006 / Accepted: 4 July 2006 Online publication: 23 August 2006

Abstract This paper describes the validation of an Isocratic HPLC method for the assay of zonisamide as bulk and in pharmaceutical dosage forms. The method employs a Waters X-Terra RP 18 (150 mm · 4.6 mm, 5 lm particle size) column with a mobile phase of acetonitrile and 0.1 M sodium dihydrogen phosphate monohydrate pH 3.0 in the ratio 18:82 (v/v) and UV detection at 240 nm. A linear response (r > 0.999) was observed in the range of 0.5–100 lg mL)1. The method shows good recoveries (average 98.9%) and the relative standard deviation intra and inter-day were found to be <0.5%. Validation parameters as specificity, robustness were also determined. The method can be used for quality control assay of zonisamide as bulk and in finished dosage forms and for stability studies as the method separates zonisamide from its degradation products and excipients.

Keywords Column liquid chromatography Zonisamide Stability testing Quality control Method validation

Introduction Zonisamide [1,2-benzisoxazole-3-methane sulfonamide] is used as an anti convulsant in patients with epileptic disorders [1, 2]. It has a benzisoxazole structure that is unrelated to other antiseizure drug. Zonisamide is well absorbed from the gastrointestinal tract [3]. Tmax is 2–6 h following the oral, single dose administration of 200–800 mg in both patients and healthy volunteers [3, 4]. Food has no impact on the absorption of zonisamide [5]. The Original DOI: 10.1365/s10337-006-0024-6 0009-5893/06/09

therapeutic range of this drug is relatively narrow; high doses often cause side effects such as drowsiness, ataxia, anorexia, decreased spontaneity, mental slowness and weight loss [2, 6, 7]. Several methods have been reported for the analysis of zonisamide using gas chromatography [8, 9], micellar electro kinetic capillary chromatography [10], enzyme immunoassay [11] and HPLC methods with UV detection [12–19] for the determination of zonisamide in human plasma. So far, to our present

knowledge, no stability indicating analytical method available for the determination of zonisamide as bulk and in pharmaceutical dosage forms in literature. It was felt necessary to develop a stability indicating LC method for the determination of zonisamide as bulk and in pharmaceutical dosage forms.

Experimental Material and Reagents Samples of zonisamide (Fig. 1) and zonisamide impurities were received from Process R&D, Dr. Reddy’s Laboratories, Hyderabad, India. Zonisamide capsules were received from formulation R&D, Dr. Reddy’s Laboratories, Hyderabad, India. Zonisamide 100 mg capsules weight about 376 mg contains 100 mg of active pharmaceutical ingredient (API) i.e. zonisamide, microcrystalline cellulose and Hydrogenated vegetable oil. HPLC grade acetonitrile was purchased from Merck, Darmstadt, Germany. Analytical grade sodium dihydrogen phosphate monohydrate was purchased from Rankem, Mumbai, India. Ortho phosphoric acid purchased from S.A Fine Chemicals, Mumbai, India. High purity water was prepared by using Millipore Milli Q plus purification system.

Equipment The M/s Waters HPLC System with a photo diode array detector was used for

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CH2SO 2NH2 N O Fig. 1. Chemical structure of zonisamide

the method development and force degradation studies. The out put signal was monitored and processed using Waters Empower Net Working Software. The LC System used for method validation was Agilent 1100 series LC system with variable wavelength detector (VWD). The out put signal was monitored and processed by using Waters Empower Networking Software on Pentium computer (Digital equipment co.).

Chromatographic Conditions The chromatographic column used was a Waters 150 mm · 4.6 mm X-Terra RP18 column with 5 l particle size. The mobile phase was acetonitrile and 0.1 M sodium dihydrogen phosphate monohydrate, pH adjusted to 3.0 with orthophospharic acid in the ratio 18:82 v/v. The flow rate of the mobile phase was 1.5 mL min)1. The column temperature was maintained at 25 C and the detection wavelength of 240 nm. The injection volume was 10 lL. 0.1 N methanolic HCl (8.5 mL of concentrated HCl in 1,000 mL of water) was used as diluent.

Preparation of Standard Solution The stock solution of zonisamide reference substance (1.0 mg mL)1) was prepared in diluent. The working standard solution (50 lg mL)1) was obtained by dilution of the stock solution in diluent. The overlay chromatogram of Diluent and Standard is shown in Fig. 2.

Preparation of Sample Solution Twenty capsules were weighed and the content transferred into a clean and dry mortar and the empty capsules shells weighed and the average weight of capsules content determined and the content were mixed. Then equivalent to 100 mg

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of drug was transferred into a 100 mL volumetric flask, 70 mL of the diluent was added and kept on rotary shaker for 10 min to disperse the material completely and sonicated for 20 min and diluted to 100 mL with diluent. The resulting solution was centrifuged at 3,000 rpm for 5 min. Supernant solution was taken 5 mL and diluted to 100 mL with diluent. Filtered about 2 mL by using 0.45 l nylon 66 membrane filter. The overlay chromatogram of placebo and test of zonisamide were shown in Fig. 3.

Specificity Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities [20]. Chromatographic runs of placebo solution and force degradation studies were performed in order to provide an indication of the stability indicating properties and specificity of the method. The degradation samples were prepared by transferring capsules powder equivalent to 100 mg of zonisamide in to a 100 mL volumetric flask. The stress conditions employed were acid, base, neutral and oxidant media, heat, moisture and light. After the degradation treatments were completed, the samples were allowed to equilibrate to room temperature, neutralized with acid or base (when necessary), and diluted with diluent to 50 lg mL)1. The samples were analyzed against a freshly prepared control sample (with no degradation treatment) and evaluated for peak purity by using photo diode array detector. Specific conditions were described below.

Effect of Acid, Alkaline and Neutral Hydrolysis Ten milliliter of the 1.0 mg mL)1 zonisamide test solution were transferred to a flask and 5 mL of 5 N HCl, 5 N NaOH or water respectively added. The acidic solution was refluxed for 6 h at 80 C; the alkaline solution was refluxed for 2 h at 80 C; and the neutral solution was refluxed for 12 h at 80 C. After that all samples were allowed to equilibrate to room temperature, neutralized with base or acid as appropriate and then diluted with diluent to 50 lg mL)1.

Effect of Oxidation A 1.0 mg mL)1 zonisamide test solution was prepared from the Capsules. Five milliliter of this solution was added to 5 mL of 1% H2O2 and refluxed for 1 h at 80 C. After that, the solution was diluted with diluent to 50 lg mL)1 and filtered about 2 mL by using Nylon 66 membrane filter.

Effect of Moisture and Heat Thin layers of capsules powder were distributed over a glass plate to evaluate the effect of moisture and heat. The plate were stored at 25 C, 90% relative humidity for about 168 h. A similar sample was kept in a oven at 105 C for 12 h. After specified time the samples were prepared in diluent as per the sample preparation.

Effect of UV and Visible Light The photochemical stability of the drug product was also studied by exposing the drug product to 1,200 K lux of visible light and 200 W h m)2 of UV light by using photo stability chamber. After exposure the samples were prepared in diluent as per the sample preparation.

Method Validation Precision

Assay of method precision (intra-day precision) was evaluated by carrying out six independent assays of test sample of zonisamide capsules against qualified reference standard. The percentage of R.S.D of six assay values obtained was calculated. The intermediate precision (inter-day precision) of the method was also evaluated using two different analysts, different HPLC systems and different HPLC columns in different days in the same laboratory. Linearity

Linearity test solutions for assay method prepared from stock solution at ten concentration levels in the range of about 0.5–100 lg mL)1 (corresponding to 1–200% of assay nominal sample

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concentration). The peak area versus concentration data was performed by least-squares regression analysis. Accuracy

A study of recovery of zonisamide from spiked placebo was conducted. Samples were prepared by mixing placebo with zonisamide raw material equivalent to about 50, 75, 100, 125, 150% of the assay nominal sample concentration. Sample solutions were prepared in triplicate for each spike level as described in the sample preparation. The percentage of recovery was calculated.

Fig. 2. Overlay chromatogram of diluent and zonisamide standard preparation

Robustness

To determine the robustness of the developed method, experimental conditions were purposely altered. One factor at a time was changed to estimate the effect. Thus, five replicate injections of standard solution were injected under each parameter change. The flow rate of the mobile phase was 1.5 mL min)1. To study the effect of flow rate on the tailing factor for zonisamide peak and the R.S.D for peak area of zonisamide from replicate injections of standard, it was changed by 0.2 mL min)1 steps from 1.3 to 1.7 mL min)1. The effect of pH of buffer in mobile phase on the tailing factor for zonisamide peak and the R.S.D for peak area of zonisamide from replicate injections of standard was studied by varying ±0.2 pH units. The effect of column temperature on tailing factor for zonisamide peak and the R.S.D for peak area of zonisamide from replicate injections of standard was studied at 20 and 30 C instead of 25 C. The effect of organic phase composition (acetonitrile) in mobile phase on the tailing factor for zonisamide peak and the R.S.D for peak area of zonisamide from replicate injections of standard was studied by changing ±10% of the organic phase composition.

Solution Stability and Mobile Phase Stability

The solution stability of zonisamide in the assay method was carried out by leaving both the test solutions of sample and reference standard in tightly capped volumetric flasks at room temperature for 48 h. The same sample solutions was

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Fig. 3. Overlay chromatogram of zonisamide capsules placebo and test

assayed for 12 h interval up to the study period. The mobile phase stability was also carried out by assaying freshly prepared sample solutions against freshly prepared reference standard solutions for 12 h interval for 48 h. Mobile phase prepared was kept constant during the study period. The % R.S.D of assay of zonisamide was calculated for the study period during mobile phase stability and solution stability experiments.

Results and Discussion Method Development and Optimization The HPLC procedure was optimized with a view to develop a stability indicating assay method. Pure drug along with its

related impurities were injected and run in different solvent systems. Our preliminary trials using different compositions of water, acetonitrile and methanol on different reversed phase stationary phases did not give good peak shape and the separation between zonisamide and zonisamide known impurities in a C-18 column. Introduction of buffer (sodium dihydrogen phosphate) improved the peak shape of zonisamide in a C-18 Column. When the mobile phase consisting 0.1 M sodium dihydrogen phosphate monohydrate buffer (buffer pH adjusted to 3.0 with orthophospharic acid) and acetonitrile in the ratio 82:18 v/v early elution (retention time was about 5 min) of zonisamide and the separation between zonisamide and zonisamide known impurities were observed on Waters X-Terra RP-18 Column with a flow rate of 1.5 mL min)1.

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Table 1. Results of precision of test method Sample number

peek area and concentration of the analyte.

Assay of zonisamide as % of labeled amount

1 2 3 4 5 6 Mean RSD

Analyst-1 (intra-day precision)

Analyst-2 (inter-day precision)

98.7 98.7 98.8 99.1 99.0 99.0 98.9 0.2%

98.6 98.6 99.0 98.8 99.0 98.1 98.7 0.3%

Accuracy

The percentage recovery of zonisamide in pharmaceutical dosage forms ranged from 97.8 to 99.1 (Table 2). Robustness

Table 2. Recovery results of zonisamide in pharmaceutical dosage forms Spike level (%)

Average ‘mg’ added

Average ‘mg’ found

Mean % recovery

Percentage R.S.D

50 75 100 125 150

49.997 74.967 99.85 124.81 149.76

48.997 73.543 98.552 122.314 147.364

98.0 98.1 98.7 98.0 98.4

0.2 0.2 0.3 0.1 0.1

In all the deliberate varied chromatographic conditions carried out i.e. flow rate, column temperature, pH of the buffer in mobile phase and organic phase composition in mobile phase, the tailing factor and the % R.S.D for the zonisamide peak area from the five replicate injections of standard was found to be with in the acceptable limits, illustrating the robustness of the method (Table 3). Solution Stability and Mobile Phase Stability

Table 3. Results of robustness study Parameter

Flow rate Column temperature pH (±0.2 units of the set pH) Mobile phase composition

Observed value Variation

Tailing factor

R.S.D for five injections of standard (%)

1.3 mL min)1 1.7 mL min)1 20 C 30 C pH 2.8 pH 3.2 90% Organic 110% Organic

1.0 1.0 1.0 1.0 1.1 1.1 1.0 1.0

0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.0

Method Validation Precision

Method repeatability (intra-day precision) was evaluated by assaying six samples, prepared as described in the sample preparation. The mean % assay and percentage R.S.D for assay values were found to be 98.9 and 0.2%, respectively, which is well within the acceptance criteria i.e. assay value should be between 97.0 and 103.0% and R.S.D should be not more than 2.0%. The intermediate precision (inter-day precision) was performed by assaying six samples prepared by different analyst, different HPLC system and different HPLC column in different days as described in the sample preparation. The mean % assay and percentage R.S.D for assay values were

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found to be 98.9 and 0.3%, respectively. The results of intra-day precision and inter-day precision were evaluated with respect to student’s t test and found that t-test was passed. The result shows that good precision of the method (Table 1).

Linearity

Linear calibration plat for assay method was obtained over the calibration ranges tested, i.e. 0.5–100 lg mL)1 and the correlation co-efficient was found to be greater than 0.999. Linearity was checked over the same concentration range for three consecutive days. The percentage of R.S.D values of the slope and Y intercept of the calibration curves was 3.2 and 5.8, respectively. The result shows that an excellent correlation existed between the

The R.S.D of assay of zonisamide during solution stability and mobile phase stability experiments was within 1%. The solution stability and mobile phase stability experiments data confirms that sample solutions and mobile phase used during assay determination was stable up to 48 h. Results of Force Degradation Studies

All the stressed samples prepared, were injected into the HPLC system with photodiode array detector as per the described chromatographic conditions. Degradation was not observed in light exposure and water hydrolysis. In heat, moisture, acid hydrolysis and oxidative conditions degradation were found to be very less. Only in base hydrolysis significant degradation was observed. All degradant peaks were resolved from zonisamide peak in the chromatograms of all stressed samples. The chromatograms of the stressed samples were evaluated for peak purity of zonisamide using Waters Empower Networking Software. For all forced degradation samples, the purity angle (the weighted average of all spectral contrast angles calculated by comparing all spectra in the integrated peak against the peak apex spectrum) found to be less than threshold angle (the sum of the purity noise angle and solvent angle, the purity noise angles across the integrated peak) and there was no purity flag (the purity flag is an indication of

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Table 4. Table of results of specificity Stress condition

Drug product

Refluxed with 5 N HCI solution for about 6 h at 80 C Refluxed with 5 N NaOH solution for about 2 h at 80 C Refluxed with 1% H2O2 for about 1 h at 80 C Refluxed with purified water for about 12 h at 80 C Exposed to Visible light for about 1,200 K lux Exposed to UV light both at shorter and longer wavelengths for 200 W h m)2 Dry heated for about 12 h at 105 C Exposed to humidity at 25 C, 90% RH for about 7 days

% Degradation

Purity angle

Purity threshold

Purity flag

0.25 5.02 1.54 Nil Nil Nil 0.22 0.41

0.060 0.201 0.114 0.057 0.099 0.105 0.059 0.097

0.261 0.315 0.310 0.264 0.306 0.310 0.256 0.311

No No No No No No No No

spectral homogeneity, compares the purity angle with the purity threshold) for the zonisamide peak (Table 4). This indicates that there is no interference from degradants in quantitating the zonisamide in capsules. Thus, this method is considered to be ‘‘Stability Indicating’’. The resulting chromatogram and purity plot of base stressed sample is shown in Fig. 4.

Conclusion An isocratic reversed phase HPLC method has been developed and validated for the determination of zonisamide as bulk drug and in pharmaceutical formulations. This chromatographic assay fulfilled all the requirements to be identified as reliable and feasible method, including accuracy, linearity, recovery and precision data. It is a highly specific and precise analytical procedure and its chromatographic run time of 7 min allows the analysis of a large number of samples in a short period of time. Therefore, this HPLC method can be used as a routine sample analysis.

Acknowledgments The authors wish to thank the management of Dr. Reddy’s Group for supporting this work. The authors wish to acknowledge the Bulk R&D and Formulation R&D Group for providing the samples for our research.

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Fig. 4. Chromatogram and purity plot of base stressed zonisamide capsules test

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