STABILITY INDICATING LIQUID CHROMATOGRAPHIC METHOD FOR THE QUANTITATIVE DETERMINATION OF RUFINAMIDE IN PHARMACEUTICAL DOSAGE FORMS

Rufinamide is chemically known as 1[(2, 6difluorophenyl) methyl]-1H-1,2,3-triazole-4 carboxamide with molecular formula C10H8F2N4O and molecular weight 238.19 g/mol as shown in Figure 1. Rufinamide is an antiepileptic drug approved by the US Food and Drug Administration as adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in children 4 years and older and adults. Lennox-Gastaut syndrome consists of a variety of treatment-resistant seizures and is most common among paediatric patients 1 . The mechanism of action of Rufinamide is unknown but it is presumed to involve stabilizat ion of the sodium channel inactive state, effectively keep ing the ion channels closed. It is believed to prolong the refractory period of voltage-dependent sodium channels, making neurons less likely to fire 2 .


INTRODUCTION
Rufinamide is chemically known as 1-[(2, 6difluorophenyl) methyl]-1H-1,2,3-triazole-4 carbo xamide with molecular formu la C 10 H 8 F 2 N 4 O and molecular weight 238.19 g/mo l as shown in Figure 1. Rufinamide is an antiepileptic drug approved by the US Food and Drug Admin istration as adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in children 4 years and older and adults. Lennox-Gastaut syndrome consists of a variety of treatment-resistant seizures and is most common among paediatric patients 1 . The mechanism of action of Rufinamide is unknown but it is presumed to involve stabilizat ion of the sodium channel inactive state, effectively keep ing the ion channels closed. It is believed to prolong the refractory period of voltage-dependent sodium channels, making neurons less likely to fire 2 .

Figure 1: Chemical Structure of Rufinami de
Very few methods are reported in the literature regarding the clinical studies and no stability indicating method is available in the official compendia using HPLC for analysing Rufinamide in dosage forms. Analytical methods for Rufinamide fro m pharmaceutical dosage form should be developed and validated. To date, all analytical methods described in literature for the determination of Rufinamide in biological fluids involve liquid chro matography 3-7 and liquid chro matography-mass spectrometry methods 8-9. Quality control of pharmaceutical products requires identification and quantification of the active ingredient and its impurities for safety and efficacy reasons. Impurit ies and potential degradation products that may exist in med icines can change the chemical, pharmacological and toxico logical properties of the product. Since pharmacopoeias do not describe a suitable method for the determination of Rufinamide in pharmaceutical formu lations, in the present work we developed simple, rap id and accurate reverse phase liquid chromatographic method for the determination of Rufinamide tablets as an alternative method. Apart from this, it can be used for assays of Rufinamide in biological flu ids or in pharmacokinetic investigations.

ABS TRACT
A selective, specific and sensitive stability-indicating high-performance liquid chromatographic method was developed and validated for the determination of Rufinamide in tablet dosage forms. Reversed-phase chromatography was performed on Shimadzu M odel CBM -20A/20 Alite, equipped with SPD M 20A prominence photodiode array detector using C18 column (250 mm × 4.6 mm, 5 μm) with a flow rate of 1.0 mL/min. Detection wavelength was 210 nm and temperature was 30°C. Linearity was observed in the concentration range of 0.01-160 μg/mL with regression equation y = 112887 x + 35285 with correlation coefficient of 0.9998. The LOQ and LOD were found to be 0.0086 μg/mL and 0.0028 μg/mL respectively. The percentage relative standard deviation in precision and accuracy studies was found to be less than 2%. Rufinamide was subjected to stress conditions of degradation in aqueous solutions including acidic, alkaline, oxidation, photolysis and thermal degradation and it was found that the drug is highly resistant towards all degradations as the decomposition was less than 1.5%. The developed method was validated with regard to linearity, accuracy, precision, selectivity and robustness and t he method was found to be precise, accurate, linear and specific. As the proposed LC method achieved satisfactory resolution between Rufinamide, its degradation products, intermediate product possibly present in Rufinamide drug substance and other impurities in the end product before refining in the final step of synthetic process, it can be employed as a stability indicating one, used for the synthetic process control and determination of Rufinamide in pharmaceutical preparations.
Rufinamide is available as tablets with brand names Pr BANZEL ™ ® as tablets (Label claim: 200 and 400 mg) and BANZEL ® as tablets (Label claim: 200 and 400 mg ) as well as oral suspension (Label claim: 40 mg/ mL) . All chemicals were of analytical grade and used as received.

Chromatographic Condi tions
Isocratic elution was performed using methanol and water (52:48, V/ V) with flow rate 1.0 mL/ min. 20 µL of sample was injected into the HPLC system.

Preparation of Rufinami de Stock Soluti on
Rufinamide stock solution (1000 μg/ mL) was prepared by accurately weighing 25 mg of Rufinamide in a 25 mL amber volu metric flask and making up to volume with mobile phase. Working solutions for HPLC injections were prepared on a daily basis from the stock solution in a solvent mixture of methanol and water (52:48, V/ V) (mobile phase). Solutions were filtered through a 0.45 μm memb rane filter prior to injection.

Preparation of Sample Solutions
Twenty tablets from each brand ( Pr BANZEL ™ ® and BANZEL ® ) were procured, weighed and crushed to a fine powder. Powder equivalent to 25 mg Rufinamide was accurately weighed into a 25 ml volu metric flask and made up to volume with mobile phase. The contents of the volumetric flask were sonicated for 30 min to enable complete dissolution of Rufinamide. The solution was filtered and the filtrate was diluted with mobile phase. 20 μL of these solutions were injected into the system and the peak area was recorded fro m the respective chro matogram.

Forced Degradation Studies/Specificity
The study was intended to ensure the effective separation of Rufinamide and its degradation peaks of formulat ion ingredients at the retention time of Rufinamide. Separate portions of drug product and ingredients were exposed to the following stress conditions to induce degradation. Forced degradation studies were performed to evaluate the stability indicating properties and specificity of the method 10 . All solutions for use in stress studies were prepared at an initial concentration of 1 mg/ mL of Rufinamide and reflu xed for 30 min at 80 ºC. A ll samp les were then diluted in mobile phase to give a final concentration of 80 μg/ mL and filtered before injection.

Aci dic and Alkaline Degradati on
Acid decomposition was carried out in 0.1 M HCl at a concentration of 1.0 mg/ mL Rufinamide and after reflu xation for 30 min at 80 ºC the stressed sample was cooled, neutralized and diluted with mobile phase to give a final concentration of 80 μg/mL and filtered before injection.
Similarly stress studies in alkaline conditions were conducted using a concentration of 1.0 mg/ mL in 0.1 M NaOH and reflu xed for 30 min at 80 ºC. After cooling the solution was neutralized and diluted with mobile phase to give a final concentration of 80 μg/ mL and filtered before injection.

Oxi dati ve Degradati on
Solutions for oxidative stress studies were prepared using 3% H 2 O 2 at a concentration of 1 mg/ mL of Rufinamide and after reflu xat ion for 30 min at 80 ºC on the thermostat the sample solution was cooled and diluted with the mobile phase to give a final concentration of 80 μg/ mL and filtered before inject ion.

Thermal Degradati on
For thermal stress testing, the drug solution (1 mg/ mL) was heated in thermostat at 80 ºC for 30 min, cooled and diluted with the mob ile phase to give a final concentration of 80 μg/ mL and filtered before inject ion.

Photol ytic Degradati on
The drug solution (1 mg/ mL) for photo stability testing was exposed to UV light for 4 hours UV light chamber (365 n m) and diluted with the mobile phase to give a final concentration of 80 μg/ mL and filtered before inject ion.

Method Vali dation
The method was validated for the following parameters : system suitability, linearity, limit of quantitation (LOQ), limit of detection (LOD), precision, accuracy, selectivity and robustness 11 .

Linearity
Linearity test solutions for the assay method were prepared fro m a stock solution at different concentration levels of the analyte (0.01-160 µg/ mL). 20 µL of each solution was injected in to the HPLC system and the peak area of the chromatogram obtained was noted.
The solutions ext racted fro m the marketed formu lat ions were in jected in to the HPLC system and the peak area of the chromatograms was noted. The analytical curve was evaluated on three different days. The peak area vs. concentration data was analyzed with least squares linear regression. The slope and y-intercept of the calibration curve was reported.

Precision
The intra-day precision of the assay method was evaluated by carrying out 9 independent assays of a test sample of Rufinamide at three concentration levels (20, 40 and 80 µg/mL) (n=3) against a qualified reference standard. The %RSD o f three obtained assay values at three different concentration levels was calculated. The interday precision study was performed on three different days i.e. day 1, day 2 and day 3 at three different concentration levels (20, 40 and 80 µg/mL) and each value is the average of three determinations (n=3). The % RSD of three obtained assay values on three different days was calculated.

Accuracy
The accuracy of the assay method was evaluated in triplicate at three concentration levels (80, 100 and 120%), and the percentage recoveries were calcu lated. Standard addition and recovery experiments were conducted to determine the accuracy of the method for the quantification of Rufinamide in the drug product. The study was carried out in triplicate at 18, 20 and 22 µg/mL. The percentage recovery in each case was calculated.
Sensitivity/ Li mit of quanti ficati on (LOQ) and limit of detection (LOD) The limit of quantification (LOQ) and limit of detection (LOD) were based on the standard deviation of the response and the slope of the constructed calibration curve (n=3), as described in International Conference on Harmonizat ion guidelines Q2 (R1) 11 . Sensitivity of the method was established with respect to limit of detection (LOD) and LOQ for Ru finamide. LOD and LOQ were established by slope method as mentioned below.

× standard deviation of y-intercept
Slope of the calibration curve LOQ= 10 × standard deviation of y-intercept Slope of the calibration curve LOD and LOQ were experimentally verified by inject ing six replicate injections of each impurity at the concentration obtained from the above formu la.

Robustness
The robustness of the assay method was established by introducing small changes in the HPLC conditions which included wavelength (208 and 212 n m), percentage of acetonitirile in the mobile phase (54 and 50) and flow rate (0.9 and 1.1 mL/ min). Robustness of the method was studied using six rep licates at a concentration level of 20 µg/mL of Rufinamide.

Solution Stability and Mobile Phase Stability
The solution stability of Rufinamide in the assay method was carried out by leaving both the sample and reference standard solutions in tightly capped volumetric flasks at room temperature for 48 h. The same sample solutions were assayed at 12 h intervals over the study period. The mobile phase stability was also assessed by assaying the freshly prepared sample solutions against freshly prepared reference standard solutions at 12 h intervals up to 48 h. The prepared mobile phase remained constant during the study period. The % RSD of the Rufinamide assay was calculated for the mobile phase and solution stability experiments. An additional study was carried out using the stock solution by storing it in a tightly capped volumetric flask at 4 ºC.

RES ULTS AND DISCUSS ION
No stability indicating method is available in the official compendia using HPLC for analysing Rufinamide in dosage forms till now. The present proposed method is simp le, precise and accurate in comparison to the reported methods in the literature (Table 1).
A reversed-phase chromatographic technique was developed to quantitate Rufinamide at 210 n m. Methanol was chosen as an organic modifier in the mobile phase. Satisfactory resolution was achieved with use of a mixture of methanol and water (52:48, V/ V) as demonstrated in Figure 2. C8 and C18 colu mns were first evaluated as stationary phase for the separation of Rufinamide. C18 column was adopted for the analysis because it provided a better separation of the analytes.  The present stability-indicat ing method for the determination of Rufinamide in pharmaceutical formulat ions is specific because the drug peak was well separated even in the presence of degradation products. Overall, the data demonstrated that the e xcip ients and the degradation products did not interfere with the Rufinamide peak, indicating the selectivity of the method. The complete separation of the analytes was accomplished in less than 10 min and the method can be successfully applicable to perform long-term and accelerate stability studies of Rufinamide formulat ions.

HPLC Method Devel opment and Opti mization
Initially the stressed samples were analyzed using a mobile phase consisting of water: acetonitrile (70:30, v/v) at a flow rate of 1.0 mL/ min. Under these conditions, the resolution and peak symmetry were not satisfactory, so the mobile phase was changed to methanol: water (52:48, V/ V) with a flow rate of 1.0 mL/ min under wh ich peaks were well resolved with good symmetry and sharpness. Therefore, mobile phase containing methanol: water (52:48, V/ V) was chosen for the best chromatographic response for the entire study.

System Suitability
The system suitability test was performed to ensure that the complete testing system was suitable for the intended application. The parameters measured were peak area, retention time, tailing factor, capacity factor and theoretical plates. In all measurements the peak area varied less than 2.0%, the average retention time was 5.5 ± 0.05 minutes. The capacity factor was more than 2, theoretical plates were 9873 (mo re than 2000) and tailing factor was 1.22 (less than 2) for the Rufinamide peak. The proposed method offers high sensitivity and Rufinamide can be detected accurately. In all the cases, the Rufinamide peak was well separated fro m the degradation products.

Linearity
The calibration curve for Rufinamide was linear over the concentration range of 0.01-160 μg/ mL. The data for the peak area of the drug in corresponds to the concentration was treated by linear regression analysis (Table 2) and the regression equation for the calibration curve (Figure 3) was found to be y = 112887 x + 35285 with correlat ion coefficient of 0.9998.  precision was calculated by assaying three samples of each at three different concentration levels (20, 40 and 80 µg/mL) on three different days. The % RSD range was obtained as 0.32-0.40 and 0.57-0.64 for intra -day and inter-day precision studies respectively (Table 3). Because the stability of standard solutions can also affect the robustness of analytical methods, the stability of standard solutions of the drug substance used in this method was tested over a long period of time. One portion of a standard solution was kept at room temperature and the other portion was stored under refrigeration at approximately 4ºC and the content of these solutions was regularly co mpared with that of freshly prepared solutions.
No change in drug concentrations were observed for solutions stored under refrigeration. But it is recommended that the sample and standard solutions must therefore, be freshly prepared in amber colored flasks to protect from light.

Accuracy
The method accuracy was proven by the recovery test. A known amount of Rufinamide standard (10 μg/ mL) was added to aliquots of samples solutions and then diluted to yield total concentrations as 18, 20 and 22 μg/mL as described in Table 4. The assay was repeated over 3 consecutive days. The resultant % RSD was 0.83 (<2.0 %) with a recovery 97.20-97.83 %.

Li mit of Detection and Li mit of Quantification
The LOQ and LOD were determined based on the 10 and 3.3 times the standard deviation of the response, respectively, divided by the slope of the calibration curve. The LOQ and LOD were found to be 0.0086 μg/ mL and 0.0028 μg/ mL respectively.

Robustness
The robustness of an analytical procedure refers to its ability to remain unaffected by small and deliberate variations in method parameters and provides an indication of its reliab ility for routine analysis 10 . The robustness of the method was evaluated by assaying the same sample under different analytical conditions deliberately changing fro m the original condition. The detection wavelength was set at 208 and 212 n m (± 2 n m), the ratio of percentage of methanol: water in the mobile phase was applied as 54:48 and 50:50 (± 2 %, V/ V), the flow rate was set at 0.9 and 1.1 mL/ min (± 0.1 mL/ min). The results obtained from assay of the test solutions were not affected by varying the conditions and were in accordance with the results for original conditions. The % RSD value of assay determined for the same sample under original conditions and robustness conditions was less than 2.0% indicating that the developed method was robust.

Selectivity/Specificity
The specificity of the developed method was determined by injecting sample solutions (80 μg/mL) wh ich were prepared by forcibly degrading under such stress conditions as heat, light, o xidative agent, acid and base under the proposed chromatographic conditions. The stability indicating capability of the method was established from the separation of Rufinamide peak fro m the degraded samples derived fro m the software. The degradation of Rufinamide was found to be very similar for both the tablets and standard.

Solution Stability and Mobile Phase Stability
The %RSD of the assay of Rufinamide fro m the solution stability and mobile phase stability experiments was within 2%. The results of the solution and mobile phase stability experiments confirm that the sample solutions and mobile phase used during the assays were stable up to 48 h at room tempe rature and up to 3 months at 4ºC.

Analysis of Commercial Formulations (Tablets)
The proposed method was applied to the determination of Rufinamide tablets Pr BANZEL ™ ® and BANZEL ® and the result of these assays yielded 96.74-97.10 % respectively with RSD < 2.0 %. The result of the assay (Table 5) indicates that the method is selective for the assay of Rufinamide without interference fro m the excipients used in these tablets. The typical chromatograms for Rufinamide obtained fro m the ext racted marketed formu lat ions were shown in Figure 4a and 4b.

Forced Degradation Studies
Rufinamide standard and tablet powder was found to be quite stable under dry heat conditions. Typical chromatograms obtained following the assay of stress ed samples are shown in Figure 5a-5e.  A very slight decomposition was seen on exposure of Rufinamide drug solution to acidic (1.36), alkaline (0.58) and oxidation (0.81). During the oxidative degradation two major degradants were observed at 2.452 mins and 2.784 mins without interfering the elution of drug peak (5.534 mins) and the percentage of drug decomposition was found to be 0.81 % indicat ing that the drug is highly resistant towards oxidation. Rufinamide has undergone thermal (0.16) and UV degradation (0.77) very slightly i.e less than 1.0 %. Table 6 summarises the data of degradation studies .

CONCLUS ION
The proposed stability-indicat ing HPLC method was validated as per ICH guidelines and applied for the determination of Rufinamide in pharmaceutical dosage forms. The method was found to be accurate, precise, robust and specific as the drug peak did not interfere with the extra peaks aroused during the forced degradation studies. At the same time the chromatographic elution step is undertaken in a short time (< 6 min ). No interference fro m any components of pharmaceutical dosage form or degradation products and therefore the method can be successfully applied to perform long-term and accelerated stability studies of Rufinamide formu lations. In conclusion, the high sensitivity, good selectivity, accuracy and reproducibility of the proposed method is suitable for quality control analysis of complex pharmaceutical preparation containing Rufinamide. The reduction of acetonitrile consumption is one of the best solutions to the current global acetonitrile shortage and will safeguard against future risk.