CHEMOMETRIC APPROACH FOR RP-HPLC DETERMINATION OF DRONEDARONE USING RESPONSE SURFACE METHODOLOGY

CHEMOMETRIC APPROACH FOR RP-HPLC DETERMINATION OF DRONEDARONE USING RESPONSE SURFACE METHODOLOGY

Dasari Vasavi Devi ^{* 1}, Dugasani Swarnalatha ² and Gopireddy Venkata Subba Reddy ³

Jawaharlal Nehru Technological University ¹, Anantapur - 515002, Andhra Pradesh, India.

Annamacharya College of Pharmacy ², Rajampeta - 516126, Andhra Pradesh, India.

Jawaharlal Nehru Technological University Anantapur College of Engineering ³, Pulivendula - 516390, Andhra Pradesh, India.

ABSTRACT: The present study depicts the assessment of class III antiarrhythmic drug dronedarone in its drug substance and drug product. Response surface randomized central composite quadratic design has been employed for the optimization of method parameters using reverse-phase high-performance liquid chromatography (RP-HPLC) on Kromasil C18 250 × 4.6 mm, 5 µ with UV detection at 289 nm. The ranges of the independent variables used for the optimization were flow rate 0.9 to 1.1, column temperature 28 °C to 33 °C and composition of buffer in the mobile phase is 55 to 65%. The influence of these independent variables on the output responses: retention time, asymmetric factor, theoretical plates, plate height and capacity factor were evaluated. The five responses were simultaneous optimized by using central composite design. Optimum conditions chosen for the assay were flow rate of 0.945 ml/min, temperature 31.3 °C and buffer: Acetonitrile has taken in the ratio 61.93: 38.07 respectively. The retention time of dronedarone is 2.356 minutes with the employment of the optimum conditions given by the design experiments. All the system suitability parameters were satisfied. Further the method has been validated by the regulatory guidelines framed by the ICH. The method was found linear in the concentration range of 25-150 µg/mL with a regression coefficient of 0.999. The limit of detection and limit of quantification were found to be 0.58 µg/mL and 1.75 µg/mL respectively. The method was found to be simple, linear, accurate, precise and robust. Therefore, it can be used for routine quality control of dronedarone in its tablet dosage form.

RP-HPLC, Design of experiments, Central composite design, Dronedarone

INTRODUCTION: Dronedarone is a class III antiarrhythmic drug recently approved by the US FDA in 2009 for the treatment of nonpermanent atrial fibrillation and atrial flutter ¹.

The chemical name is N- [2- butyl- 3[4- [3-(dibutylamino) propoxy] benzoyl]- 1-benzofuran-5-yl] methanesulfonamide hydrochloride according to IUPAC. Its molecular formulae are C₃₁H₄₄N₂ O₅S.HCl with molecular weight of 593.22 g/mol ².

Chemically, it is a benzofuran derivative containing a heterocyclic compound which is a structural analog of Amidarone. DRO reduces the toxic effects in Amidarone by replacing the iodine group with a methane sulfonyl group ³. Due to reduced lipophilicity, it has lower toxicity and superior pharmacokinetic characteristics than the amidarone belonging to the same class III antiarrhythmic drug ⁴. DRO is crystalline in nature with a melting point of 149-153 °C ³, with white to a practically white, non-hygroscopic fine powder. It is a new active substance and there is no official pharmacopoeial procedure available ⁵.

Literature overviews confess that drug estimated by different analytical strategies like spectrophotometric ⁶, HPLC ^7-16 and LC-MS ^{17, 18} in bulk drugs and formulation of Dronedarone. There are a few techniques available in the literature for the estimation of dronedarone. Quality is paramount in the pharmaceutical industry as people’s lives are directly dependent on the quality of medicine given to them for the treatment of diseases. As analytical development is a key part of pharmaceutical development and quality control, hence the incorporation of the quality by design to the analytical method development was introduced by regulatory bodies. QbD is an idea of quality recently introduced in the pharmaceutical industry and its usage has been the object of specific ICH guidelines ^{19, 20, 21, 22} (Q8 to Q10). As QbD is a systematic methodology of method development, one of the results of utilizing QbD ideas is a good understanding of parameters influencing analytical method performances related to the information.

QbD has been defined as “a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management” whose main goals are to improve method quality and increase operational flexibility ¹⁹. Hence, in this paper, an application of chemometric concepts to the development of an RP-HPLC method for an anti-arrhythmic drug substance and drug product as described. The proposed method was simple, sensitive, stability-indicating, accurate and more robust than the previously reported ones.

MATERIALS AND METHODS:

Chemicals and Reagents: Dronedarone reference standard was obtained from MSN Laboratories (Hyderabad). Acetonitrile, orthophosphoric acid, methanol, water were of HPLC grade and purchased from Rankem Chemicals. And also Hydrochloric acid, hydrogen peroxide, and Sodium hydroxide of analytical grade were employed for the stability studies. The tablet formulation (Multaq 400 mg film-coated tablets) were purchased from the local market and used for the analysis of drug product.

Instrumentation: A Waters Acquity HPLC with binary solvent manager, equipped with a Tunable UV detector and auto sampler was used. Data collection, signal monitoring and data processing were assisted by Empower 2 software. In addition, an electronic balance (BL-220H; Shimadzu Corporation), a pH meter (ELICO® LI 120), a sonicator (PCi, Mumbai), hot air oven, UV chamber has been employed in this study.

Software Employed: Design-Expert 11.0.4.0 trail version (Stat-Ease Inc., USA) has been used for the experimental design, data analysis and desirability function calculations. The rest of the calculations for the analysis were performed with the help of Microsoft Excel 2007 software (Microsoft, USA).

Standard Solution Preparation: Accurately Weighed and transferred 25 mg of Dronedarone, working Standards into a 25 ml clean dry volumetric flask, add 10 ml of diluent, sonicated for 10 min and makeup to the final volume with diluent (1000 µg/ml dronedarone). 1 mL from the above stock solution was taken into a 10 mL volumetric flask and made up to 10 mL which contains 100 µg/ml dronedarone.

Preparation of Mobile Phase: The mobile phase used was 0.1% orthophosphoric acid and acetonitrile in the ratio of 61.93: 38.07 respectively. The buffer has been prepared by taking accurately 1 mL of orthophosphoric acid in a 1000 mL of volumetric flask add about 3/4^th with HPLC grade water and sonicate to degas and make up the final volume with water.

Diluents Preparation: The diluents have been prepared by using 100 mL of acetonitrile and 100 mL of water, i.e., 1:1 ratio, respectively.

Sample Solution Preparation: Accurately weighed equivalent weight of the combination (Multaq) powder sample transfer into a 100 ml volumetric flask, 25 ml of diluents was added and sonicated for 25 min; further the volume was made up with diluents and filtered by HPLC filters (4000 µg/ml dronedarone). 0.25 ml of filtered sample stock solution was transferred to a 10 ml volumetric flask and made up with diluents (100 µg/ml dronedarone). The label claim of dronedarone is 400mg.

Preliminary Study:

Screening of Columns and Mobile Phase: In RP-HPLC, with the employment of design experiments initially screened for columns, mobile phase, and wavelength. In this panorama, BDS 250, Discovery 250 and Kromosil 250 columns were screened. Latter selection of proper mobile phase has been done with various organic modifiers such as methanol, acetonitrile, orthophosphoric acid and potassium dihydrogen phosphate in various proportions. The entire region was scanned over the range of UV and the wavelength was selected at 289 nm with the help of a PDA detector. From these initial screening, the Kromasil® C18 HPLC column and the mobile phase consisting of OPA buffer and acetonitrile in the ratio of 60:40 respectively have been taken for further optimization studies.

Optimization of Method by Experimental Design: The method optimization was done using the Design expert 11.0.4.0 trail version (State-Ease Inc., USA) software. Central composite design under the category of response surface methodology (RSM) has been employed to design a set of experimental runs by concerning the three independent variables i.e., flow rate, mobile phase ratio and temperature. Here, the ranges of the independent variables were entered along with their actual levels in Table 1. In the optimization experiments, with the help of central composite design, the effects of three study parameters were simultaneously evaluated to assess the effect on the 5 responses with the help of 20 experimental runs. The five responses which are dependent variables taken as were retention time, asymmetric factor, theoretical plates, plate height and capacity factor.

TABLE 1: FACTORS CHOSEN IN CENTRAL COMPOSITE DESIGN

Chromatographic Conditions: The final conditions optimized by the software include 0.94 ml/min. flow rate, 61.93% of buffer in the mobile phase and 31.3 °C column temperature. The RP-HPLC analysis was performed on Kromasil® C18 HPLC column (5 µm particle size, 250 × 4.6 mm) using a mobile phase of acetonitrile and OPA buffer (38.07: 61.93 v/v) with the injection volume 10 µl at 289 nm using PDA detector. The comparison of the initial HPLC method and the optimized HPLC method from design expert experiments are drawn in Table 2.

TABLE 2: A COMPARISON OF INITIAL HPLC METHOD, OPTIMIZED HPLC METHOD FROM DESIGN EXPERT EXPERIMENTS

Preparation of Samples for Stability Studies: The stability studies have been performed to realize to what extent the drug is stable at the accelerated conditions. This was done with the assistance of ICH specifications. Dronedarone was subjected to stress under acidic, basic, oxidative, hydrolytic, thermolytic and photolytic conditions.

Oxidative Studies: To 1 ml of stock solution of dronedarone 1 ml of 10% hydrogen peroxide (H₂O₂) was added separately. The solutions were kept for 30 min at 60 °C. For the HPLC study, the resultant solution was diluted to obtain (100 ppm) solution and 10.00 µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Solution for Acid Degradation Studies: To 1 ml of stock solution Dronedarone 1ml of 2N Hydrochloric acid was added and refluxed for 30 min at 60 °C. The resultant solution was diluted to obtain (100ppm) solution and 10.00 µl solutions were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Solution for Alkali Degradation Studies: To 1 ml of stock solution dronedarone 1 ml of 2N sodium hydroxide was added and refluxed for 30 min at 60 °C. The resultant solution was diluted to obtain (100ppm) solution, and 10.00 µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Dry Heat Degradation Studies: The standard drug solution was placed in an oven at 105 °C for 6 h to study dry heat degradation. For the HPLC study, the resultant solution was diluted to (100 ppm) solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Photo Stability studies: The photochemical stability of the drug was also studied by exposing the (1000 ppm) solution to UV Light by keeping the beaker in UV Chamber for 7 days or 200 Watt-hours/m in photostability chamber. For the HPLC study, the resultant solution was diluted to obtain (20 ppm & 400 ppm) solutions and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Neutral Degradation Studies: Stress testing under neutral conditions was studied by refluxing the drug in water for 6 h at a temperature of 60 ºC. For the HPLC study, the resultant solution was diluted to (100ppm) solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Assessment of Validation Parameters: According to ICH guidelines, an analytical strategy was known to be validated in the event that it has been assessed through characteristics such as precision, accuracy, linearity, the limit of detection, the limit of quantification and robustness. The characteristics of analytical technique ought to be within prescribed limit and characterized standards to confirm its accuracy and authenticity ²³.

Precision: It is the closeness of agreement between a series of measurements obtained from multiple testing of a homogenous sample under endorsed conditions. Precision includes repeatability and intra laboratory repeatability; both were performed for six replicates at a concentration of 100 µg/ml dronedarone.

Accuracy: The accuracy of the method was determined by computing the percentage recovery of dronedarone. By applying the standard addition technique for the drug recovery studies have been done at three distinct concentrations such as 50, 100, and 150% of label claim. At each level, ICH suggestion for performing accuracy was minimum triplicate at every concentration and results retrieved were compared.

Linearity: Linearity was established from 25 to 150% of working standard concentration using a minimum of six calibration levels (25, 50, 75, 100, 125 and 150%) having a range of 25 to 150 ppm of dronedarone. The calibration curve was plotted as the concentration of the reference standard of substance against peak area and the linearity of the method was evaluated by regression analysis.

LOD and LOQ: The limit of detection and limit of quantification of the method were accessed from the following formulae:

LOD = 3.3 × (σ/S)

LOQ = 10 × (σ/S)

Where σ = standard deviation of response and S = slope obtained from calibration curves of linear study.

Robustness: A few parameters like flow rate, mobile phase ration, and temperature were changed deliberately for the robustness evaluation using the HPLC method. One factor was changed at one time to estimate the effect. Each factor selected was changed at three levels (-1, 0, +1) with respect to the optimized parameters. The robustness of the method was done at the concentration level 100 ppm of dronedarone.

RESULTS AND DISCUSSION:

Optimization of Central Composite Design (CCD): The optimization of analytical factors was carried out using design expert software. Twenty experimental runs were generated from the three factors and five responses by using the quadratic model under random response surface central composite quadratic design Table 3.

TABLE 3: CENTRAL COMPOSITE DESIGN MATRIX FOR FACTORS AND RESPONSES

The effects of independent variables on the responses for the 20 experimental runs are depicted in Table 4.

TABLE 4: SUMMARY RESULTS FOR RESPONSES IN QUADRATIC MODEL

The polynomial equations for the response generated by ANOVA are depicted below:

Retention time (t_R Dro) = + 2.35 - 0.2484A + 0.0411B - 0.0022*C - 0.0037AB + 0.0008*AC -0.0002*BC + 0.0391*A² + 0.0021*B² - 0.0083*C²

Asymmetric facto r= + 1.38 -0.0051*A + 0.0071*B + 0.0147*C + 0.0037*AB - 0.0213 AC + 0.0087*BC - 0.0049*A² - 0.0297 B² - 0.0138*C²

Theoretical plates = + 7113.15 - 275.45*A + 229.21*B - 91.21*C

Plate height = + 6.03354-9.78909*A + 0.007108*B + 0.066152*C - 0.0075AB + 0.003AC- 0.00002BC + 3.83377A² + 0.000077B² - 0.001135C²

Capacity factor = + 2.13-0.4044*A + 0.0556*B -0.0124*C + 0.0187*AB + 0.0212*AC - 0.0213*BC + 0.0803*A² + 0.0255*B² - 0.0028*C²

The above equations in terms of coded factors can be used to make predictions about the response for given levels of each factor. By default, the high level factors are coded as +1 and the low levels of the factors are coded as -1. The coded equation is useful for the identification of relative impact of the factors by comparing the factors coefficients. The obtained model from the design expert was validated by using ANOVA. The Derringer’s desirability shown in the Fig. 1 which was one.

The perturbation plots are constructed to evaluate the effect of the variables on retention time, asymmetric factor, theoretical plates, capacity factor, and plate height of dronedarone Fig. 2.

FIG. 1: BAR GRAPH SHOWING DESIRABILITY VALUES

FIG. 2: PERTURBATION PLOTS SHOWING THE EFFECT OF INDEPENDENT VARIABLES ON RESPONSES

The chromatogram of standard dronedarone at optimized conditions obtained by the design was shown in Fig. 3.

FIG. 3: CHROMATOGRAM OF DRUG SUBSTANCE

The optimized chromatographic conditions include flow rate 0.945 ml/min, column temperature 31.303 °C and a mixture of 0.1% OPA buffer and Acetonitrile in the ratio of 61.93: 38.07 as mobile phase, respectively. The system suitability parameters obtained are quite satisfactory for these optimum conditions and they are within the limit.

Stability Studies: The reference sample was subjected to various stress levels and the stability of the drug was observed. The results of stressed studies Table 5 revealed that the method is stability-indicating.

TABLE 5: SUMMARY OF STABILITY STUDIES

Validation of the Method: According to the ICH guidelines the optimized method has been validated. The calibration curve was found to be linear over the range of 25-150 μg/mL of dronedarone Table 6. As shown in Table 6, the regression analysis of the calibration curve of the drug was found to be 0.999. The detection limit and quantification limit for dronedarone were found to be 0.58 and 1.75 μg/mL respectively. The precision data representing both repeatability and intermediate precision are summarized in Table 6. The % RSD values for both repeatability and precision were less than 2% which indicates that the proposed method is precise. The results for validation and system suitability parameters are shown in Table 6. The results for robustness are presented in Table 7, which shows that change in conditions like flow rate, mobile phase ratio and column temperature did not significantly affect the recoveries, peak area and retention time of the drugs indicating that the proposed method was robust. The % RSD was calculated, which was found to be in the permissible limit.

The accuracy of the proposed method was evaluated by calculating the recovery studies of the test drug at three different concentration levels (50%, 100%, 150%) by the standard addition method. A known amount of dronedarone was added to prequalified sample solution and three replicates of each concentration were injected in developed chromatographic conditions. The mean percentage recovery of dronedarone was varied between and indicating that the developed method was found to be accurate. The % recovery results were shown in Table 8.

TABLE 6: VALIDATION SUMMARY & SST PARAMETERS

TABLE 7: ROBUSTNESS STUDIES RESULTS

TABLE 8: ACCURACY STUDIES

* % recovery from triplicate determination

 Application of the Method: The developed and validated method has been employed for the estimate of the dronedarone content in a commercially available brand of the tablet containing 400 mg of dronedarone (Multaq 400 mg). The potency of the tablet formulation sample was found to be 99.80% Table 9 and the system suitability parameters of a drug product are shown in Table 10. The amount measured was in good agreement with the label claims. The results of the assay indicated that the method was selective for analysis of dronedarone without interference from the excipients Fig. 4.

TABLE 9: ASSAY RESULTS OBTAINED BY THE PROPOSED METHOD FOR THE DRUG IN PHARMCEUTICAL PREPARATION

[(Multaq Tablet, Label Claim 400 mg Dronedarone): (n=6)]

TABLE 10: SYSTEM SUITABILITY PARAMETERS OF DRUG PRODUCT

FIG. 4: CHROMATOGRAM OF DRUG PRODUCT

CONCLUSION: A fruitful, simple, robust chemometric approach by the RP-HPLC method proved to be a valuable approach in optimizing methods for dronedarone. A comparison of experimental results with predicted results illustrated the preciseness of the design. The stability of the developed technique was shown by exposing the drug to different stress conditions. The acquired results revealed that the stability-indicating power of the strategy. Further validation was performed in consistence with ICH guidelines and the robustness of the method was checked by changing three chromatographic parameters. The validation result indicates strategy meets the regulatory aspects; henceforth concerning the method done, it very well may be utilized in the routine quality control examination of the pharmaceutical formulation.

ACKNOWLEDGEMENT: The authors are very thankful to the management of Annamacharya College of Pharmacy, for providing the facilities for working, technical support and discussions.

CONFLICTS OF INTEREST: The authors have declared no conflicts of interest.

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    How to cite this article:

    Devi DV, Swarnalatha D and Reddy GVS: Chemometric approach for RP-HPLC determination of dronedarone using response surface methodology. Int J Pharm Sci & Res 2020; 11(1): 255-63. doi: 10.13040/IJPSR.0975-8232.11(1).255-63.

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