RP-HPLC ANALYSIS METHOD OF BISOPROLOL FUMARATE IN A NEW TABLET FORMULATION

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1 Med. Surg. J. Rev. Med. Chir. Soc. Med. Nat., Iaşi 2018 vol. 122, no. 3 PHARMACY ORIGINAL PAPERS RP-HPLC ANALYSIS METHOD OF BISOPROLOL FUMARATE IN A NEW TABLET FORMULATION Alina Diana Panainte 1, Mădălina Vieriu 1*, Gladiola Țântaru 1, M. Apostu 1, Nela Bibire 1, Liliana Avasilcăi 2, Ionela Daniela Morariu 2 Grigore T. Popa University of Medicine and Pharmacy Iasi Faculty of Pharmacy 1. Department of Analytical Chemistry 2. Department of Environmental Chemistry * Corresponding author. vieriu_madalina@yahoo.com RP-HPLC ANALYSIS METHOD OF BISOPROLOL FUMARATE IN A NEW TABLET FORMULATION (Abstract): Aim: Development of a new HPLC method for the quantitative determination of bisoprolol fumarate in a novel sustained release tablet formulation. Material and methods: The sustained release matrix tablets with bisoprolol fumarate were formulated using an innovative combination of hydroxypropyl methylcellulose and Precirol ATO5 as excipients. The HPLC separation was done using an Agilent Eclipse XDB-C18 column (150 mm 4.6 mm, 5 µm) and a mobile phase consisting of acetonitrile 10 mm and ph 4.5 phosphate buffer solution (10:90, v/v) at a flow rate of 1.0 ml/min. The method was validated according to ICH guidelines in terms of accuracy, precision, linearity and robus t- ness. Results: Bisoprolol fumarate was separated in less than 4 minutes with good resolution and without any tailing effect or excipient interference. The limit of detection and the limit of quantification were 2.79 and 3.07 µg/ml, respectively. The relative standar d deviation evaluated for the precision of the method was lower than 1%. Conclusions: The HPLC method developed for the determination of bisoprolol fumarate in a novel sustained release tablet formulation was specific and simple and it could also be used for in vitro studies. Keywords: HPLC METHOD, BISOPROLOL FUMARATE, PRECIROL, HYDROXYPRO- PYL METHYLCELLULOSE, NEW TABLET FORMULATIONS. Hypertension continues to be one of the leading causes of death in the world. The management of hypertension requires longterm treatment that may result in poor patient compliance with conventional dosage forms due to greater frequency of drug administration (1). For this disease the most suitable administration route is the oral route. The advantages of oral administration and oral dosage forms are convenience, ease of use and lower costs. Bisoprolol fumarate (BF) is a synthetic β 1 -selective adrenoceptor blocking agent used extensively in the management of hypertension, angina pectoris, cardiac arrhythmias and myocardial infarction. It may be used alone or in combination with other antihypertensive agents, particularly thiazide diuretics (2, 3). A number of factors might affect the pharmacokinetic profile of BF and thereby contribute to the changes in therapeutic efficacy or modify 634

2 RP-HPLC analysis method of bisoprolol fumarate from a new tablet formulation the adverse events profile. A quick and efficient way to eliminate these problems is the development of an appropriate dosage form in order to improve patient compliance. The release rate of BF, a highly water-soluble drug can be modulated using hydroxypropyl methylcellulose and Precirol ATO5 as excipients (4, 5, 6). In order to increase the bioavailability of a drug, one of the options found in the arsenal of formulators today is melt granulation. In general, melt granulation is done by melting an appropriate polymer (Precirol) and then homogenizing the active ingredient and other formulation components into the melt (4). The aim of the procedure is the homogeneous distribution of the active ingredient in the resulting solid, which can be processed further into a solid dosage form. The advantage of melt granulation technique in comparison to conventional granulation is that no water or organic solvents are needed. Because there is no drying step, the process is less time consuming and uses less energy than wet granulation (5). Over the last decade, high performance liquid chromatography (HPLC) has proven to be the predominant technology used in pharmaceutical laboratories worldwide in order to analyze various drug substances. The aim of the study was the development and validation of a Reversed Phase- High Performance Liquid Chromatography (RP-HPLC) method for the determination of BF, formulated into new oral tablets with modified release. Thorough analysis of specialized literature revealed that there were numerous determination methods of bisoprolol in bulk, tablet dosage forms and biological samples, such as HPLC (7, 8), wave voltammetry (9, 10), UV-VIs spectrophotometry (11, 12), LC-MS/MS (13, 14). The reported methods are sensitive, but they are quite complicated and time consuming, thus unsuitable for routine analysis, and require expensive or sophist i- cated instruments. MATERIAL AND METHODS The substances used were: bisoprolol fumarate % reference substance (Unichem Laboratories LTD India), HPLCgrade acetonitrile (Merck Germany), hydrogen peroxide, ortho-phosphoric acid (Chemical Company Romania), sodium hydroxide, hydrochloric acid (Merck Germany). All chemicals and solvents used in the assay procedures were of analytical reagent grade. Apparatus. An Agilent Technologies 1100 High Performance Liquid Chromatograph equipped with Diode Array Detector, a Kern 770 analytical balance and a Cole Parmer ultrasonic bath were used. Preparation of modified release tablets. The 20 mg bisoprolol fumarate modified release matrix tablets had been formulated previously using an innovative combination of hydroxypropyl methylcellulose and Precirol ATO5 as excipients through melt granulation technique (4, 6). Chromatographic conditions.the chromatographic separation of bisoprolol was performed using isocratic elution on an Agilent Eclipse XDB-C18 reverse phase stainless steel column (150 mm x 4.6 mm, 5 µm). The temperature of the column and auto sampler was maintained constant at 25 C. A 50 µl sample loop injector was used. The detection was done spectrophotometrically at 227 nm. The mobile phase was a mixture of acetonitrile and ph 4.5 phosphate buffer solution (10:90, v/v) at a flow rate of 1 ml/min Preparation of stock and standard solutions: A 100 µg/ml BF stock solution was prepared using Milli-Q water. Aliquots of 635

3 Alina Diana Panainte et al. the stock solutions were diluted using mobile phase to yield BF standard solutions in the concentration range 5-80 µg/ml. Procedure for quantitative determination of BF from the original 20 mg tablets: 20 tablets were accurately weighed, crushed and finely powdered. An amount of powder equivalent to 10 mg BF was weighed and transferred into a 50 ml volumetric flask with methanol and ultrasonicated for 15 minutes. The solutions were filtered and further diluted using mobile phase to achieve the final concentration of 50 μg/ml BF. The described method has been validated for the assay of BF according to ICH guidelines (15). System suitability test of the HPLC system was performed through six replicate injections of BF standard solution. The capacity factor, tailing factor, theoretical plates and symmetry were also evaluated (16). Linearity, LOD and LOQ: The standard solution was injected in triplicate into the HPLC system and calibration graphs were obtained by plotting the area of the peaks versus corresponding concentration levels. Regression data analysis was performed using least squares linear regression for the statistical analysis. Limit of detection (LOD) and limit of quantification (LOQ) were calculated using the equations: LOD = 3.3 standard deviation/ slope and LOQ = 10 standard deviation/slope (17, 18). Precision study was performed to evaluate the intra- and inter-day variation. It was carried out by estimating the corresponding responses three times on the same day and on three different day (first, second and fifth day) for three different concentration levels of BF (25, 50, 75 μg/ml). The accuracy of the analytic method was assessed as the percentage relative error. The recovery was assessed at three different concentration levels (15). The robustness was determined by analyzing the same sample under a variety of conditions, such as: variations in flow rate, detection wavelength and mobile phase composition. Stability studies indicated that the samples were stable while kept at room temperature and 4 C for 12 (short-term) and 48 hours (long-term). RESULTS AND DISCUSSION The objective of the chromatographic method was to separate the peaks of BF and its impurities in a short run time with good system suitability parameters. The majority of the ionizable pharmaceutical compounds can be very well separated on C18 (17), thus, bisoprolol could be satisfactorily separated using reversed phase chromatography. Several parameters like ph and the composition of the mobile phase, flow rate, column temperature, wavelength were examined for the optimization of HPLC analysis of bisoprolol. According to the results it was established that the optimum mobile phase was a mixture of acetonitrile and ph 4.5 phosphate buffer solution (10:90, v/v) and the retention time for the pharmaceutical substance was 3.52 min (fig. 1). Detection wavelength was established at 227 nm (fig. 2). Fig. 1. Retention time 636

4 RP-HPLC analysis method of bisoprolol fumarate from a new tablet formulation summarized below (tab. II). Fig. 2. BF absorption spectra System suitability was assessed by injecting six replicates of standard solution into the apparatus. It was established through six replicate injections of standard solution that the tailing factor was less than 2.0 (18, 19) and the theoretical plates were greater than 5,000 (tab. I). TABLE I System suitability parameters Capacity factor 8.02 Tailing factor 1.72 Theoretical plates 8400 Symmetry 0.91 For the linearity study the graph (fig. 3) showed a good linearity in the 5-80 μg/ml BF concentration range with a correlation coefficient (r) of The statistical parameters of the developed method are Retention time Linear range Fig. 3. Calibration curve TABLE II Statistical parameters Parameter Value Limit of detection Limit of quantification 3.52 min 5-80 μg/ml 2.79 μg/ml 3.07 μg/ml Slope Intercept Correlation coefficient (r) Values for accuracy and precision of the method are listed in table III. All experimental results are in the range of the acceptability for accuracy ( %). The % RSD values found in precision study showed that the proposed method provides acceptable intra- and inter-day variations for BF determination. TABLE III Accuracy and precision studies Precision Accuracy Theoretical Intra-day Inter-day conc. (μg/ml) Mean recovered Mean % conc. of BF recovery Mean*±SD % RSD Mean*±SD % RSD ± ± ± ± ± ± * average concentration (μg/ml) for three determinations 637

5 Alina Diana Panainte et al. The results of robustness study (tab. IV) showed that there were no significant changes in the chromatographic pattern when the above modifications were made in the experimental conditions, proving that the method was robust. The % RSD of bisoprolol fumarate was lower than 2.0 %. The results of the stability studies are given the percent ratios within the acceptance range of % (tab. V). TABLE IV Robustness Study Parameter Optimized Variations Retention time (min) Conclusion 0.8 ml/min 3.59 robust Flow rate (±0.2 ml/min) 1.0 ml/min 1.0 ml/min 3.52 robust 1.2 ml/min 3.33 robust Detection wavelength (±5 nm) Mobile phase composition (acetonitrile: ph 4.5 phosphate buffer solution) (±0.5 %) Added, μg ml -1 Room temperature (12 h) 227 nm 10:90 (v/v) 223 nm 3.52 robust 227 nm 3.52 robust 230 nm 3.58 robust 5:95 v/v 3.61 robust 10:90 v/v 3.52 robust 15:85 v/v 3.55 robust TABLE V Stability studies of BF in solution Intra-day Refrigerator (4 C, 12 h) Inter-day Room temperature (12 h) Refrigerator (4 C, 12 h) ± ± ± ± ± ± ± ± ± ± ± ±2.66 The validated method was applied to the determination of BF in original tablet formulations. The result of the assays (n = 6) are presented in table VI. No interference of the tablet excipients was detected. TABLE VI Analysis of BF in developed tablets Label claim % found ± SD RSD (%) (mg/tablet) (n=6) ± ± CONCLUSIONS We have developed a new HPLC method with UV detection for the estimation of bisoprolol fumarate in sustained release tablets. The advantages of our method are short analysis time, a simple procedure for sample preparation, specificity, and ability to separate the drug from formulation excipients. No extraction procedure is necessary, and the mobile phase composition is quite simple to prepare and is economical. The method is suitable for routine analysis of BF in bulk or in tablet dosage forms and can be also used during in vitro studies. 638

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