EXPERIMENTAL RESEARCH FOR THE DETERMINATION OF BI- SOPROLOL FUMARATE IN BULK AND DOSAGE FORMS USING LIQUID CHROMATOGRAPHY

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1 Rev. Med. Chir. Soc. Med. Nat., Iaşi 2017 vol. 121, no. 2 PHARMACY ORIGINAL PAPERS EXPERIMENTAL RESEARCH FOR THE DETERMINATION OF BI- SOPROLOL FUMARATE IN BULK AND DOSAGE FORMS USING LIQUID CHROMATOGRAPHY Alina-Diana Panainte, Gladiola Ţântaru, Nela Bibire*, M. Apostu, Mădălina Vieriu Grigore T. Popa University of Medicine and Pharmacy Iasi Faculty of Pharmacy Department of Pharmaceutical Sciences (I) *Corresponding author. nelabibire@yahoo.com EXPERIMENTAL RESEARCH FOR THE DETERMINATION OF BISOPROLOL FUMARATE IN BULK AND DOSAGE FORMS USING LIQUID CHROMATOGRAPHY (Abstract) Aim: To develop a simple, economic, feasible, and effective RP-HPLC method for the quantitative determination of bisoprolol in bulk and pharmaceutical formulations. Material and methods: Chromatographic separation was achieved on a Zorbax SB-C18 chromatographic column. The mobile phase was a mixture of methanol and 0.02M potassium acetate (40:60) with a 1 ml/min flow rate. The UV detection was done at 225 nm. The method was validated in accordance with the current ICH guidelines in terms of linearity, limit of detection, limit of quantification, precision, accuracy, recovery, and system suitability. Results: The retention time for bisoprolol was 2.38 minutes. A linear response was observed over the concentration range 5-80 μg/ml of bisoprolol fumarate. The limit of detection and the limit of quantitation for bisoprolol fumarate were 1.15 and 3.81μg/mL, respectively. The precision of the method was satisfactory and the relative standard deviations did not exceed 1%. The accuracy of the method was proved and the mean recovery of drug was in the range %. Conclusions: The proposed method proved to be selective, simple, precise, accurate, and it was successfully applied for the determination of bisoprolol in tablets. Keywords: ANTIHYPERTEN- SIVE, BISOPROLOL, CHROMATOGRAPHIC ANALYSIS. Bisoprolol fumarate (BF), chemically known as (±)-1-[4-[[2-(1-methylethoxy) ethoxy]methyl]phenoxy]-3-[(1-methylethyl) amino]-2-propanol(e)-2-butenedioate, is a synthetic, beta1-selective adrenoceptor blocking agent used in the treatment of hypertension. The two substituents present in the para position of the benzene ring might be the reason for its β1-adrenergic receptor selectivity. BF is used alone on in combination with other drugs to treat hypertension. BF is well absorbed following oral administration. The absolute bioavailability for a 10 mg dose is greater than 80%. Absorption is not influenced by meals. The first pass metabolism of bisoprolol fumarate is less than 20% and it is eliminated through renal and non-renal pathways, as about 50% of the dose is found unchanged in the urine and the remainder is eliminated as inactive metabolites (1-3). Chemical analysis methods are widely used in pharmaceutical practice to ensure 433

2 Alina-Diana Panainte et al. the quality and safety of drugs. Quality control should be achieved through faster, more reliable and easily achievable processes. Several analytical methods for the determination of BF in bulk, dosage forms, and human plasma have been proposed, but they are devaluated by long retention times and the use of expensive chemicals (4-9). Hence, the aim of the present study was to develop a simple, economic, feasible, and effective RP-HPLC method for quantitative determination of BF in bulk and pharmaceutical formulations. MATERIAL AND METHODS Instrumentation and chromatographic conditions: The analysis of BF was carried out using an Agilent 1100 system equipped with a photodiode array detector. A reversed-phase HPLC column Zorbax SB- C18, 3 x 100 mm, 3.5 μm particle size was used. The column and autosampler temperature were maintained at 25 C. The chromatographic separation of BF was achieved by isocratic elution. The mobile phase used for the analysis was a mixture of methanol and 0.02M potassium acetate (40:60 v/v). A 0.45 μm membrane filter was used to filter and degas the mobile phase. The injection volume was 25 μl. The output signal was monitored and integrated at 225 nm. Chemicals and Reagents: All solvents used were analytical grade. Milli-Q water, methanol, potassium acetate were obtained from Merck (Darmstadt, Germany). Pharmaceutical grade BF was produced by Unichem Laboratories LTD, India. Two tablet formulation of 5 mg BF (A) and 10 mg (B) were procured from the local pharmacies. Only class A standard volumetric glassware was used. Standard solution preparation: 100 μg/ml BF standard stock solution was prepared by transferring 10 mg of BF reference standard into a 100-mL volumetric flask using 25 ml methanol. The solution was diluted up to the volume with mobile phase. Extraction procedure for dosage forms: Twenty tablets labeled to contain 5 and 10 mg of BF were individually weighed, mixed and finely powdered. The amount of powder equivalent to one tablet was accurately weighed, transferred into 100 ml volumetric flask using the mobile phase and then sonicated for 15 minutes. The sample solution was filtered using 0.45 μm filter. RESULTS AND DISCUSSION To optimize the analysis method, a number of parameters (organic modifier, flow rate) have been investigated. Different mobile phases were used to achieve the best separation and resolution. BF was eluted with the optimum peak shape (fig. 1) achieved when using a mixture of methanol and 0.02M potassium acetate (40:60, v/v). The UV detection was set at 225 nm. System suitability tests, evaluating column efficiency (N), tailing factor, resolution, were done to prove that the chromatographic system was adequate for the analysis (tab. I). These tests were carried out on freshly prepared BF standard stock solutions. Fig. 1. Retention time of BF 434

3 Experimental research for the determination of bisoprolol fumarate in bulk and dosage forms using liquid chromatography TABLE I System suitability parameters Retention time 3.33 Area 2678 Tailing factor 1.20 Symmetry 0.74 Theoretical plates (N) 4210 Resolution 1.11 Linearity study: The standard stock solution was diluted to prepare three sets of BF standard solutions in the 5-80 μg/ml concentration range. Each set was analyzed to plot a calibration curve (fig. 2). To evaluate the linearity of the method (tab. II), standard deviation (SD), slope, intercept, regression coefficient (r2), limit of detection and quantification were calculated (10-12). The precision of the method was studied for a 45 μg/ml BF solution through six replicate determinations (9). The percentage relative standard deviation (RSD%) was lower than 1.0% proving the high repeatability of the new method (tab. III). The accuracy of the proposed method was established by means of the standard addition technique, by adding known amounts of standard substance to the 5µg/mL solution to reach three different concentration levels (25%, 100% and 125%) (10). The accuracy of the developed method for BF determination ranged from 99.74% to % (tab. IV). As to the robustness studies, deliberate changes in the flow rate of mobile phase (± 0.2 ml/min) and column temperature (± 2 C) were made to evaluate the impact on the method. These small and deliberate variations had no significant effect on the measured system suitability parameters (tab. V). The proposed method was successfully applied for the simultaneous determination of BF in tablets without interference from the excipients and without prior separation (tab. VI). Fig. 2. Calibration curve TABLE II Linearity studies of the method Regression coefficient (r 2 ) Correlation coefficient (r) Standard Error (SE) Intercept Slope LOD (µg/ml) 1.15 LOQ (µg/ml) 3.81 Theoretical concentration (µg/ml) 45 TABLE III Precision of the method Peak area Calculated concentration (µg/ml) RSD %

4 Alina-Diana Panainte et al. Spiked level % TABLE IV Accuracy of the method Concentration of BF (μg/ml) Added Found Recovery % Mean TABLE V Robustness Parameter Value Area Theoretical Plates Tailing factor Temperature 25-2 C C Flow rate ml/min ml/min Label claim (mg/tablet) TABLE VI Experimental results of BF assay in tablets Concentration founds Recovery RSD n=3 (mg/tablet) (%) (%) Obliquity A (5) B (10) CONCLUSIONS The RP-HPLC method we have developed offers various advantages, including minimal sample prepping before chromatographic separation, enough sensitivity for quantitative analysis of bisoprolol fumarate in pharmaceutical products, good precision and accuracy. The method involves the use of easily available, inexpensive laboratory reagents and it can thus be used for routine analysis and quality control. REFERENCES 1. DiNicolantonio JJ, Fares H, Niazi AK, et al. β-blockers in hypertension, diabetes, heart failure and acute myocardial infarction: a review of the literature. Open Heart 2015; 2: e Fares H, Lavie CJ, Ventura HO. Vasodilating versus first-generation B-blockers for cardiovascular protection. Postgrad Med 2012; 124:

5 Experimental research for the determination of bisoprolol fumarate in bulk and dosage forms using liquid chromatography 3. Trobec KC, Grabnar I, Kos MK, Vovk T, Trontelj J, Anker SD, Rosano G, Lainscak M. Bisoprolol pharmacokinetics and body composition in patients with chronic heart failure: a longitudinal study. Eur J Clin Pharmacol 2016; 72(7): Athota RV, Jagarlapudi SK, Singampalli MR. Stability indicating RP-HPLC method for simultaneous assay of bisoprolol and hydrochlorothiazide in combined tablet dosage form. I J Pharm Tech Resch 2016; 9(7): Ulu ST, Aydoğmuş Z. An HPLC method for the determination of bisoprolol in human plasma and its application to a pharmacokinetic study. J Chromatogr Sci 2012; 50(7): Joshi SJ, Karbhari PA, Bhoir SI, Bindu K, Das C. RP-HPLC method for simultaneous estimation of bisoprolol fumarate and hydrochlorothiazide in tablet formulation. J Pharm Biomed Anal 2010; 52: Pește G, Oniscu C, Vlase A. Experimental research for determination of bisoprolol fumarate in human plasma samples using liquid chromatography- tandem mass spectrometry (LC-MS/MS) technique. Rom Biotech Lett 2010; 15(2): Induri M, Bhagavan Raju M, Rajendra Prasad Y. Validated and stability indicating liquid chromatography method for quantification of bisoprolol fumarate in tablet dosage form. Int J Pharm 2012; 2(1): Panainte AD, Vieriu M, Ţântaru G, Apostu M, Bibire N. Fast RP-HPLC method for the determination of bisoprolol. Rev Med Chir Soc Med Nat (Med Surg J) 2016; 120(3): ICH Topic Q2B. Validation of Analytical Procedures (CPMP/ICH/281/95). London: The European agency for the evaluation of Medicinal Products, Oprean R, Rozet E, Dewe W. Ghid de validare a procedurilor analitice. Cluj Napoca: Editura Medicală Universitară Iuliu Haţieganu, Hortolomei M, Ochiuz L, Popovici I, Timofte D, Petrescu DC, Ghiciuc CM. Development and validation of the highperformance liquid chromatography method for the quantitative determination of erythromycin in dermo-preparations. Rev Med Chir Soc Med Nat Iasi 2015; 119(4): NOUTĂȚI NEWS STRUCTURE-BASED DESIGN, SYNTHESIS AND ANTITUMORAL EVALUATION OF ENULOSIDES The purpose of this research is to designe and obtaine in high yields a new class of antitumoral agents with lower structural complexity. The general strategy for the synthesis of enulosides is based on the use of glycals as starting materials. From the pharmacological point of view, some synthetic enulosides exhibited activity against Mycobacterium tuberculosis H37Rv and they can also act as glycosidase inhibitors. All compounds exhibited high antitumoral activity in micromolar range against the tested tumor cell lines. (Santos JAM, Santos CS, Almeida CLA, et al. Structure-based design, synthesis and antitumoral evaluation of enulosides. European Journal of Medicinal Chemistry 2017, 128: ). Oana Dragostin 437

METHOD DEVELOPMENT AND VALIDATION BY RP-HPLC FOR ESTIMATION OF ZOLPIDEM TARTARATE

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Ramalakshmi et al. SJIF Impact Factor 6.647 Volume 7, Issue 2, 1010-1018 Research Article ISSN 2278 4357 METHOD DEVELOPMENT AND VALIDATION BY RP-HPLC