Simultaneous determination of bisoprolol fumarate and rosuvastatin calcium in a new combined formulation by validated RP-HPLC European Journal of Chemistry 10 (1) (2019) 52-56 European Journal of Chemistry View Journal Online View Article Online Simultaneous determination of bisoprolol fumarate and rosuvastatin calcium in a new combined formulation by validated RP-HPLC Marco Mounir Zaki 1,*, Nada Sayed Abdelwahab 1, Adel Ahmed Ali 2 and Souty Mounie Zaki Sharkawi 3 1 Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt marcomounir11@yahoo.com (M.M.Z.), nadasayed2003@yahoo.com (N.S.A.) 2 Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt adelahmed.ceutics@yahoo.com (A.A.A.) 3 Pharmacology and Toxicology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt drsoutyph@yahoo.com (S.M.Z.S.) * Corresponding author at: Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt. Tel: +2.122.7027144 Fax: +2.082.2317950 e-mail: marcomounir11@yahoo.com (M.M. Zaki). 10.5155/eurjchem.10.1.52-56.1815 Received: 20 November 2018 Received in revised form: 13 December 2018 Accepted: 18 December 2018 Published online: 31 March 2019 Printed: 31 March 2019 A simple, specific, and precise RP-HPLC method was developed and validated for simultaneous determination of Bisoprolol fumarate (BIS) and Rosuvastatin calcium (ROS) in new formulated tablets. The developed RP-HPLC method depended on chromatographic separation using C18 column (150×4.6 mm, 0.5 μm) with mobile phase consisted of acetonitrile and 0.05 aqueous solution of orthophosphoric acid at the ratio of 65:35 % (v:v) with a flow rate of 1 mL/min and UV detection was carried out at 230 nm. Factors affecting the developed methods were studied and optimized and the retention times for BIS and ROS were found to be 2.758 and 4.974 min, respectively. Linearity of the proposed method was observed over a concentration range 0.2-50 μg/mL for each of BIS (r = 0.9999) and ROS (r = 0.9998). The proposed method was successfully applied for the determination of the studied drugs in their bulk powder, laboratory prepared mixtures and in the formulated tablets. The developed method is the first chromatographic method for determination of those drugs and showed no significant difference when compared with the reported methods. Tablets RP-HPLC Bisoprolol Rosuvastatin Chromatography Novel formulation Cite this: Eur. J. Chem. 2019, 10(1), 52-56 Journal website: www.eurjchem.com 1. Introduction Bisoprolol, (RS)-1-[4-[[2-(1-methylethoxy)ethoxy]methyl] phenoxy]-3-[(1-methylethyl)amino]propan-2-ol (Figure 1), is a cardioselective beta blocker used mainly for treatment of hypertension [1,2]. Rosuvastatin, (3R,5S,6E)-7-[4-(4-fluoro phenyl)-2-(N-methylmethanesulfonamido)-6-(propan-2-yl) pyrimidin-5-yl]-3,5-dihydroxyhept-6-enoic acid (Figure 1), working as lipid lowering drug by inhibition of hydroxyl methyl glutaryl coenzyme A (HMG CoA) reductase [3]. The combination of BIS and ROS is not official in any of the commonly known pharmacopoeias. While this combination is fruitful for the treatment of patients with multiple cardio- vascular diseases, such as hypertension with/or susceptible to atherosclerosis, or hypercholesterolemia. This combination was the invention related to Bondjers et al. [4] as United States Patent Application Publication No.: US 2003/0060477A1 Pub. Date: Mar. 27, 2003. O HO HN O O HO O OH O (a) N NNS OO F OH OOHOH 2 Ca (b) Figure 1. Chemical structure of (a) Bisoprolol fumarate and (b) Rosuvastatin calcium. ABSTRACT RESEARCH ARTICLE KEYWORDS European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2019 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. http://dx.doi.org/10.5155/eurjchem.10.1.52-56.1815 http://dx.doi.org/10.5155/eurjchem.10.1.52-56.1815 https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.10.1.52-56.1815&domain=pdf&date_stamp=2019-03-31 http://www.eurjchem.com/ http://dx.doi.org/10.5155/eurjchem.10.1.52-56.1815 mailto:marcomounir11@yahoo.com mailto:nadasayed2003@yahoo.com mailto:adelahmed.ceutics@yahoo.com mailto:drsoutyph@yahoo.com mailto:marcomounir11@yahoo.com http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.10.1.52-56.1815&domain=pdf&date_stamp=2019-03-31� Zaki et al. / European Journal of Chemistry 10 (1) (2019) 52-56 53 The literature survey revealed that no any chroma- tographic method was reported for the simultaneous estimation of BIS and ROS in combination. While only one spectrofluorimetric method was found for their determination in those tablets [5]. On the other hand, determination of each drug was reported either alone or in other combinations. BIS was selectively determined by several methods like non aqueous potentiometric titration [1], spectrophotometric [6- 9], spectrofluorimetric [9], and voltammetric methods [10]. Also, it was analyzed by different chromatographic methods such as HPTLC [11-13], HPLC [14-17], UPLC [18,19], and LC- MS/MS methods [20,21]. Rosuvastatin calcium was determined either alone or in its different combined dosage forms by several methods including titrimetric [22], different spectrophotometric [23-29], spectrofluorimetric [30], HPTLC [31-34] and HPLC [35-38] methods. This research paper presents, for the first time, RP-HPLC method for the separation and quantification of BIS and ROS. The developed method was successfully applied for resolving the both drugs in a single run using a single detection wavelength which promotes application of the developed method in further quality control studies of the proposed drugs. 2. Experimental 2.1. Apparatus The instrument used was an HPLC Agilent 1260 Infinity (Germany), equipped with an Agilent 1260 infinity preparative pump (Model No. G1361A), Agilent 1260 infinity diode array detector VL (Model No. G131SD), Agilent 1260 infinity thermostated column compartment (Model No. G1316A), and Agilent 1260 infinity preparative autosampler (Model No. G2260A). The stationary phase was a Zorbax Eclipse plus C18 column (150×4.6 mm id, 5 μm particle size, United States). A Sonix TV SS-series ultrasonicator (United States) was also used. Nylon 66 membrane syringe filter for filtration of the formulation solution, (Npore, Ghaziabad, India) was used. 2.2. Materials 2.2.1. Chemicals and reagents All chemicals and solvents used throughout this work were of analytical grade and were used without further purification such as methanol; acetonitrile were HPLC grade (SDS, France), while orthophosphoric acid and deionized water were purchased from El-NASR Pharmaceutical Chemicals Co., Abu-Zabaal, Cairo, Egypt. For tablets formu- lation, the used Avecil PH 101, pharmaburst, aerosil, magnesium stearate, and lactose monohydrate were purc- hased from Sigma-Aldrich Company, Egypt [5]. 2.2.2. Pure standard Bisoprolol fumarate (BIS) was kindly supplied by AMOUN Pharmaceutical Co. Egypt. While, Rosuvastatin calcium (ROS) was kindly supplied by Astrazeneca Co., Cairo, Egypt. 2.2.3. Standard solutions The stock standard solutions of BIS and ROS were prepared in the concentration of 1000 μg/mL in methanol. Then, two working standard solution of each BIS and ROS (100 then 10 μg/mL) were prepared from their respective stock standard solutions (1000 μg/mL) in methanol. All stock standard solutions were freshly prepared on the day of analysis and stored in the refrigerator to be used within 24 h. 2.3. Procedures 2.3.1 Laboratory prepared mixtures Different laboratory prepared mixtures containing different ratios of BIS and ROS were prepared by accurately transferring different volumes of each from their respective working solutions of each drug into 10 mL-glass volumetric flasks and diluting to volume using the mobile phase 2.3.2. Tablets formulation The tablets were formulated using, the pure drugs, Avecil PH 101, Pharmaburst, Aerosil, magnesium stearate, and lactose monohydrate with suitable ratios to get the final tablet weight 150 mg according to reference [5]. By using mortar and pestle, the calculated amounts of the drug and excipients were mixed then the calculated amount of magnesium stearate was added. Single punch machine was used for the preparation of tablets by concave 8 mm punch and die set. 2.4. Method validation 2.4.1. Linearity and range Accurately measured aliquots equivalent to 2-500 μg/mL of each drug were separately transferred from their respective working standard solutions (100 and 10 μg/mL) into two series of 10 mL glass volumetric flasks and diluted to volume with mobile phase. Triplicate 20 μL injections were made for each concentration. Chromatographic separation was performed on the C18 column at 25 °C, and the effluent was UV-scanned at 230 nm. Isocratic elution consisting of acetonitrile and 0.05% aqueous solution of orthophosphoric acid at the ratio of 65:35 % (v:v) was used with a flow rate of 1 mL/min and UV detection at 230 nm. The peak areas were recorded, and calibration curves relating the obtained integrated peak areas to corresponding concentrations were constructed. 2.4.2. Accuracy The accuracy of the proposed method was assessed by analyzing the samples with different concentrations of BIS and ROS within their linearity ranges by the developed method. The concentrations of both drugs were calculated from their corresponding regression equations, and the mean recoveries were calculated. 2.4.3. Precision Repeatability was evaluated by assaying three concent- rations of each drug (1, 10, and 20 μg/mL) three times intraday. Intermediate precision was evaluated by assaying the three chosen concentrations of each drug in triplicate on 3 successive days using the procedure stated in the Linearity and range section and SD values were calculated. 2.4.4. Specificity The specificity of the chromatographic method was ascertained by application of the developed method to the laboratory-prepared mixtures containing different ratios of BIS and ROS, following the procedure stated in the Linearity and range section. Also, specificity was confirmed by calculating system suitability testing parameters, such as capacity factor, resolution, and selectivity factor for the separated peaks. 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.1.52-56.1815 54 Zaki et al. / European Journal of Chemistry 10 (1) (2019) 52-56 Table 1. Data of average weight, thickness, diameter, friability, and hardness of ten formulated tablets. Parameters Mean * S.D. Uniformity of weight (mg) 150.50 1.24 Tablet thickness (mm) 3.03 0.005 Tablet diameter (mm) 8.06 0.01 Friability % fine 0.370 0.104 Hardness (Kg) 8.02 0.227 * Average of ten determinations. Table 2. Results of analysis of laboratory prepared mixtures and assay of the formulated tablets content by applying the proposed method and application of standard addition technique. Sample BIS ROS Mean±SD, Lab. prepared mixtures a 99.74±0.563 100.11±0.727 %Recovery±SD, Formulated tablets b 100.20±0.934 100.65±0.856 Standard addition a 98.50±0.424 99.80±0.704 a Average of 3 determinations. b Average of 10 tablets determinations. 2.4.5. Sensitivity Sensitivity of the method was established with respect to LOD and LOQ for both drugs. The LOD and LOQ were established by the slope method using the lower part of the calibration curves and the slope of the regression equations as mentioned below: Standard deviation of the response Slope of the calibrat LOD = ion c 3.3× urve (1) Standard deviation of the response Slope of the calibrati LOQ on = 10× curve (2) 2.4.6. Robustness Robustness is the capacity of a method to remain unchanged with small changes in method parameters, e.g., changes in acetonitrile (±1%) and mobile phase flow rate (±0.1 mL/min). The effect of these changes on retention time (tR) values were recorded and expressed as RSD. 2.4.7. System suitability testing parameters Parameters such as resolution (RS), peak asymmetry, selectivity factors (α), and capacity factor (k') were calculated to test the overall system performance. 2.4.8. Application to the formulated tablets The content of 20 formulated tablets were powdered and mixed well. An amount of the powdered tablets equivalent to 100 mg of each drug was accurately weighed and transferred to 100 mL glass volumetric flask, 75 mL methanol was added and the prepared solution was ultra-sonicated for about 30 minutes then cooled well; the volume was completed with methanol to get 1000 μg/mL stock solution and, then filtered. Calculated dilutions were made to obtain concentrations of both BIS and ROS in their linearity ranges. Then, the procedure illustrated under linearity was followed. 3. Results and discussion Utilization of drug combinations to treat cardiovascular conditions that does not correspond well with availability of combination of fixed-dose products containing these agents. Chronic conditions such as dyslipidemia and hypertension are frequently coexisting and also they are common causes of coronary or ischemic heart diseases [39]. The aim of our work is to assist in further quality control and clinical studies for the use of the new combination that improve the patient compliance. 3.1. Evaluation of the formulated tablets The formulated tablets were evaluated according to the quality control criteria [40-42]. Table 1 summarizes the acceptable results values concerning weight uniformity, thickness and diameter, friability, and hardness for ten randomly chosen tablets. The content uniformity of those tablets was tested, and the results summarized in Table 2 were found to be within the official acceptable range for the tablets content analysis [1,14]. 3.2. Method developments and optimization Our main goal of establishing this RP-HPLC method was to provide rapid and specific tool for further quality control analysis of BIS and ROS. Various isocratic mobile phase systems were tried on many reversed phase columns C8 and C18 with different length and successfully attempts were reached upon using C18 column (150×4.6 mm, 0.5 μm). Several trials were applied beginning with methanol and water at first with different ratios to separate the two studied drugs with reasonable retention time with sharp peaks but it was noticed they took long time to be completely eluted especially ROS, even after increasing methanol ratios. So methanol had to be replaced by acetonitrile: water (40:60), (50:50), (60:40), and (65:35) which somewhat enabled to elute both of the drugs in earlier time upon increasing the ratio of acetonitrile but with broad peaks which affected peaks symmetry. In order to enhance the separation and peaks shapes for each drug the effect of pH was tested and it was found that both drugs response well to acidic medium, so acetic acid, formic acid, and orthophosphoric acid were tested. Successful attempts were obtained by using 0.05% aqueous solution of orthophosphoric acid in place of using neutral water to reach the optimum mobile phase consisting of acetonitrile: 0.05% aqueous solution of orthophosphoric acid (65:35, v:v) with flow rate 1 mL/min. Several wavelengths were tested (220, 230, 242, and 254 nm); the most suitable wavelength for detection was 230 nm, at which high sensitivity of both cited drugs with minimum detector noise was obtained. Upon using those optimum chromatographic conditions, satisfactory separation of BIS and ROS were obtained and eluted at 2.758 and 4.974 min, respectively, as shown in Figure 2. 3.3. Method validation Validation of the proposed method was performed according to ICH guidelines [43]. The linearity of the proposed methods was evaluated, and linearity was proved in the range of 0.2-50.0 μg/mL for each drug. Regression and analytical parameters are shown in Table 3. 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.1.52-56.1815 Zaki et al. / European Journal of Chemistry 10 (1) (2019) 52-56 55 Table 3. Assay and validation parameters obtained by applying the proposed methods. Parameters RP-HPLC BIS ROS Range (μg/mL) 0.2-50 0.2-50 Slope 379.28 578.74 Intercept 245.40 139.75 Correlation coefficient 0.9999 0.9998 Accuracy (Mean recovery %) 99.50 100.02 Precision (SD) Repeatability 0.325 0.195 Intermediate Precision 0.645 0.425 LOQ (μg/mL) 0.20 0.20 LOD (μg/mL) 0.07 0.07 Robustness (RSD%) * Acetonitrile ratio ±1% 0.548 0.452 Flow rate ±0.2 mL/min 0.348 0.445 * Change in retention time. Table 4. System suitability testing parameters of the HPLC method. ROS BIS Parameters 4.94 4.94 Resolution 2.08 2.08 selectivity (α) 1.00 1.05 Tailing factor (T) 6.106 2.940 Capacity factor (K') 2456.52 1452.10 Column efficiency (n) 0.006 0.010 HETP * * HETP = Height equivalent to the theoretical plate (cm/plate). Table 5. Statistical comparison between the results obtained by the proposed method and the reported methods. Item BIS, Found% ROS, Found% HPLC Reported [15] HPLC Reported [35] Pure drug 101.00±1.200 100.11±1.140 100.09±0.750 99.96±1.020 N 6 6 6 6 Student t-test (2.228) * 0.943 - 0.108 - F-Value (5.050) * 1.107 - 1.857 - * The values in the parenthesis are corresponding theoretical value at degree of freedom p = 0.05. [15] Reported method for determination of BIS by HPLC on cyano column (4.6×250 mm, 5 μm) with the isocratic mobile phase of 0.1 M aqueous phosphate buffer, acetonitrile and tetrahydrofuran (85:10:5, v:v:v) at a flow rate of 1.0 mL/min. The UV detection was carried out at 225 nm. [35] Reported method for determination of ROS by HPLC on C18 column (250×4.6 mm, 0.5 µm) using acetonitrile and 1 % acetic acid in water (80:20, v:v) with a flow rate of 1 mL/min and UV detection at 252 nm. Figure 2. HPLC chromatogram of bisoprolol fumarate and rosuvastatin calcium using acetonitrile: 0.05% aqueous solution of orthophosphoric acid (65:35, v:v) at 230 nm. Good percentage recoveries were obtained when testing method accuracy and the results are given in Table 3. Accuracy was further assessed by applying the standard addition technique on the formulated tablets for which good results were obtained, revealing the good accuracy of the proposed method and proving that excipients did not interfere Table 2. The proposed method provided acceptable intra- and inter- day variation, indicating their acceptable precision, and that they are suitable for the quality control (QC) of the suggested components. Good standard deviation (SD) values were obtained, Table 3. Specificity of the proposed method was evident from the HPLC chromatograms in Figure 2. Also, specificity of the methods was proven from the good recovery percentages obtained when they were applied for the determination of BIS and ROS laboratory-prepared mixtures, Table 2. Moreover, the good results obtained when this method was applied for analysis of the formulated tablets, Table 2, confirmed that there was no interference from excipients. Low values of LOD and LOQ, as shown in Table 3, proved the high sensitivity of the developed method. The method was found to be robust, and small changes in the studied parameters did not lead to significant changes in tR values or the area or symmetry of the peaks, Table 3. When system suitability testing parameters were evaluated, acceptable values were obtained, Table 4. Finally, when the statistical comparison of the results obtained by the proposed method and the reported methods [15,35] were carried out, the values of the calculated t- and F- revealed that there was no significant difference with respect to both accuracy and precision between the proposed method and the reported ones, Table 5. 4. Conclusion The successful combination of two drugs has been formulated in tablets for treating coexisting cardiovascular problems. 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.1.52-56.1815 56 Zaki et al. / European Journal of Chemistry 10 (1) (2019) 52-56 The new formulation has passed all quality control criteria with acceptable results regarding the mean and standard deviation values. Also, the presented work afford rapid, accurate, and specific RP-HPLC method for simulta- neous determination of the cited drugs in pure forms and in lab prepared mixture with high sensitivity which was proved by the small values of LOD 0.07 μg/mL for each drug. Moreover, it was applied on the formulated tablet and it was no interference from excipients this assures the accuracy of the method with average mean recovery of 99.5 and 100.02% for BIS and ROS, respectively. Very good separation of the drugs with reasonable retention times which will be the corner stone for further routine quality control procedure or any clinical studies on those drugs. Disclosure statement Conflict of interests: The authors declare that they have no conflict of interest. Author contributions: All authors contributed equally to this work. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. ORCID Marco Mounir Zaki http://orcid.org/0000-0001-8379-6865 Nada Sayed Abdelwahab http://orcid.org/0000-0002-0700-7542 Adel Ahmed Ali http://orcid.org/0000-0002-1328-5987 Souty Mounir Zaki Sharkawi http://orcid.org/0000-0002-4662-5917 References [1]. The British Pharmacopoeia, Volumes II and III, Her Majesty’s Stationery Office, London, UK. 2009. [2]. Sweetman, S. C.; Martindale, The complete drug reference, 2009. Thirty sixth editions, Pharmaceutical Press, Chicago, 2009. [3]. United States Food and Drug Administration. Crestor (rosuvastatin calcium) Tablets. Prescribing Information Label. 2012; Available online: http://www.accessdata.fda.gov/drugsatfda_docs/label/2012/02136 6s026lbl.pdf (accessed on 10 December 2018). [4]. Bondjers, G.; Wiklund, O.; Wikstrand, J. Patent application number US 10/220, 790 Publication number US20030060477 A1, Publication date Mar 27, 2003. Available online (3 july 2017) at https://www.google.com/patents/US20030060477?cl=en10 [5]. Abdelwahab, N. 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(ed.), Lea- Febiger, Philadelphia, pp. 412, 1986. [43]. International Conference on Harmonization Q2 (R1), Validation of Analytical Procedures: Text and Methodology, ICH Harmonized Tripartite Guideline, 2005. Copyright © 2019 by Authors. This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.1.52-56.1815 http://orcid.org/0000-0001-8379-6865 http://orcid.org/0000-0002-0700-7542 http://orcid.org/0000-0002-1328-5987 http://orcid.org/0000-0002-4662-5917 http://www.accessdata.fda.gov/drugsatfda_docs/label/2012/021366s026lbl.pdf http://www.accessdata.fda.gov/drugsatfda_docs/label/2012/021366s026lbl.pdf https://www.google.com/patents/US20030060477?cl=en10 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Apparatus 2.2. Materials 2.2.1. Chemicals and reagents 2.2.2. Pure standard 2.2.3. Standard solutions 2.3. Procedures 2.3.1 Laboratory prepared mixtures 2.3.2. Tablets formulation 2.4. Method validation 2.4.1. Linearity and range 2.4.2. Accuracy 2.4.3. Precision 2.4.4. Specificity 2.4.5. Sensitivity 2.4.6. Robustness 2.4.7. System suitability testing parameters 2.4.8. Application to the formulated tablets 3. Results and discussion 3.1. Evaluation of the formulated tablets 3.2. Method developments and optimization 3.3. Method validation 4. Conclusion Disclosure statement ORCID References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: