Simultaneous determination of metformin, captopril, lisinopril, and enalapril by RP-HPLC: its applications in dosage formulations and in human serum M. Saeed Arayne, Najma Sultana, M. Hashim Zuberi, Farhan Ahmed Siddiqui & Urooj Haroon Medicinal Chemistry Research ISSN 1054-2523 Med Chem Res DOI 10.1007/s00044-013-0501-z 1 23 Your article is protected by copyright and all rights are held exclusively by Springer Science +Business Media New York. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication. 1 23 Author's personal copy MEDICINAL CHEMISTRY RESEARCH Med Chem Res DOI 10.1007/s00044-013-0501-z ORIGINAL RESEARCH Simultaneous determination of metformin, captopril, lisinopril, and enalapril by RP-HPLC: its applications in dosage formulations and in human serum M. Saeed Arayne • Najma Sultana • M. Hashim Zuberi • Farhan Ahmed Siddiqui Urooj Haroon • Received: 15 October 2012 / Accepted: 16 January 2013 Ó Springer Science+Business Media New York 2013 Abstract A simple, rapid, isocratic, high-performance liquid chromatography (HPLC) method has been developed for the first time for simultaneous determination of Metformin and ACE inhibitors (lisinopril, captopril, enalapril, and its degradable product) in bulk drugs, pharmaceutical products and in human serum. The separation was performed on a PurospherÒ Star RP-18 endcapped (250 mm 9 4.6 mm id) column using methanol–water as mobile phase 50:50 (v/v) and 60:40 (v/v) as diluent. The pH of mobile phase was adjusted to 3.2 with ortho-phosphoric acid, flow rate was adjusted to 1 mLmin-1 at room temperature (25 °C) and analytes peaks were observed using UV detector at 218 nm. The retention times and LOD of lisinopril, enalapril, enalapril diketopiperazine, and captopril were 2.24, 2.63, 3.82, 4.28, and 4.78 min and were 0.028, 0.044, 0.20, 0.016, and 0.145 lgmL-1 respectively. The method was validated according to ICH guidelines. The linearity of the method was studied over the concentration range of 5–50 lgmL-1 for metformin M. S. Arayne  M. H. Zuberi  F. A. Siddiqui (&) Department of Chemistry, University of Karachi, Karachi 75270, Pakistan e-mail: farhanchemist@hotmail.com; farhanchemist@gmail.com M. H. Zuberi Department of Environmental Studies, Sindh Madressatul Islam University, Karachi, Pakistan U. Haroon Department of Chemistry, Federal Urdu University for Arts, Science and Technology, Karachi, Pakistan N. Sultana Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, Karachi, Pakistan and 2.5–250 lgmL-1 for the ACE inhibitors, where it demonstrated good linearity with r = 0.9998, 0.9979, 0.9997, and 0.9987 (n = 6), respectively. The developed method was successfully applied to quantitate metformin, lisinopril, captopril, and enalapril in pharmaceutical formulations and human serum. Keywords Metformin  Captopril  Enalapril  Lisinopril  ACE inhibitors and RP-HPLC Introduction Diabetes mellitus and hypertension are the most common diseases of this era. Patients with diabetes have a much higher rate of hypertension than would be expected in the general population. The inhibitors of the angiotensin-converting enzyme are widely used for the treatment of mild-to-moderate hypertension and heart failure, either alone or in conjunction with other drugs (Cocolas et al., 1998). Reduction in blood pressure helps to prevent diabetic complications. Barring contraindications, angiotensin-converting enzyme (ACE) inhibitors (Fig. 1) are considered first-line therapy as they provide additional benefits (Vaughan et al., 1997; Pasquale et al., 1997). Metformin (MF) 1,1-Dimethylbiguanide (Fig. 1) is the drug of choice for treating type 2 diabetes, particularly obese patients. It helps to reduce LDL cholesterol and triglyceride levels, and may aid in weight loss. In year 2008, MF is one of the only two oral anti-diabetics in the World Health Organization Model List of Essential Medicines (Medicines, 2007). Captopril (CAP) is (2S)-1-[(2S)-2Methyl-3-sulfanylpropanoyl] pyrrolidine-2-carboxylic acid (Fig. 1), it is widely prescribed as antihypertensive drug. Enalapril (ENA), an ACE inhibitor used in the treatment of 123 Author's personal copy Med Chem Res CH3 H N N NH2 H 3C NH determination of these drugs in pharmaceutical formulation and in human serum. Robust studies were completed to insure continuous performance of the methods in diverse analytical environments. NH Metformin Experimental COOH O Chemicals and reagents N HS H CH 3 Captopril H 3C O O HOOC CH 3 N N H O Enalapril MF hydrochloride (NeodiparÒ 250 mg), CAP (CaprilÒ 5 mg), LIS (LameÒ 5 mg), and ENA (CortecÒ 5 mg) reference standards were kindly supplied by Sonaphy Aventis Limited, Efroze Chemical industries (Pvt.) Ltd., Atco Laboratories (Pvt.) Ltd., and Nabi Qasim Industries (Pvt.) Ltd., respectively, and tablets were purchased from local market. Analytical grade phosphoric acid (85 % pure), and HPLC grade methanol were purchased from Merck Marker Ltd. Water was bi-distilled and deionized by Stedec CSW-300. Drug-free human serum was obtained from the National Institute of Cardiovascular Disease, Karachi, Pakistan (NICVD). Instrumentation and chromatographic conditions Lisinopril Fig. 1 Chemical structures of Metformin and ACE inhibitors hypertension and some types of chronic heart failure is (2S)1-[(2S)-2-{[(2S)-1-ethoxy-1-oxo-4-phenylbutan-2-yl] amino}propanoyl]pyrrolidine-2-carboxylic acid (Fig. 1). ENA was the first member of the group of ACE inhibitors having two carboxylic acid groups attached. Lisinopril (LIS) (2S)-1-[(2S)-6-Amino-2-[[(1S)-1-carboxy-3-phenylpropyl] amino]hexanoyl]pyrrolidine-2- carboxylic acid (Fig. 1) is an orally active ACE inhibitor effective in lowering blood pressure, renovascular hypertension and congestive heart failure. It is a lysine analog of enalaprilat Lancaster and Todd, 1998) and is not significantly metabolized in humans and is excreted unchanged in urine. The literature survey revealed few analytical reports for the analysis of these drugs individually (European Pharmacopoeia, 2001; Amini et al., 1999; Bahmaei et al., 1997; Hillaert and Bossche, 1999; Huang et al., 2006; Shimada et al., 1982; Tajerzadeh and Hamidi, 2001; Anzenbacherová et al., 2001; United States Pharmacopoeia, 2006; Al-Momani, 2001; Dinç et al., 2005; Wong and Charles, 1995; Sagirli and Ersoy, 2004; Najma et al., 2011; Arayne et al., 2010, 2009). Since MF and ACE inhibitors are generally coprescribed drugs, no chromatographic method reported for their simultaneous determination and quantitation. The focus of the present work described herein was to develop selective and sensitive HPLC methods for the 123 The liquid chromatographic system consisted of Shimadzu model LC-10 AT VP pump, Rheodyne manual injector fitted with a 20 lL loop, a Shimadzu model SPD-20AV variable wavelength UV–Visible detector (Shimadzu Corporation, Kyoto, Japan). Chromatographic system was integrated via Shimadzu model CBM-102 Communication Bus Module. Analysis was performed on a PurospherÒ Star RP-18 endcapped (25 cm 9 4.6 mm id) analytical reversed phase column. Class GC-10 software was used for data acquisition. The mobile phase consisted of methanol–water 50:50 (v/v), pH was adjusted to 3.2 with phosphoric acid (85 %) and 60:40 (v/v) as diluent. Prior to delivering into the system, it was filtered through 0.45 lm filter and degassed using an ultrasonic bath. The analysis was carried out under isocratic conditions using a flow rate of 1.0 mL min-1 at room temperature. Chromatograms were recorded at 218 nm (isobestic point) was considered satisfactory, permitting the detection of all drugs with adequate sensitivity. Method validation Development of optimum mobile phase In order to develop an RP-HPLC method initially, different ratios of methanol, acetonitrile, and water were investigated for simultaneous estimation of MF and ACEI (CAP, ENA, and LIS). The best separation was obtained at mobile phase composition of methanol and water 50:50 (v/v) with a pH 3.2 (± 0.2) and 60:40 (v/v) as diluent. Flow rate Author's personal copy Med Chem Res Stock solutions of MF 100 lgmL-1 were prepared by dissolving 10 mg of active drug in 100 mL flask and 250 lgmL-1 of ACE inhibitors (CAP, ENA and LIS) by dissolving 25 mg in 100-mL flask. These solutions were sequentially diluted to give working solution at concentrations in the ranges 1.25–25.0 lgmL-1 for MF and 12.5–250 lgmL-1 for ACEIs with diluent (60:40 v/v) for preparation of calibration curves. is the most simple and precise among other reported methods. This system is quite robust. Other ODS columns and chromatographic systems have been tested with minimal effect on the resolution of the analytes. PurospherÒ Star RP-18 endcapped (25 cm 9 4.6 mm id) column is recommended because it demonstrated ruggedness and reproducibility in this assay. A typical reference chromatogram, MF and ACEIs (LIS, CAP, ENA and its degraded product (ENA diketopiperazine) of pure standard mixture using PurospherÒ Star RP-18 endcapped (250 mm 9 4.6 mm) column at flow rate 1.0 mL min-1, is shown in Fig. 2. The optimum wavelength selected was 218.0 nm at which much better detector response for each drug was obtained. The retention times for the investigated drugs (pharmaceutical formulations) were found to be MF 2.24, LIS 2.63, ENA (d) 3.82, ENA 4.28, and CAP 4.78 min. Typical chromatograms are shown in Fig. 3 pharmaceutical formulation, Fig. 4 human serum containing all the drugs and Fig. 5 blank human serum. Preparation of formulation samples Linearity and calibration curve Individual tablets (10 each) were pulverized using a mortar and pestle, and completely transferred to a 100-mL conical flask. The volume was adjusted with diluent and the flask was mechanically shaken for 5 min. Stock solutions were sequentially diluted to give working solution at concentrations in the ranges 1.25–25.0 lgmL-1 for MF and 12.5–250 lgmL-1 for ACE inhibitor. The samples were filtered through a 0.45-lm membrane filter. The amount of MF and ACE inhibitors per tablet was calculated from the related linear regression equations. The linearity of the method was determined using of MF and ACE inhibitors standard solutions in the presence of human serum at five concentration levels in the range of 1.25–25.0 and 12.5–250 lgmL-1, respectively (n = 3). Least-square regression calibration curves were constructed by plotting mean peak areas of MfCl and ACEIs as a function of the drug concentration in the standard working solution. The linearity and regression calibration selection was based on peak parameters (height, asymmetry, tailing), baseline drift and run time. Internal standard was not used, as there was no extraction in the simultaneous determination of MF and ACE inhibitors in human serum and in pharmaceutical dosage forms. Individual drug solutions (100 lgmL-1) were injected into the sample loop of 20 lL. Elution pattern and resolution parameters were studied as a function of pH. Standard solution preparations Preparation of serum sample The Plasma samples were stored in the freezer at -14 °C and allowed to thaw at room temperature before processing. The plasma samples were centrifuged at 3,000 rpm for 6–7 min. An aliquot (1.0 mL) was pipetted into a 10-mL polypropylene test tube and acetonitrile (2.0 mL). The mixture was vortex mixed briefly, and after standing for 5 min at room temperature, the mixture was centrifuged at 3,000 rpm for 5 min. The supernatant obtained was filtered through a 0.45 lm filter. Serum thus obtained was mixed in the ratio of 1:1 with drug solutions and was then injected into the HPLC system. Results and discussion Specificity and robustness The proposed LC method for the simultaneous determination of MF and ACEI in human serum and dosage forms Fig. 2 Typical chromatogram of 1 Metformin, 2 lisinopril, 3 enalapril diketopiperazine, 4 enalapril, and 5 captopril 123 Author's personal copy Med Chem Res Fig. 5 Typical chromatogram of drug-free human serum Fig. 3 Typical chromatogram of 1 Metformin, 2 lisinopril, 3 enalapril diketopiperazine, 4 enalapril, and 5 captopril in dosage formulation to the true value and the precision as the degree of that similarity (ICH Guideline, 2005; Green, 1996). Reproducibility of the method was performed as inter-day and intra-day accuracy and precision in pharmaceutical dosage forms by injecting six replicates of three concentrations (80, 100, and 120 %) into the system. Coefficient of variance (CV) and percentage recovery of each drug were also evaluated as shown in Table 3. Recovery studies To study the interference of formulation additives, analytical percent recovery experiment was performed by adding known amounts of pure drug to the pre-analyzed samples of commercial dosage forms (Table 4). The percent analytical recovery values were calculated as follows: % Recovery ¼ ½ðCv  Cu Þ=Ca   100; Fig. 4 Typical chromatogram of 1 Metformin, 2 lisinopril, 3 enalapril diketopiperazine, 4 enalapril, and 5 captopril in human serum where Cv was the total drug concentration measured after standard addition, Cu, drug concentration in the formulation and Ca was the drug concentration added to the formulation. Precision and accuracy for all the drugs were performed separately in the presence of excipients and formulations. curves equation are represented in Tables 1 and 2, respectively. Limit of detection (LOD) and limit of quantitation (LOQ) Accuracy and precision The limits of Detection (LOD) and Quantitation (LOQ) were calculated in accordance with the 3.3(r/s) and 10(r/s) criteria respectively, where r is the standard deviation of the peak area (for six replicates) for the sample and s is the The accuracy of an analytical method is defined as the similarity of the results obtained by the analytical method 123 Author's personal copy Med Chem Res Table 1 Linearity of MF and ACE inhibitors by proposed method Injected Concentration (lgmL-1) Recovered Concentration (lgmL-1) % Recovery % RSD n=6 Table 2 Statistical regression analysis of metformin and ACE inhibitors Drugs Raw material Human Serum Regression equation Lisinopril R 2 R2 Regression equation 12.5 12.62 100.96 1.34 Metformin 20,085x ? 4,150.4 0.9994 20,337x ? 1,406.4 0.9997 25 25.49 101.96 1.76 Lisinopril 8,285.6x -3,307.7 0.9996 8,286x - 8,867.1 0.9985 50 49.2 98.4 0.89 Enalapril 4,186x ? 3,072.5 0.9999 100 101.2 101.2 1.05 Captopril 3,515.4x - 828.47 0.9998 3,508x ? 1,252.4 150 200 148.3 197.8 98.86 98.9 0.64 0.97 250 251.8 100.72 1.11 92.96 1.34 Table 3 Accuracy and precision in pharmaceutical formulations 97.76 0.57 Concentrations (lgmL-1) 0.9999 4,195x - 687 x is the concentration of MF and ACEIs in lgmL area ratio -1 0.9999 and y is the peak Enalapril 12.5 11.62 25 24.44 50 51.2 100 98.66 150 148.95 99.3 0.47 Metformin 200 199.3 99.65 1.08 250 251.8 100.72 1.29 102.4 98.66 12.62 25 50 Recovery (%) Day 1 Day 2 Day 3 8.00 0.75 1.11 1.02 100.53 10.00 0.98 0.99 0.89 99.96 12.00 1.09 0.85 0.74 99.60 1.34 Captopril 12.5 CV (n = 6) 0.82 100.96 1.44 Captopril 24.95 99.8 0.96 80.00 0.61 1.67 1.45 98.63 50.38 100.76 0.84 100 150 100.88 151.1 100.88 100.73 1.52 1.05 100.00 120.00 0.94 0.73 1.15 1.44 1.62 1.81 99.88 102.36 Enalapril 200 200.9 100.45 0.46 80.00 1.23 1.98 2.30 99.56 250 248.9 99.56 0.91 100.00 0.86 0.76 1.79 101.13 120.00 0.91 1.34 1.99 98.92 Metformin 1.25 1.28 102.4 0.91 5.5 5.42 98.54 1.24 80.00 1.44 1.52 2.59 102.88 5 4.87 97.4 1.32 100.00 1.99 1.07 1.31 100.06 10 9.92 99.2 1.47 120.00 0.88 0.97 1.69 99.38 15 14.56 97.06 1.84 20 20.51 102.55 0.66 25 24.38 97.52 0.72 slope obtained from calibration curve equation (ICH Guideline, 2005; Green, 1996). Using the parameters mentioned above, LOD and LOQ were estimated to be 0.028, 0.044, 0.20, and 0.145 lgmL-1 while LOQ were 0.085, 0.136, 0.60, and 0.441 lgmL-1 for MF, LIS, ENA, and CAP respectively. Application to human serum A spiked serum sample with MF and ACEIs corresponding to the intermediate concentration level of the calibration points was prepared by adding 4 mL of the drug solution containing MF (10 lgmL-1) and ACEI (100 lgmL-1) to Lisinopril 4 mL of drug-free human serum. This spiked serum was made up to 10 mL with diluent. No interference was observed in the analysis as shown in Figs. 4 and 5. Conclusion The proposed method, in addition to its novelty for determining four active ingredients at single wavelength is sufficiently rapid, simple, sensitive as well as precise and accurate that complies with the ICH guidelines [22]. Low LOD 0.028, 0.044, 0.20, and 0.145 lgmL-1 for MF, LIS, ENA, and CAP, respectively, were obtained. No interference of extra pharmaceutical ingredients and human serum was observed in the assay and was successfully applied in raw materials and pharmaceutical formulations. Linearity, accuracy, precision, limit of detection and quantification, 123 Author's personal copy Med Chem Res Table 4 Recovery of metformin and ACE inhibitors (standard addition method) Concentration of drug in formulations (lgmL-1) Concentration Total C.V. of drug added concentration (lgmL-1) of drug found (lgmL-1) % Analytical recovery Metformin 5 10 14.88 0.1356 102.93 10 10 20.17 0.5256 99.64 15 10 25.04 0.4545 99.86 0.3984 99.66 Captopril 50 100 99.8 100 100 200.11 1.1057 100.11 150 100 251.85 0.6832 50 100 100 100 100.66 200.25 0.2703 101.32 0.8667 100.25 150 100 248.77 1.1426 50 100 100.95 0.2202 101.9 100 100 199.65 1.8885 99.65 150 100 249.85 1.8757 99.9 98.99 Enalapril 99.18 Lisinopril and specificity were established. 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