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Development and Validation of RP-HPLC Method for Simultaneous Estimation of Glimepiride and Sildenafil Citrate in Rat Plasma-Application to Pharmacokinetic Studies DOI 10.1055/s-0033-1349865 Drug Res 2013; 63: 510–514 For personal use only. No commercial use, no depositing in repositories.
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510 Original Article
Development and Validation of RP-HPLC Method for Simultaneous Estimation of Glimepiride and Sildenafil Citrate in Rat Plasma-Application to Pharmacokinetic Studies Authors Affiliations
Key words ▶ glimepiride ● ▶ sildenafil citrate ● ▶ RP-HPLC ● ▶ rat plasma ●
A. S. Tripathi1, A. P. Dewani2, P. G. Shelke3, R. L. Bakal2, A. V. Chandewar3, P. M. Mazumder4 1
Department of Pharmacology, P. Wadhwani College of Pharmacy, Maharashtra, India Department of Quality Assurance, P. Wadhwani College of Pharmacy, Maharashtra, India 3 Department of Pharmaceutical Chemistry, P. Wadhwani College of Pharmacy, Maharashtra, India 4 Department of Pharmaceutical Sciences, Birla Institute of Technology, Jharkhand, India 2
Abstract
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A simple and sensitive method was developed for simultaneous estimation of Glimepiride (GLIM) and Sildenafil citrate (SIL) in rat Plasma by reverse phase high performance liquid chromatography (RP-HPLC). The drug samples were extracted by liquid-liquid extraction with 300 μl of acetonitrile and 5 ml of diethyl ether. Chromatographic separation was achieved on C18 column using methanol: water (85:15 v/v) as
Abbreviations
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received 25.01.2013 accepted 03.05.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1349865 Published online: July 24, 2013 Drug Res 2013; 63: 510–514 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence Asst. Prof. A. S. Tripathi Department of Pharmacology P. Wadhwani College of Pharmacy Dhaman Gaon Road Yavatmal Maharashtra 45001 India Tel.: + 91/758/8891 155 Fax: + 91/723/2238 747
[email protected] shloksk@rediffmail.com
Glimepiride Sildenafil citrate Diode-array detection % RSD
GLIM SIL DAD % Relative standard deviation
Introduction
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Glimepiride, 3-ethyl-4-methyl-N-(4-[N-((1r,4r)4-methylc yclohex ylcarbamoyl)sulfamoyl] phenethyl)-2-oxo-2,5-dihydro-1 H -pyrrole-1carboxamide, is an antidiabetic drug. The drug provokes a brisk release of insulin from pancreas by acting on the so called sulfonylurea receptor (SURI) present on the pancreatic β cell membrane. GLIM causes depolarization by reducing the conductance of ATP sensitive K + channel which enhances Ca2 + influx causing degranulation and results in increased rate of insulin secretion at any glucose concentration [1]. Sildenafil citrate, 1-[4-ethoxy-3-(6,7-dihydro1-methyl-7-oxo-3-propyl-1H- pyrazolo[4,3–d] pyrimidin-5-yl) phenylsulfonyl] – 4 methylpiperazine, primarily indicated in the treatment of erectile dysfunction [2]. It acts by inhibiting cGMP-specific phosphodiesterase type 5, an
Tripathi AS et al. HPLC Method Glimepiride and Sildenafil … Drug Res 2013; 63: 510–514
mobile phase at a flow rate of 1 ml/min and UV detection at 230 nm. The retention time of GLIM and SIL was found to be 2.5 and 4.0 min respectively with total run time of 7 min. The developed method was validated for accuracy, precision, linearity and recovery. The method was linear and found to be acceptable over the range of 100–12 000 ng/ml. The method was successfully applied for the analysis of rat plasma sample for application to pharmacokinetic.
enzyme that promotes degradation of cGMP, which regulates blood flow in the penis. The chemical structures of GLIM and SIL are shown ▶ Fig. 1. in ● Literature reveals that SIL and GLIM having the role for the management of renal failure and blood glucose in diabetic nephropathy respectively [3]. Thus there is possibility of drug-drug interaction between two, so it is necessary to estimate these drugs in plasma. Till date no any literature reports the simultaneous estimation of SIL and GLIM in plasma. Literature reports the estimation of GLIM and SIL separately in the human plasma and biological samples viz. The estimation of GLIM separately by micellar electrokinetic capillary chromatography with diode-array detection (DAD) or ultraviolet detection [4], high performance liquid chromatography (HPLC) with DAD [5], ultraviolet detection detection [6], UV detection by derivative spectrophotometry [7], liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) [8–11], an HPLC method for the quantification of GLIM in tablets [12], the determination of related substances in glimepiride [13], an method for quantification of cis-isomer of GLIM by normal phase chromatography [14] and the quantification of cis-isomer of GLIM in a bulk drug substance by reverse-phase chromatography [15].
Original Article 511
Fig. 1 Chemical structure of a GLIM b SIL.
Fig. 2 HPLC trace of GLIM and SIL using Ultraviolet detection at 230 nm. a Blank plasma sample b Quality control standard (5000 ng/mL).
Even several high-performance liquid chromatographic (HPLC) methods have been reported for the determination of SIL separately in biological samples. Gas chromatography – mass spectrometry (GC/MS) [16], micellar electrokinetic chromatography [17], liquid chromatography – mass spectrometry (LC/MS) [18, 19] as well as liquid chromatography – tandem mass spectrometry (LC/MS/MS) [20–22] methods have been reported. Liquid chromatography – mass spectrometry and liquid chromatography – tandem mass spectrometry (LC/MS/MS) are expensive and thus unavailable in many laboratories. High-performance liquid chromatographic methods with UV detection have been reported for the simultaneous determination of SIL and its active metabolite [23–27].
Experimental
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Instrumentation A double beam UV-Visible spectrophotometer, model UV-2401 PC (Japan) with 10 mm matched quartz cell was used. The HPLC instrument consisted of Thermo separation product quaternary gradient equipped with pump spectra system P-4000 having inline membrane degasser. Detector was a UV visible detector belonging to spectra system UV 1000. Rheodyne 9 725 injector with 20 μl loop. All the data was processed using Data Ace software. Separation was achieved using a Prontosil C18 stationary phase (150 × 4.6 mm i. d. 5 μm particle size) and
Tripathi AS et al. HPLC Method Glimepiride and Sildenafil … Drug Res 2013; 63: 510–514
512 Original Article
Sr. No. 1 2 3
Nominal Concentration (ng/mL)
Recovered amount (ng/mL)
GLIM
SIL
GLIM
100 400 800
100 400 800
95 385 793.28
Accuracy ( %)
SIL
GLIM
97.5 390 796.8
95 % 96.25 % 99.16
SIL 97.5 % 97.5 % 99.6 %
Inter-day Precision Sr. No.
Concentration of drug
Table 1 Results of Accuracy studies.
Peak Area SIL
%RSD
Peak Area GLIM
%RSD
2.09 2.04 0.92
79.57 ± 0.95 190.38 ± 1.16 376.45 ± 1.04
1.19 1.23 0.76
Table 2 Results of precision studies (Inter-day).
solution (ng/mL) 1 2 3
100 400 800
29.3 ± 0.52 56.54 ± 0.66 205.43 ± 0.60
Sr. No.
Concentration of drug
Peak Area SIL
1 2 3
100 400 800
Intra-day Precision %RSD
Peak Area GLIM
%RSD
Table 3 Results of precision studies (Intra-day).
solution (ng/mL) 28.5 ± 0.45 57.24 ± 0.66 203.13 ± 0.45
2.01 2.03 0.98
Table 4 The percentage extraction recovery of measurement of GLIM and SIL from plasma. Nominal concentration (ng/mL) 100 400 800
Mean % Recovery GLIM
SIL
91.2 86.4 89.7
89.3 88.5 89.4
the analytical column was protected by a Phenomenex C18 guard column (4 mm × 2.0 mm, i. d.).
Materials and reagents Glimepiride was gifted generously by Zim laboratories pvt. ltd., Nagpur, India. Sildenafil citrate was gifted by Ajanta Pharmaceuticals pvt. ltd. Aurangabad, India. HPLC grade methanol belonged to Spectrochem pvt. ltd. Mumbai, India. Water HPLC grade belonged to Loba chem., Mumbai, India.
Animal Healthy Male Sprague-Dawley rats (250–300 gm) were used for the pharmacological screening. The animals were housed in polypropylene cages with wire mesh top and husk bedding and maintained under standard environmental conditions (25 ± 20 C, relative humidity 60 ± 5 %, light- dark cycle of 12 h each) and fed with standard pellet diet (Trimurti feeds, Nagpur) and water ad libitum, were used for the entire animal study. The rats were housed and treated according to the rules and regulations of CPCSEA and IAEC. The protocols for all the animal studies were approved by the Institutional Animal Ethical Committee (IAEC). CPCSEA registration no.- (650/02/C/CPCSEA/08).
Chromatographic conditions All determinations were carried out at room temperature. The isocratic separation of compounds was carried out by using mobile phase consisting of methanol: water (85:15 v/v). The flow rate was maintained at 1 ml min − 1 monitoring of analytes
81.61 ± 0.83 189.58 ± 1.14 381.35 ± 1.08
1.11 1.13 0.66
was carried out at 230 nm using a UV detector. The volume of injection was 20 μl. The mobile phase was filtered through 0.45 μm membrane filter and degassed by ultrasonification before using it for chromatographic analysis.
Preparation of stock and standard solutions The stock solution of GLIM and SIL (1.0 mg/mL) was prepared together and diluted with methanol to give standard solutions of 100–12 000 ng/mL. Standard calibration samples were prepared daily by spiking 100 μL of drug-free plasma with 100 μL of appropriate GLIM and SIL standard solution to achieve final concentrations of 100–12 000 ng/mL for plasma.
Preparation of quality control samples The concentrations of GLI and SIL were 100, 400, and 800 ng/mL in plasma to represent low, middle, and high quality controls, respectively. Appropriate volumes from stock solution of GLI and SIL were added to normal rat plasma to get low, middle and high quality control samples and stored at − 20 °C. The quality control samples were taken out from storage for analysis to determine intra- and inter-day precision and accuracy.
Extraction procedure Blood samples were collected into the tubes containing disodium EDTA and centrifuged at 3 000 rpm for 10 min. A 100 μL plasma was spiked with 100 μL standard solution of GLIM and SIL the mixture was added with 300 μL of acetonitrile and 5 ml of diethyl ether, tubes were shaken and subjected to centrifugation for 10 min at 3 000 rpm. The clear supernatant layer was transferred into another conical glass tube and organic layer was evaporated completely at room temperature. After evaporation the residue was reconstituted in 1.0 mL mobile phase and a 20 μL aliquot and was injected in developed chromatographic conditions.
Application of the assay The above method was successfully applied for the Pharmacokinetic studies of GLIM and SIL citrate in Healthy rats (n = 8). Rats
Tripathi AS et al. HPLC Method Glimepiride and Sildenafil … Drug Res 2013; 63: 510–514
Original Article 513
were housed with free access to food and water. The rats were housed on wire bottom cages, fasted overnight with free access to water before administration of drugs. After a single oral administration of 0.5 mg/kg of GLIM and 2.5 mg/kg of SIL, 0.5 ml of blood samples were collected from retro orbital plexus sinus at 0.5, 1, 2, 4, 6, 12 and 24 h time-points. Plasma was separated by centrifugation and stored at − 20 °C until analysis. Aliquots 100 μL of plasma samples were processed as discussed in section 2.6 and were subjected to HPLC analysis for GLIM and SIL concentrations. The pharmacokinetic parameters were calculated with a NonCompartmental model using Kinetica TM Soft-ware (version 4.4.1 Thermo Electron Corporation, U.S.A). Each value is expressed as Mean ± SD.
Accuracy Accuracy studies were performed for GLIM and SIL in terms of recovery studies. For this 100, 400 and 800 ng/mL of both drugs in plasma were injected and % recovery and % RSD were calcu▶ Table 1. lated shown in ●
Precision Inter-day and intra-day precision studies were done by injecting 3 serial dilutions in developed chromatographic conditions (n = 6). For precision studies 100, 400 and 800 ng/mL were injected (n = 6). Peak areas were calculated for % RSD values, results for inter-day ▶ Table 2, 3 respectively. and intra-day precision are shown in ●
Extraction recovery Results and Discussion
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Method validation Selectivity and specificity Blank plasma was studied for endogenous interference. A repre▶ Fig. 2a. sentative chromatogram of the plasma blank is shown in ● No additional peaks of endogenous substances were observed. ▶ Fig. 2b shows the chromatograms of calibration standard con● taining 100 ng/mL of GLIM and 100 ng/mL of SIL in plasma.
Linearity and limit of quantitation Linear calibration curves with correlation coefficients greater than 0.9999 were obtained over the concentration range 100– 12 000 ng/mL for GLIM and SIL in plasma. The coefficient of regression i. e., r = 0.9924 for GLIM and r2 = 0.9996 for SIL. The results shown that within the concentration range indicated there was an excellent correlation between peak area ratio and each concentration of GLIM and SIL. The limit of quantitation, defined as the lowest concentration analyzed with an accuracy of ± 15 % and a co-efficient of variation < 15 %, was 100 ng/mL for the determination of GLIM and 100 ng/mL for SIL in plasma.
Extraction recovery of GLIM and SIL were determined by comparing peak areas obtained from extracted plasma samples with those found by extracting blank matrices through the extraction procedure and spiking with a known amount of GLIM and SIL. The results showed that the mean extraction recoveries of GLIM and SIL were > 85 % at concentrations of 100, 400 and 800 ng/ml ▶ Table 4). Different organic extraction solvents respectively (● were evaluated in the experiment, including Methanol, Acetonitrile, Chloroform and Diethylether. Diethyl ether and acetonitrile combination proved to be the most efficient in extracting GLIM and SIL from plasma and had a small variation in extraction recoveries over the concentration range. Averaged for 6 measurements at each concentration level (n = 6); % recovery = (response of extracted spike)/(response of postextracted spike) × 100.
Application of the analytical method in pharmacokinetic studies The described method was applied to a pharmacokinetic study in rats. After a single oral administration of GLIM (0.5 mg/kg) and SIL (2.5 mg/kg) to rats, plasma concentrations were determined over a period of 24 h after administration. The mean serum concentration-time curve after an oral dose of GLIM (0.5 mg/kg) and ▶ Fig. 3 and the main pharmacokiSIL (2.5 mg/kg) is shown in ● ▶ Table 5. The Cmax of netic parameters are summarized in ● GLIM detected in the rats was 8.6 μg/ml, and the Tmax was 4 h. Whereas the Cmax of SIL detected in the rats was 2.19 μg/ml, and the Tmax was 4 h.
Conclusion
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Fig. 3 Mean serum concentration-time profile of GLIM and SIL after oral administration of 0.5 mg/kg GLIM and 2.5 mg/kg of SIL in rats.
In the present study a simple, accurate and precise method was developed for simultaneous estimation of GLIM and SIL by HPLC. The developed method was simple employing water and not a buffer as component of mobile phase. The developed method was short with elution of both GLM and SIL less than 5 min and specific with no interferences of blank matrix interfering the quantification of both GLIM and SIL. The developed method was applied successfully for pharmacokinetic studies of GLIM and
Table 5 The main pharmacokinetic parameters of mean drug serum concentration time curve (mean ± SD, n = 6) of GLIM and SIL in rats after single oral administration of 0.5 mg/kg of GLIM and 2.5 mg/kg of SIL. Drugs
AUCo–t (ug.h/ml)
AUC0–∞ (ug.h/ml)
C max (ug)
Tmax (h)
Kel
T1/2 (h)
GLIM SIL
35.57 ± 0.329 2.19 ± 0.052
36.98 ± 0.04 3.1 ± 0.02
8.6 ± 0.1 0.8 ± 0.05
4h 1h
0.19 ± 0.05 0.10 ± 0.01
3.6 6.93
Tripathi AS et al. HPLC Method Glimepiride and Sildenafil … Drug Res 2013; 63: 510–514
514 Original Article
SIL in rats. The applicability of method suggests its further application for bioequivalence, bioavailability and drug interaction studies.
Acknowledgement
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We are grateful to P. Wadhwani College of Pharmacy,Yavatmal (MS), India for providing the financial support for this work.
Conflict of Interest
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No
References 1 Tripathi KD. Essential of medical pharmacology, jaypee brothers medical publisher(p) ltd. 6th edition 2009; 266 2 Vardi M, Nini A. Phosphodiesterase inhibitors for erectile dysfunction in patients with diabetes mellitus. Cochrane Database Syst Rev 2007; 1: CD002187 doi:10.1002/14651858.CD002187. pub3. PMID 17253475 3 Jeong KH, Lee TW, Ihm CG et al. Effects of sildenafil on oxidative and inflammatory injuries of the kidney in streptozotocin-induced diabetic rats. Am J Nephrol 2009; 29: 274–282 4 Roche ME, Oda RP, Lawson GM et al. Capillary Electrophoretic Detection of Metabolites in the Urine of Patients Receiving Hypoglycemic Drug Therapy. Electrophoresis 1997; 18: 1865–1874 doi:10.1002/ elps.1150181024 5 Jingar JN, Rajput SJ, Dasandi B et al. Development and Validation of LC-UV for Simultaneous Estimation of Rosiglitazone and Glimepiride in Human Plasma. Chromatographia 2008; 67: 951–955 doi:10.1365/ s10337-008-0633-3 6 Song YK, Maeng JE, Hwang HR et al. Determination of Glime-piride in Human Plasma Using Semi-Microbore High-Performance Liquid Chromatography with Column switching. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 2004; 810: 143–149 7 Khan IU, Aslam F, Ashfaq M et al. Determination of Glimepiride in Pharmaceutical Formulations Using High-Performance Liquid Chromatog-raphy and First-Derivative Spectrophotometric Methods. Journal of Analytical Chemistry 2009; 64: 171–175 doi:10.1134/ S1061934809020130 8 Salem II, Idrees J, Al Tamimi JI. Determination of Glimepiride in Human Plasma by Liquid Chromatography Electrospray Ionization Tandem Mass Spectro- metry. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences. 2004; 799: 103–109 doi:10.1016/j.jchromb.2003.10.024 9 Kim H, Chang KY, Lee HJ et al. Determination of Glimepiride in Human Plasma by Liquid Chromatography — Electrospray Ionization Tandem Mass Spectrometry. Bulletin of the Korean Chemical Society 2004; 25: 109–114 doi:10.5012/bkcs.2004.25.1.109 10 Kim H, Chang KY, Park CH et al. Determination of Glimepiride in Human Plasma by LC-MS-MS and Comparison of Sample Preparation Methods for Glimepiride. Chromatographia 2004; 60: 93–98 doi:10.1365/s10337-004-0351-4
11 Yuzuak N, Ozden T, Eren S et al. Determination of Glimepiride in Human Plasma by LC-MS-MS. Chromatographia 2007; 66: 165–168 doi:10.1365/s10337-007-0368-6 12 Pawar SP, Meshram GA, Phadke MU. Simultaneous LC Estimation of Glimepiride and Metformin in Glimepiride Immediate Release and Metformin Sustained Release Tablets. Chromatographia 2008; 68: 1063–1066 doi:10.1365/s10337-008-0802-4 13 Lehr KH, Damm P. Simultaneous Determination of the Sulphonylurea Glimepiride and Its Metabolites in Human Serum and Urine by High-Performance Liquid Chromatography after PreColumn Derivatization. Journal of Chromatography-Biomedical Applications 1990; 526: 497–505 doi:10.1016/S0378-4347(00)82531-1 14 The United States Pharmacopeia (USP) and the National Formulary (NF). The Official Compendia of Standards 2006; 29: 1001 15 Pathare DB, Jadhav AS, Shingare MS. RP-LC Determination of the CisIsomer of Glimepiride in a Bulk Drug Substance. Chromatographia 2007; 66: 639–641 doi:10.1365/s10337-007-0356-x 16 Saisho K, Scott KS, Morimoto S et al. Extraction and determination of sildenafil (Viagra) and its N-desmethyl metabolite in rat and human hair by. GC-MS. Biol Pharm Bull 2001; 24: 1384–1388 17 Nevado JJB, Flores JR, Penalvo GC et al. Determination of sildenafil citrate and its main metabolite by sample stacking with polarity switching using micellar electrokinetic chromatography. J Chromatogr A 2002; 953: 279–286 18 Weinmann W, Bohnert M, Wiedemann A et al. Post-mortem detection and identification of sildenafil (Viagra) and its metabolites by LC/MS and LC/MS/MS. Int J Legal Med 2001; 114: 252–258 19 Dumestre-Toulet V, Cirimele V, Gromb S et al. Last performance with VIAGRA®: post-mortem identification of sildenafil and its metabolites in biological specimens including hair sample. Forensic Sci Int 2002; 126: 71–76 20 Eerkes A, Addison T, Naidong W. Simultaneous assay of sildenafil and desmethylsildenafil in human plasma using liquid chromatography – tandem mass spectrometry on silica column with aqueous organic mobile phase. J Chromatogr B 2002; 768: 277–284 21 Kim J, Ji H, Kim S et al. Simultaneous determination of sildenafil and its active metabolite UK-103,320 in human plasma using liquid chromatography – tandem mass spectrometry. J Pharm Biomed Anal 2003; 32: 317–322 22 Wang L, Wang J, Cui Y et al. Liquid chromatographic – tandem mass spectrometric method for the quantitation of sildenafil in human plasma. J Chromatogr B 2005; 828: 118–121 23 Hyland R, Roe EGH, Jones BC et al. Effect of P-glycoprotein modulation on the clinical pharmacokinetics and adverse effects of morphine Br. J Clin Pharmacol 2000; 51: 239–248 24 Cooper JDH, Muirhead DC, Taylor JE et al. Development of an assay for the simultaneous determination of sildenfail (viagra) and its metabolite (UK-103,320) using automated sequential trace enrichment of dialysates and high-performance liquid chromatography. J Chromatogr B 1997; 701: 87–95 25 Jeong CK, Lee HY, Jang MS et al. Narrowbore high-performance liquid chromatography for the simultaneous determination of sildenafil and its metabolite UK-103,320 in human plasma using column switching. J Chromatogr B 2001; 752: 141–147 26 Liaw J, Chang TW. Determination of transdermal sildenafil in nude mouse skin by reversed-phase high-performance liquid chromatography. J Chromatogr B 2001; 765: 161–166 27 Guermouchea MH, Bensalah K. Solid phase extraction and liquid chromatographic determination of sildenafil and N-demethylsidenafil in rat serum with basic mobile phase. J Pharm Biomed Anal 2006; 40: 952–957
Tripathi AS et al. HPLC Method Glimepiride and Sildenafil … Drug Res 2013; 63: 510–514