Simultaneous estimation of Rosiglitazone Maleate, Glimepride and Amlodipine Besylate in Tablet Dosage formulation by RP-HPLC

 

Rajeev K. Singh¹*, Pushpendra Sharma²

¹Research Scholar, Department of Chemistry, Sri Satya Sai University of Technology and Medical Sciences, Opp. Oil-fed Plant, Bhopal-Indore Road, Sehore, Madhya Pradesh-466001, India.

²Sri Satya Sai University of Technology and Medical Sciences, Opp. Oil-fed Plant, Bhopal-Indore Road, Sehore, Madhya Pradesh-466001, India.

 

ABSTRACT:

A simple, rapid and precise reversed-phase high performance liquid chromatography method for simultaneous estimation of rosiglitazone maleate, glimepride and amlodipine besylate in a marketed tablet dosage form has been developed and validated. At optimum condition was performed on a C₁₈ column by ratio of mobile phase Methanol: Water: Ortho Phosphoric Acid (75:25:0.2) with maintain of pH 4.5±0.01 at 1.0 mL/min. Selected wavelength at 230 nm and sample volume injected 20 µL at ambient temperature. Total run time was 10 min; rosiglitazone maleate, glimepride and amlodipine besylate were eluted with retention time of 1.75, 2.22 and 6.483 respectively. The developed method was validated as per ICH guideline. Validation revealed that method is specific, rapid, accurate, precise, reliable and reproducible.

 

 

 

 

 

INTRODUCTION:

Rosiglitazone chemically 5-[[4-[2-(methyl-2-pyridinylamino) ethoxy] phenyl] methyl 2,4-thiazolidinedione is used as anti-diabetic drug from the thiazolidinedione class used in the management of type-2 diabetics. It is selective agonist for nuclear peroxisome proliferator-activated receptor-gamma (PPARγ). It binds to PPARγ, which, in turn, activates insulin-responsive gene that regulate carbohydrate and lipid metabolism. It exerts its principal effect by lowering insulin resistance in peripheral tissue, but an effect to lower glucose production by the liver also has been reported. It tends to reverse insulin resistance by stimulating GLUT₄ expression and translocation: entry of glucose into muscle and fat is improved^1-4.

 

Glimepiride chemically 3-ethyl-2,5-dihydro-4-methyl-N-[2-[4-[[[[(trans-4-ethylcyclohexyl)amino]carbonyl] amino] sulphonyl] phenyl] ethyl]-2-oxo-1H-pyrrole-1-carboxamide.Glimepiride causes hypoglycaemia by stimulating insulin release from pancreatic β cells. Its effects in the treatment of diabetes, however, are more complex. The acute administration of sulfonylureas to type II diabetes mellitus patients increases insulin release from the pancreas. Sulfonylureas may further increase insulin levels by reducing hepatic clearance of the hormone. In the initial months of sulfonylurea treatment, fasting plasma insulin levels and insulin responses to oral glucose challenges are increased. With chronic administration, circulating insulin levels decline to those that existed before treatment, but, despite this reduction in insulin levels, reduced plasma glucose levels are maintained. A direct extrapancreatic action of sulfonylureas to increase insulin receptors on target cells and to inhibit gluconeogenesis in liver has been suggested, but not proven^5-8.

 

Amlodipine chemically 2-[(2-aminoethoxymethyl]-4-(2-chloro-phenyl)-3-ethoxycarbonyl-5-Methoxycarbonyl-6-methyl-1,4-dihydropyridine is used as antihypertensive drug. Its involved in excitation-contraction coupling in the heart differ from those in vascular smooth muscle in that a portion of the two inward currents is carried by sodium ion (Na⁺) through the fast channel in addition to that carried by calcium ion (Ca²⁺) through the slow channel. In the sinoatrial (SA) and atrioventricular (AV) nodes, depolarization is largely dependent on the movement of Ca²⁺ through the slow channel. Within the cardiac myocyte, Ca²⁺ binds to troponin, the inhibitory effect of troponin on the contractile apparatus is relieved, and actin and myosin interact to cause contraction. Thus, Ca²⁺ channel blockers can produce a negative inotropic effect. The greater degree of peripheral vasodilation seen with the dihydropyridines is accompanied by sufficient baroreflex-mediated increase in sympathetic tone to overcome the negative inotropic effect^9-13. The chemical structure of Rosiglitazone maleate, Glimepiride and Amlodipine besylate are shown in fig 1. Several assay techniques have been described for quantitative determination of Rosiglitazone maleate, Glimepiride and Amlodipine besylate in individual and in combination. The UV Spectroscopy determination^14-25, UV and HPLC determination^26-30, HPLC determination^27-34, HPTLC determination^35-40.

 

 

Fig 1 (a) Chemical Structure of Rosiglitazone Maleate

 

Fig 1 (b) Chemical Structure of Glimepiride

 

Fig 1 (c) Chemical Structure of Amlodipine besylate

 

MATERIAL AND METHODS:

Rosiglitazone maleate and Glimepiride were supplied by Zydus Research Center, Ahmedabad, India as gift sample under batch number WSG01566 and WSG00976 respectively. Amlodipine besylate were supplied by Cadila Pharmaceuticals, Ahmedabad under batch number 3AM003. All the solvents and reagents used for the analysis were of HPLC grade. While the solvents used for thin layer chromatography were of analytical reagent grade (AR), the HPLC grade water was obtained from Millipore DirectQ3, SSSUTMS, Sehore.

 

Instrumentation and Chromatographic conditions:

For the chromatographic analysis, Shimadzu HPLC system was used that was equipped with a solvent delivery module {LC-10 AT VP}, Rheodyne manual injector 7725i fitted with 20 µL loop and UV detector {SPD-10 A VP}. The separation was achieved on RP C₁₈ column {250 x 4.6 mm, 5 micron particle size} using methanol: water: ortho phosphoric acid (75: 25: 0.2, v/v/v) as mobile phase. The pH of mobile phase was adjusted to 4.5 with the help of liquid ammonia. The flow rate was kept at 1 mL/min and the peaks were integrated by UV detector at 230 nm. Operation, data acquisition and analysis were performed using Spinchrom 1.7 software.

 

Method development

Solubility of drugs:

The solubility of rosiglitazone maleate, amlodipine besylate and glimepiride was determined in different solvents like acetonitrile, methanol, isopropranol, absolute ethanol, water and mobile phase. All the three drugs were soluble in the mobile phase {methanol: water: ortho-phosphoric acid, (75: 25: 0.2); pH 4.5}. Rosiglitazone maleate and amlodipine besylate were soluble even at higher concentration of 3 mg/mL; while 10 mg of glimepiride was soluble at 15 mL of mobile phase, i.e. solubility of glimepiride in mobile phase was found to be 666.67 µg/mL.

 

Selection of mobile phase:

Different mobile phases were tried to optimize the best mobile phase that shall give better peak shape, better resolution and shorter retention time. Changing the mobile phase composition doesn’t provide good resolution between amlodipine besylate and rosiglitazone maleate. Therefore, in order to resolve the rosiglitazone maleate and amlodipine; alteration of pH was done.

 

Selection of pH:

The mobile phase that eluted the drugs well with less tailing and good peak shape was taken to optimize the effect of pH in order to select the best pH condition. The mobile phase selected as the optimized mobile phase was the mixture of methanol: water: ortho-phosphoric acid in the ratio of 75: 25: 0.2, v/v/v. Further, the pH of mobile phase was adjusted to 4.5, 5.0 and 5.5, and the injection of drugs individually as well as in mixture was analysed and response was observed in context of retention time, resolution and peak shape. At pH 5.5, the Rosiglitazone maleate and Amlodipine besylate had the same retention time. Therefore when the mixture of both drugs was given, they combined together and gave a single peak. Both the drugs were resolved at pH 5.0 and 4.5; while no significant difference was found in the retention time of glimepiride at pH 4.5, 5.0 and 5.5. pH 4.5 was selected as best pH condition because the resolution between rosiglitazone maleate and amlodipine besylate was high as compared to pH 5.0. At pH 4.0, the rosiglitazone was poorly retained and had short retention time and thus appeared at the retention time of void volume.

 

Selection of modifier:

Different peak modifiers (acidic and basic) were used to select the best analytical condition. Triethylamine and tetrahydrofuran were not found satisfactory as the peak shapes were broad and tailing was more. Further using acidic modifiers increased the peak height and the peak shape also became better with lesser tailing. Ortho-phosphoric acid was selected over glacial acetic acid because glacial acetic acid produced some negative peaks. After the selection of peak modifier, the concentration of the modifier was determined and the drugs were individually and in mixture were analysed using 0.2 and 0.5 % of peak modifier. The mobile phase used was 75% methanol, 25% water: 0.2% ortho-phosphoric acid; pH was adjusted to 4.5 with the help of liquid ammonia. The flow rate was kept at 1.0 mL/min and wavelength was set at 230 nm. There was no significant change observed in the peak shape and peak area of rosiglitazone maleate, amlodipine besylate and glimepiride. But the retention time of glimepiride was affected significantly and was increased as the concentration of modifier was increased. Therefore, 0.2% modifier was selected over 0.5%.

 

Selection of analytical wavelength:

The UV absorbance scan was used to determine the lambda maxima (λ_max) of rosiglitazone maleate, amlodipine besylate and glimepiride. On the basis of overlain spectra of the drugs, the wavelength 230 nm was selected for the optimisation of mobile phase, pH and peak modifiers. After optimisation of mobile phase, pH and peak modifiers; the effect of wavelength was studied keeping other parameters unchanged. The analysis of all the three drugs was done at wavelength 220, 230 and 240 nm. 230 nm wavelengths were selected as best detection wavelength as all the three drugs responded well as compared to the response at other wavelengths.

 

Method validation:

The method validation was carried out according to the ICH guideline and the parameters for validation were selected on the basis of availability of resources and limitations.

 

Specificity:

The specificity of the method was determined by comparing the chromatograms of blank solution (mobile phase), solution with tablet excipients (lactose, magnesium stearate, dextrose, carboxymethyl cellulose and talc) and standard drug solution. Representative chromatograms were generated and compared with the chromatogram of individual drugs. It was observed that no extraneous peaks were eluted at the same retention time of drug. Also, the peaks of other two drugs were eluted at different retention time as that of parent drug. The method was thus found to be specific for the analysis of rosiglitazone maleate, amlodipine besylate and glimepiride.

 

Fig 2 Chromatogram of blank sample (mobile phase)

 

Fig 3 Chromatogram of tablet excipients

 

Interaction studies:

The interaction study was done by comparing the peak area of individual drug with the peak area of the same drug in mixture at same concentration. The difference of ±20% in peak area of individual drug versus (v/s) peak area of drug in mixture is often acceptable. The data of analysis for interaction study revealed that the proposed method doesn’t have any interaction with the drug solutions. Also, the drugs are stable in the mixture of other two drugs as the difference in peak area was found to be in acceptable range of ±20%.

 

Table 1: Interaction study data for Rosiglitazone Maleate

Conc. (µg/mL)	Mean area of rosiglitazone	Mean area of rosiglitazone in mixture	% difference (individual v/s mixture)
0.25	6.7799	5.5406	-18.28
0.5	11.8769	10.380367	-12.60
1.0	21.996067	20.6377	-6.176
2.0	41.9937	41.2209	-1.84
4.0	81.501333	80.920233	-0.713
8.0	162.44753	164.8869	+1.502
16.0	326.00237	337.09487	+3.403

 

Table 2: Interaction study data for Amlodipine Besylate

Conc. (µg/mL)	Mean area of amlodipine	Mean area of amlodipine in mixture	% difference (individual v/s mixture)
0.25	5.5508667	4.6691	-15.886
0.5	10.0201	9.0027667	-10.153
1.0	19.082333	18.1699	-4.782
2.0	36.883	35.169767	-4.655
4.0	73.042333	70.699533	-3.207
8.0	143.42803	142.6353	-0. 533
16.0	290.03173	290.93653	+0.312

 

Table 3: Interaction study data for Glimepiride

Conc. (µg/mL)	Mean area of Glimepiride	Mean area of Glimepiride in mixture	% difference (individual v/s mixture)
0.25	3.5824	3.4465333	-3.793
0.5	6.5526333	6.5482333	-0.067
1.0	13.3377	12.7859	-4.137
2.0	25.8657	25.3195	-2.112
4.0	51.260633	50.746767	-1.003
8.0	102.305	100.95227	-1.322
16.0	204.28773	205.82187	+0.751

 

Linearity and Range:

The linearity of compounds was evaluated by the analysis of working standard solutions of rosiglitazone, amlodipine and glimepiride of seven different concentrations (0.25-16 µg/mL). Injections of all concentrations (0.25, 0.5, 1, 2, 4, 8 and 16 µg/mL) in triplicate were given and response was recorded for each drug. For peaks that are well resolved, both peak height and area are proportional to the concentration.²The peak area and concentration of each drug was subjected to regression analysis to calculate the calibration equations and correlation coefficients.

 

All the three drugs were found to be linear in the concentration range of 0.25-16 µg/mL. The coefficient of correlation (r²) for rosiglitazone maleate, amlodipine besylate and glimepiride were found to be 1.00, 0.9999 and 1.00, respectively. The linear equation obtained for rosiglitazone maleate, amlodipine besylate and glimepiride were found to be y = 20.249x + 1.3836, y = 18.031x + 0.7946 and 12.744x + 0.3675, respectively.

 

Fig 4 Standard curve of Rosiglitaone Maleate

 

Fig 5 Standard curve of Amlodipine Besylate

 

Fig 6 Standard curve of Glimepiride

Stability:

The stability of the drugs in solution was determined by plotting the standard curve of each drug and mixture on different days; and the results obtained on different days were compared with the results obtained on first day of analysis.In the research work, stability was determined for 14 days. The difference of ±20% in peak area of drug on 1^st day versus (v/s) peak area of drug at following days is often acceptable. The data of analysis for stability study revealed that the proposed method was stable for at least 14 days. (Table-4-6)

 

Accuracy and Precision:

Accuracy of the method was determined by comparing the results of quality control samples with the results of calibration standard curve. The accuracy was calculated as percentage bias and difference ± 2% is often recommended for estimation in bulk and ±20% for estimation in human plasma or biological fluids. The accuracy of the method was found to be in acceptable limit of less than ± 2.0 %. The precision of method was calculated as percentage relative standard deviation (% RSD); which is also known as percentage coefficient of variance (% CV). The % CV less than 5% is acceptable. (Table-7-9)

 

System precision for rosiglitazone maleate, amlodipine besylate and glimepiride was determined by comparing the standard deviation and percent coefficient of variance (%CV) of retention time, capacity factor, peak asymmetry and resolution of all three concentrations of QC samples of all the three drugs. (Table-10)

Table 4 Stability study data for Rosiglitazone Maleate up to fourteenth day

 

 

Table 5 Stability study data for Amlodipine Besylate up to fourteenth day

Conc.(µg/mL)	% area difference (1st day v/s 14th day)
0.25	-2.346
0.5	-1.683
1.0	-4.442
2.0	-1.958
4.0	-2.432
8.0	-0.708
16.0	-1.405

 

Table 6 Stability study data for Glimepiride up to fourteenth day

Conc. (µg/mL)	% area difference (1st day v/s 14th day)
0.25	-4.330
0.5	+0.367
1.0	-3.250
2.0	-2.475
4.0	-1.051
8.0	-0.473
16.0	-2.198

 

Table 7 The data of quality control analysis for Rosiglitazone Maleate

 

 

Table 8 The data of quality control analysis for Amlodipine Besylate

 

Table 9 The data of quality control analysis for Glimepiride

 

 

 

 

Table 10 System precision data for Rosiglitazone Maleate, Amlodipine Besylate and Glimepiride.

* = mean ± Standard deviation

 

Table 11 Intra-day and inter-day accuracy and precision data of ROS, AML and GLM

a - Mean ± standard deviation

b - Bias % = [(found-spiked)/spiked] x 100

 

LOD and LOQ:

The limit of detection (LOD) and limit of quantitation (LOQ) was calculated by the 3: 1 and 10: 1 ratio of the standard deviation of the peak area of lowest concentration (0.25 µg/mL): slope of the curve, respectively.⁵ The LOD and LOQ of rosiglitazone maleate, amlodipine besylate and glimepiride were determined and are given in table 5.16.

 

Table 12 LOD and LOQ of drugs

Drug	LOD (ng/mL)	LOQ (ng/mL)
Rosiglitazone Maleate	16.23	54.16
Amlodipine Besylate	19.88	66.28
Glimepiride	15.81	52.69

Fig 7 Chromatogram of Rosiglitazone Maleate in optimised conditions

 

Fig 8 Chromatogram of Amlodipine Besylate in optimised conditions

 

Fig 9 Chromatogram of Glimepiride in optimized conditions

 

Fig 10 Chromatogram of test sample containing Rosiglitazone Maleate, Amlodipine Besylate and Glimepiride in tablets

Analysis of Tablets:

Tablets were taken for testing and the aliquots were made as described in section 4.3.5. The analysis of test sample was determined at three concentration levels (high, medium and low). The triplicate injections of each concentration solution were given and average of peak area was determined. The concentration (µg/mL) of test sample was determined with the help of linear equation obtained from the calibration curve. Further, the percentage bias was calculated using the following formula:

                     Observed concentration – Claimed concentration

% bias = ----------------------------------------------------------------- X 100

                                        Claimed concentration

 

The concentration claimed for rosiglitazone maleate, amlodipine and glimepiride is 2, 2.5 and 1 mg per tablet, respectively.

 

Table 13. Estimation of three drugs in mixture of tablet

Drug	Quantity claimed (mg/tab)	Quantity found (mg/tab)	% quantity found (±SD)
Rosiglitazone Maleate	2.0	1.98	99.0 (±0.022)
Amlodipine Besylate	2.5	2.51	100.4 (±0.047)
Glimepiride	1.0	1.01	101.0 (±0.026)

SD = Standard deviation

 

RESULTS AND DISCUSSION:

The proposed method was rapid, because it takes shorter analysis time, in which the retention time of rosiglitazone maleate, amlodipine besylate and glimepiride was 2.62, 3.91 and 7.383 min., respectively. The specific study revealed the absence of any other compound in the area of interest. Also, there was no extraneous peak present and eluted at the retention time of rosiglitazone maleate, amlodipine besylate and glimepiride when the tablet excipients and blank samples were analysed. The proposed method was reproducible because results obtained with in inter-day and intra-day were in acceptable limit. The linearity was observed by linear regression equation method for rosiglitazone maleate, amlodipine besylate and glimepiride in the concentration range of 0.25, 0.5, 1, 2, 4, 8 and 16 µg/mL and the coefficient correlation (r²) were found to be 1.000, 0.9999 and 1.000, respectively. The method did not have interaction between the drugs in the solution of mixture containing rosiglitazone maleate, amlodipine and glimepiride. The difference between peak area of particular drug in single and in combination was within the acceptable limit.

 

The stability of solutions of rosiglitazone maleate, amlodipine and glimepiride was determined at intervals of 1st, 3rd, 7th and 14th day and was found to be stable up to at least 14 days. The limit of detection (LOD) for rosiglitazone maleate, amlodipine besylate and glimepiride was found to be 16.23, 19.88 and 15.81 ng/mL, respectively. The limit of quantitation (LOQ) for rosiglitazone maleate, amlodipine besylate and glimepiride were found to be 54.16, 66.28 and 52.69 ng/mL, respectively. The value of analysis of tablets obtained by the proposed method were between 99.0-101.0%, which showed that the estimation of dosage forms were accurate within the acceptance level of 95-105%.

 

CONCLUSION:

From the results, it can be concluded that the method has been successfully applied for the analysis of marketed tablets and can be used for the routine analysis of formulations containing any one of the selected drugs or their combinations without any alteration in the assay. The main advantage of the method is the common chromatographic conditions adopted for all formulations. Since the method was successfully applied for the estimation of selected drugs in bulk as well; therefore this method can also be adopted for the study of pharmaceutical release patterns of the drugs while designing the new dosage forms. The proposed method reduces the time required for switch over of chromatographic conditions, equilibration of column and post column flushing that are typically associated when different formulations are analysed by different chromatographic conditions. The simplicity, selectivity, rapidity, reproducibility and economy of the proposed method completely fulfill the objective of the research.

 

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Accepted on 31.05.2017          © RJPT All right reserved