Method Development and Validation for Simultaneous Estimation of Glimepiride and Simvastatin by using Reversed Phase High-performance Liquid Chromatography

 

Narendra Kumar Pandey, Sachin Kumar Singh*, Dipanjoy Ghosh, Rubiya Khursheed,

Rajan Kumar, Bhupinder Kapoor, Bimlesh Kumar, Ankit Awasthi

School of Pharmaceutical Sciences, Lovely Professional University, Punjab-144411, India

 

ABSTRACT:

An analytical method was developed using reverse phase high performance liquid chromatograph (RP-HPLC) system and a C-18 reverse-phase column (Nucleodur C18, 250mm × 4.6mm i.d.,5µ) for simultaneous estimation of simvastatin and glimepiride. Acetonitrile and potassium dihydrogen phosphate buffer pH 4 (75:25, v/v) were used as mobile phase. The flow rate was 1 mL min⁻¹ and the chromatogram of both drugs was detected at wavelength of 232 nm. Method was validated as per ICH Q2 (R1) guidelines. The retention times of glimepiride and simvastatin was found to be 4.726 min and 9.829 min, respectively. Both drugs have shown linearity over the concentration range 2-10µg/mL with r² of 0.997 for GLM and 0.998 for SIM. The mean percentage recovery of both the drugs was found within 98-102% at all the levels which indicated that the method was accurate. The percentage relative standard deviation was found less than 2% which indicated that method was satisfactorily précised. The LOD and LOQ were found to be 0.24 and 0.73 for simvastatin and 0.32 and 0.96 for glimepiride. The method was found to be robust as there was no significant change in response with variation in pH, flow rate and mobile phase composition. It was concluded that the developed method has passed all the validation tests and can be successfully applied to estimate the presence of both the drugs in bulk as well as in pharmaceutical formulations.

 

 

 

INTRODUCTION:

Glimepiride (GLM) is chemically 3 ethyl-4-methyl-N- {2-[4({[(4-Methyl cyclohexyl) carbonyl] amino} sulfonyl) phenyl] ethyl}-2-oxo-2, 5- dihydro-1H –Pyrrole -1- carboxamide. GLM is an oral hypoglycemic agent, primarily lowers blood glucose by stimulating the release of insulin from pancreatic beta cells. Sulfonylureas bind to the sulfonylurea receptor in the pancreatic beta-cell plasma membrane, leading to closure of the ATP-sensitive potassium channel, thereby stimulating the release of insulin^1,2.

 

Chemically simvastatin (SIM) is (1S,3R,7S,8S,8aR)-8-{2-[(2R,4R)-4-hydroxy-6-oxooxan-2-yl]ethyl}-3,7-dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl2,2-dimethylbutanoate. SIM is a specific inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the enzyme that catalyzes the conversion of HMG-CoA to mevalonate, an early and rate limiting step in the biosynthetic pathway for cholesterol^3,4. Co-administration of two drugs in fixed dose is administered to maintain the normal level of glucose and lipid in blood plasma.

 

A simple and novel analytical method is required to quantify each drug present in dosage form during dissolution studies. Till date there is no such method to quantify both the drugs simultaneously in pharmaceutical formulations using RP-HPLC. In this work a simple and sensitive method was developed and validated to estimate these drugs.

 

 

MATERIALS AND METHODS:

Materials:

Simvastatin and glimepiride were procured from Yarrow Chem, Pvt. Ltd India. All other chemicals and reagents used were of analytical grade and HPLC grade solvents were employed for the study. Triple distilled water was used throughout the study.

 

Method Development for simultaneous estimation of glimepiride and simvastatin on RP-HPLC:

The HPLC system consisted of a mobile phase delivery pump (LC-20 AD; Shimadzu, Japan), a photodiode array detector (SPDM20A; Shimadzu, Japan), a 20µL loop (Rheodyne) and LC Solution software. A C-18 reverse-phase column (Nucleodur C18, 250 mm × 4.6 mm i.d.,5µ) was utilized for estimation and separation of simvastatin (SIM) and glimepiride (GLM) in SIM-GLM mixture, using acetonitrile and potassium dihydrogen phosphate buffer pH 5 (75:25, v/v) as mobile phase. The flow rate was 1 ml min⁻¹ and detection wavelength was 232 nm. Standard solutions (2, 4, 6, 8 and 10 µg/ml) were prepared in mobile phase and analysed. The developed method was validated as per ICH Q2 (R1) guidelines.

 

Method validation:

Preparation of quality control standards:

The quality control standards were prepared at three different levels i.e., lower quality control standards (LQC), Medium quality control standards (MQC) and Higher quality control standards (HQC) of calibration curve. Hence, 6µg/mL was kept as 100% (MQC) level and 80% of 6µg/ml (i.e., 4.8 µg/ml) as LQC and 120% of 6µg/ml (i.e.7.2 µg/ml) was kept as HQC levels. All the three concentrations were prepared in plasma as well as in mobile phase.

 

Linearity and range:

The calibration curve was developed by plotting the graph between mean peak area of five replicates versus corresponding concentrations of SIM and GLM, and the regression equation was recorded.

 

Accuracy:

The accuracy of method was developed through calculation of recovery of the drug from the quality control standard solutions prepared in mobile phase and plasma. The LQC, MQC and HQC standard solutions were injected 6 times to HPLC and its mean of response was recorded. Percentage recovery was calculated by dividing the actual recovery of drug to their theoretical concentration and multiplying them by hundred. The mean of response was recorded and percentage relative standard deviation was calculated

 

                             Actual concentration recovered

Percent recovery= ––––––––––––––––––––––––– X 100

                                  Theoretical concentration

Precision:

Precision of the method was evaluated in terms of repeatability and intermediate precision. Repeatability was tested by injecting six times the samples of LQC, MQC and HQC on the same day and under same experimental conditions. The intermediate precision was evaluated by determining LQC, MQC and HQC samples six times on each of three different days (inter-day) as well as by the three different analysts (inter-analyst) under the same experimental conditions. The mean of response was recorded and percentage relative standard deviation was calculated.

 

Robustness:

In order to check the effect of small changes on robustness of the developed method, the study was carried out by varying pH of the mobile phase (3.8, 4.0 and 4.2), flow rate (0.8, 1 and 1.2 ml/min) and ratio of mobile phase  phosphate buffer: methanol as  [73:27; 75:25, and 77:23], respectively. Six replicates of medium concentration (6µg/ml) were injected and their effect on area of the peak, recovery and retention time was observed and mean of response was recorded.

 

Estimation of LOD and LOQ:

LOD and LOQ were determined by standard deviation of response (sigma) and slope of calibration curve (S). Standard deviation of Y intercepts of regression line was used as standard deviation.

 

LOD = 3.3 σ/S

LOQ = 10 σ/S

 

RESULTS AND DISCUSSION:

Selection of mobile phase for simultaneous estimation of SIM and GLM: 

For the simultaneous estimation of glimepiride and simvastatin, different trials by changing the composition of mobile phase were tried such as acetonitrile- ortho-phosphoric acid, methanol-orthophosphoric acid, Acetonitrile-water, methanol-water, acetonitrile-ammonium acetate buffer and acetonitrile- potassium dihydrogen phosphate buffer by varying the ratio and pH of the mobile phase (Fig. 1 and Fig. 2). Out of these trials 75:25 ratio of acetonitrile: potassium dihydrogen phosphate buffer (pH 4) showed better result in term of resolution and separation between two peaks and sharpness of the peaks. Since, there is a significant difference in retention time of glimepiride (4.725 min) and simvastatin (9.940 min) peaks, 75:25 ratio is selected for validation (Fig. 3).

 

Precision:

The precision of developed method was evaluated by calculating the percentage relative standard deviation for the six determinations of the LQC, MQC and HQC solutions at interday, intraday and interanalyst level under the same experimental conditions.

 

SIM

GLM

Fig. 1: Chromatogram of mixture of SIM-GLM in ACN-ortho phosphoric acid

SIM

GLM

Fig. 2: Chromatogram of mixture of SIM-GLM in methanol- ortho phosphoric acid

SIM

GLM

Fig. 3: Optimized chromatogram of SIM-GLM in ACN: KH₂PO₄ (75:25)

 

Fig. 4: Calibration curve of GLM

Fig. 5: Calibration curve of SIM

 

Table 1: Results of accuracy studies

Levels	Concentration of standard solution (μg/ml)	Concentration of sample Solution (μg/ml)	Total concentration of Solution (actual) (μg/ml)	Concentration of drug recovery from mobile phase (μg/ml) *(N=5)	Recovery (%)	Mean Recovery (%)
GLM
LQC	4.8	6	10.8	10.4±1.24	96.3	98.70
MQC	6	6	12.0	11.8±1.68	98.3
HQC	7.2	6	13.2	13.4±1.20	101.5
SIM
LQC	4.8	6	10.8	10.5±1.31	97.2	97.93
MQC	6	6	12.0	11.7±1.53	97.5
HQC	7.2	6	13.2	13.1±1.50	99.1

 

 

The observed percentage relative deviation was less than 2% for all the samples (Table 3 and Table 4). This clearly indicated that the developed method was satisfactorily précised.

 

Robustness:

Robustness of developed method was studied by varying pH of the mobile phase (3.8, 4.0 and 4.2), flow rate (0.8, 1 and 1.2 ml/min) and ratio of mobile phase (Acetonitrile: Ammonium acetate buffer pH 4.0) (73:27; 75:25, and 77:23), respectively. The observed percentage relative deviation was found less than 2% for all the samples (Table 5), indicating that the developed method was satisfactorily robust and the responses were unaffected by these changes.

 

Table 2: Results of precision studies for GLM

Parameters	Level	Concentra-tion (μg/ml)	Analytical responses (area), injections						Mean (*N=6)	SD	%RSD
			1	2	3	4	5	6
Repeatability (intraday precision)
	LQC	4.8	234538	239931	239490	240068	237295	240992	238719	2389.906	1.001138
	MQC	6	333709	334581	339807	330986	329153	325986	332370.3	4796.049	1.442983
	HQC	7.2	761009	757841	752328	754292	763383	762742	758599.2	4567.482	0.602094
Intermediate precision (interday)
Day 1	LQC	4.8	235633	240931	239360	238068	242305	243952	240041.5	3000.022	1.249793
	MQC	6	332519	335661	342107	331725	327738	326916	332777.7	5590.751	1.680026
	HQC	7.2	753849	753141	762810	751213	775361	763500	759979	9145.445	1.203381
Day 2	LQC	4.8	239177	240064	234717	239214	242260	248500	240655.3	4560.485	1.895028
	MQC	6	328849	329007	330295	329371	327389	321194	327684.2	3315.96	1.011938
	HQC	7.2	744824	753977	750359	765157	747446	750869	752105.3	7115.54	0.946083
Day 3	LQC	4.8	291262	288824	291628	295900	291569	300090	293212.2	4069.343	1.387849
	MQC	6	353207	357967	349249	345520	357674	357445	353510.3	5191.84	1.468653
	HQC	7.2	770311	770493	771935	778614	777642	775851	774141	3688.687	0.476488
Intermediate precision (inter analyst)
Analyst 1	LQC	4.8	234538	229931	229490	230168	233273	232718	231686.3	2094.004	0.90381
	MQC	6	343709	334581	330837	340676	339053	328985	336306.8	5795.342	1.723231
	HQC	7.2	764319	759337	760028	754567	759889	762139	760046.5	3261.48	0.429116
Analyst 2	LQC	4.8	239639	241176	239984	236853	243264	248516	241572	3994.146	1.653398
	MQC	6	338857	341377	338486	330375	333578	341174	337307.8	4410.651	1.307604
	HQC	7.2	754824	750367	749359	761467	754346	761735	755349.7	5295.148	0.701019
Analyst 3	LQC	4.8	250132	245360	239672	242425	238793	241163	242924.2	4216.163	1.735588
	MQC	6	351432	356134	347892	352881	351984	347644	351327.8	3204.015	0.911973
	HQC	7.2	749356	761189	760043	752346	756289	755436	755776.5	4491.501	0.59429

 

Table 3: Results of precision studies for SIM

Parameters	Level	Concentra-tion (μg/ml)	Analytical responses (area), injections						Mean (*N=6)	SD	%RSD
			1	2	3	4	5	6
Repeatability (intraday precision)
	LQC	4.8	245674	250324	245692	246231	251089	243567	247096.2	2951.589	1.19451
	MQC	6	363892	358799	359933	362236	360899	361234	361165.5	1776.224	0.491803
	HQC	7.2	439872	442234	441976	442108	440034	437994	440703	1697.959	0.385284
Intermediate precision (interday)
Day 1	LQC	4.8	257476	264007	260530	258136	255188	263039	259729.3	3000.022	1.249793
	MQC	6	361727	357938	365216	352269	353268	352254	357112	5590.751	1.680026
	HQC	7.2	446674	447270	457717	450060	462948	447006	451945.8	9145.445	1.203381
Day 2	LQC	4.8	257476	264007	260530	258136	255188	263039	259729.3	3000.022	1.249793
	MQC	6	361727	357938	365216	352269	353268	352254	357112	5590.751	1.680026
	HQC	7.2	446674	447270	457717	450060	462948	447006	451945.8	9145.445	1.203381
Day 3	LQC	4.8	323501	323849	323755	326302	321871	333086	325394	4069.343	1.387849
	MQC	6	382686	383741	377071	373265	387233	384927	381487.2	5191.84	1.468653
	HQC	7.2	470126	463550	471085	474865	473349	469357	470388.7	3688.687	0.476488
Intermediate precision (inter analyst)
Analyst 1	LQC	4.8	248964	250453	241456	249777	248788	251345	248463.8	3562.227	1.4337
	MQC	6	362887	353349	359902	363312	357998	360785	359705.5	3678.406	1.022616
	HQC	7.2	451290	449936	447755	452031	451132	447451	449932.5	1927.935	0.428494
Analyst 2	LQC	4.8	239978	248779	249933	250155	248873	251332	248175	4123.601	1.66157
	MQC	6	356334	357835	348977	360021	348886	351443	353916	4780.934	1.350867
	HQC	7.2	471133	462759	458871	453347	458873	458599	460597	5969.712	1.296081
Analyst 3	LQC	4.8	251764	247789	249977	246733	250342	251138	249623.8	1961.481	0.785775
	MQC	6	348872	357745	352246	353351	349983	348892	351848.2	3413.642	0.970203
	HQC	7.2	380031	374433	368897	367745	364338	367339	370463.8	5734.499	1.547924

 

 

Table 4: Robustness results of various parameters tested for GLM

 

Table 5: Robustness results of various parameters tested for SIM

 

Linearity and Range:

The calibration curve was developed by plotting the graph between concentration and mean area. For in-vitro studies, the calibration curve was prepared in mobile phase (as mentioned in section). The curves were found linear in the range of 2-10µg/ml with a correlation co-efficient (r²) of 0.997 for GLM (Fig.4) and 0.998 for SIM (Fig.5).

 

Accuracy:

The accuracy of the proposed method was accessed by determining the mean percentage recovery of the LOQ, MQC and HQC solutions in mobile phase. The data revealed that for all the three levels, the mean percentage recovery in mobile phase was within the fixed limits of 98-102% (Table 1). The accuracy of developed method was verified by percentage relative standard deviation which was <2%.

 

CONCLUSION:

In the present study simultaneous estimation of glimepiride and simvastatin was carried out using RP-HPLC method. The reports of validation studies reported that the method was accurate, precised, rugged and robust. This method can be successfully applied to estimate the presence glimepiride and simvastatin in various pharmaceutical formulations.

 

CONFLICT OF INTEREST:

No.

 

REFERENCES:

1.      Alakhali, K, Hassan, Y, Mohamed,  N, Mordi, MN. Pharmacokinetic of simvastatin study in Malaysian subjects. IOSR Journal of  Pharmacy 2013; 3:46-51.

2.      Yadav, SK, Mishra, S, Mishra, B. Eudragit-based nanosuspension of poorly water-soluble drug: formulation and in vitro–in vivo evaluation. AAPS PharmSciTech 2012; 13: 1031-44.

3.      Galani, VJ, Vyas, M. In vivo and in vitro drug interactions study of glimepride with atorvastatin and rosuvastatin. J Young Pharm 2010; 2: 196-200.

4.      Patrizia, G, Maria, CP, Giuseppina, G, Anna, MM, Elena, C, Simona, P, Antonietta, S, Chiara, L, and Maurizio, B. Pharmacological Actions of Statins: A Critical Appraisal in the Management of Cancer. Pharmacological Reviews 2012; 64: 106-46.

 

 

 

Received on 16.07.2019         Modified on 19.08.2019

Accepted on 30.09.2019         © RJPT All right reserved