Simultaneous  Estimation ,Validation  and Force  Degradation  Study  of Metformin Hydrochloride  and  Empagliflozin by RP-HPLC Method

 

Nachiket  S. Dighe* , Ganesh S. Shinde, Jyoti. J. Vikhe

Department of Pharmaceutical Chemistry ,Pravara Rural College of  Pharmacy, Pravaranagar MS

 

Abstract:

The Reverse Phase High performance liquid chromatography Method is one of the most sophisticated tool of the analysis. The RP-HPLC method was developed for the simultaneous estimation  of metformin and empagliflozinin bulk and Pharmaceutical dosage form. The Potassium Dihydrogenphosphate buffer was pH 3.0 and the mobile phase was optimized with consists of Methanol: Potassium Dihydrogenphosphate buffer(PH3) mixed in the ratio of 60:30 % v/v. Cosmosil C₁₈ Column (250mm x 4.6mm, Particle Size: 5micron) was used as stationary phase and retention time of Metformin HCL and Empagliflozin was found to be 5.2 min and 6.5 min respectively. The detection was carried out using UV-3000-M detector at 227nm. The solutions were chromatographed at a constant flow rate of 0.8 ml/min. the linearity range of metformin and empagliflozin were found to be from 40-200 µg/ml of metformin and 1-5µg/ml of empagliflozin. corelation coefficient value(R2)were found to be0.9991 and0.9988 for Metformin HCL and Empagliflozin . The % Recovery was found  to be 100.04% and 100.06% for Metformin HCL and Empagliflozin  respectively.  The relative standard deviation for intra day and Intra day was found to be less than 2%. The rubustness was found to be satisfactory within the range . Limit of detection was found to be 0.247µg/ml for Metformin HCL and 0.051µg/ml for empagliflozin and Limit of quantitation was found to be 0.751µg/ml for Metformin HCL and 0.157µg/ml for empagliflozin. The degradation of drug was determined under acidic , alkaline, peroxide ,photolytic and  thermal conditions .The results obtained on the validation parameters met ICH  requirements .It inferred the method found to be simple, accurate, precise and linear hence it can be employed for routine laboratory analysis with high degree of accuracy and precision.

 

 

INTRODUCTION:

Diabetes and its most abnormalities constitute a major health problem in the modern society.[1] Diabetes is a chronic , metabolic disease characterized by elevated level of blood  glucose. The most common is type 2 diabetes , usually in adults, which occurs when the body became resistance to insulin or doesn’t make enough insulin.[2]

 

Metformin (MET) is chemically named as 4 1- carbamimidamido-N, N-dimethylmethanimidamide . Its molecular formula is C₄H₁₁N5 and its molecular weight is 129.16364 g/mol. it is as shown in Fig. 1.[3] It is used as a biguanide antihyperglycemic agent used for treating non-insulin dependent diabetes mellitus (NIDDM) . It improves glycemic control by decreasing hepatic glucose production, decreasing glucose absorption, and increasing insulin-mediated glucose uptake. MET might induce weight loss and is that  the drug of alternative for obese NIDDM patients. Use of MET is associated with modest weight loss.[4]

 

 

 

 

Fig.1.Structure of Metformin HCl

 

Empagliflozin (EMPA) is chemically named as 1-chloro-4-[b-Dglucopyranos- 1-yl]-2-[4-([S]-tetrahydrofuran–3–yl-oxy) benzyl]-benzene Fig. 2. It has a molecular formula of C₂₃H₂₇ClO₇ and molecular weight is 450.912 g/mol.[5]Empagliflozin is an orally administered selective sodium glucose cotransporter-2 (SGLT-2) inhibitor, which lowers blood glucose in people with type 2 diabetes by blocking the reabsorption of glucose within the  kidneys and promoting the excretion of excess glucose in the urine [6]. The sodium glucose cotransporter 2 (SGLT2), located in the proximal tubule of the nephron, is estimated to facilitate-90% of this reabsorption [7]. It is a potent and selective competitive inhibitor of the SGLT2 protein. Sodium-glucose co-transporter 2 (SGLT2) inhibitors offer an insulin-independent mechanism for improving blood glucose levels since they promote urinary glucose excretion (UGE) by inhibiting glucose reabsorption in the kidney. In addition to glucose control, SGLT2 inhibitors are associated with weight loss and blood pressure reductions and do not increase the risk of hypoglycemia [8].

                                                                                                                                                                                              

 

Fig.2. Structure of Empagliflozin

 

 

According to the literature survey , few analytical methods are reported for analysis of  the drug individually as well as in combination with other drugs by HPLC[1-3],UPLC[8] ,UV,[9] LC/MS/MS [10]technique in pharmaceutical  dosage form. The aim of proposed study is to develop and validate analytical method for two antidiabetic drugs. Metformin HCl and Empagliflozin in pharmaceutical dosage form as per the ICH guidelines.[11]

 

MATERIAL AND METHOD:

Materials and Reagents

Table 1: Procurement of working standards

Sr. No.	Name of Drug Sample	Drug Manufacturer/Supplier
1	Metformin Hydrochloride	Gift sample obtained from a division of Aurobindo Pharma Limited, Hyderabad.
2	Empagliflozin
3	Empagliflozin+ Metformin hydrochloride (25mg/ 1000mg)	Obtained from local Pharmacy.

 

Table 2:List of Reagents and Chemicals

Sr .No.	Name	Supplied by	Specification
1	Water	In house production	HPLC Grade
2	Methanol	Merck	HPLC Grade
3	Potassium dihydrogenphosphate buffer	Merck	HPLC Grade
4	0.1N HCl	Merck	HPLC Grade
5	0.1 N NaoH	Merck	HPLC Grade
6	3% H2O2	Merck	HPLC Grade

Instruments Used

Table 3: List of apparatus /instruments used

Sr. No.	Name	Model	Manufacturer /Supplier
1	Weighing balance	PGB 100	Wenser High Precision Balance
		Max :100gm
		Min:0.001gm
2	Sonicator	WUC-4L	Wenser Ultra sonicator
		Capacity:4Liter
3	Digital PH meter	PICO+	Lab India pvt.ltd.
4	Magnetic  stirrer		Remi Equipment
5	HPLC	HPLC 3000 series	Analytical technologies

 

Table 4: HPLC Instrumental Information

Parts of Instrument	Instrument Information
System	HPLC Binary Gridient System
Model No	HPLC 3000 series
Company	Analytical Technology ltd.
Detector	UV -3000-M
Pump	P-3000-M Reciprocating (40MPa)
Column	Cosmosil C18 column (250mm x4.6mm,5µ)
Software	HPLC Workstation

 

Preparation of standard solution

Accurately weighed quantity of about 10mg of pure metformin  hydrochloride and empagliflozin working standards transferred into a 10ml clean dry volumetric flask respectively. Add diluent ,sonicated to dissolve it completely and make up the final volume with diluent(mobile phase)and filtered .from the above stock solution further five working standard solution of concentration covering the range 40-200ppm metformin hydrochloride and 1-5ppm empagliflozin  respectively .were prepared by transferring and diluting into a 10ml volumetric flask and then make upto the final volume with the same diluent (mobile phase).

 

Preparation of sample  solution

weight the approximately 10mg tablet powder of metformin HCL and empagliflozin, transferred to 10ml volumetric flask, sufficient amount of mobile phase was added and dissolved it by 20 minutes ultrasonication. Then made the volume upto the 10ml mark with the same solvent mixture to famish stock solutions contain 1000µg/ml of metformin and empagliflozin filtered with 0.45µ filter paper. From this solution appropriate dilutions of metformin HCL and empagliflozin were made to get the final concentrations. A 20µl sample was injected  under optimized chromatographic conditions and peak area  were recorded.

 

Preparation of mobile phase

Mobile phase was prepared by a mixture of 60 volumes of methanol and 30 volumes of 10mMpotassium dihydrogenphosphate buffer PH3(60:30). The mobile phase was sonicated for 10 minutes to remove gases and then filtered through 0.45µ membrane filter under vacuum filtration.

 

Selection of Analytical Wavelength

The appropriate dilution of standard stock solution of drug was prepared .The solution containing UV spectrum of 10µg/ml of metformin hydrochloride and empagliflozin. For RP-analysis , these diluted solutions were scanned in range 200-400nm. The both drugs showed considerable absorbance at 227nm so it was selected as determination wavelength.

 

Fig. No. 3 Wavelength of Metformin hydrochloride and Empagliflozin

 

HPLC  Instrumentation and Chromatographic Conditions

The analysis was performed on RP-HPLC system consists of HPLC Binary Gradient System with UV-3000-M detector equipped with a solvent deliver pump P3000-MReciprocating (40MPa), thermo scientific injector rheodyne injector (20µl capacity) and syringe Hamilton (25µl).The separation was achieved on Cosmosil C₁₈ (250mm x 4.6mm,5µ)column. The data acquisition and analysis was performed by using HPLC workstation software. The column was maintained at room temperature and the eluent was monitored at 227nm. The flow rate was keep at o.8ml/min . The mixture of methanol and potassium dihydrogenphosphate buffer PH3 in portion of 60:30%v/v was used as mobile phase .mobile phase was filtered through 0.45µm membrane filter  and sonicated before used . The injection volume was 20µl.The run time was taken as 9.74 min. All determines are carried out at ambient temperature.

 

Optimized chromatographic condition

In the proposed study the separation of metformin hydrochloride and empagliflozin was achieved by using Cosmosil C₁₈ (250mm x 4.6mm,5µ)column with mobile phase consisting of  mixture of methanol and potassium dihydrogenphosphate buffer PH3 in portion of 60:30%v/v at flow rate o.8ml/min with UV detection 227nm at ambient temperature. The retention time for metformin hydrochloride and empagliflozin were found to be  5.275 and 6.543 respectively.(fig no.4)

 

 

Fig no.4 Retention time of Metformin hydrochloride

 

 

Time	Area	Resolution	T. Plate. No	Asymmetry
6.543	1288657	0.00	8375	1.34s

Fig no. 5 Retention time of Empagliflozin

 

 

 

Time	Conc	Area	Resolution	T.Plate.No	Asymmetry
5.295	40	1650279	7.26	9505	1.05
6.621	1	86044	0.00	9860	1.06

Fig.no 6 Chromatogram of standard solution of Metformin HCL and Empagliflozin

 

RESULTS AND DISCUSSION:

Method Validation

Roubustness, Ruggedness, Limit of detection ,limit of quantification parameters as described in ICH guidelines.

 

System suitability

It is the checking of a system to ensure system performance before or during the analysis of unknown. System suitability and chromatographic parameters were validated such a resolution, theoretical plates ,and the tailing factor was calculated. The results are given in table 5.

 

Table 5:System suitability parameter for Metformin hydrochloride and Empagliflozin

System suitability parameter	Metformin hydrochloride	Empagliflozin
Retention time	5.295	6.621
Theoretical plate no.	9505	9860
Tailing factor	1.05	1.06
Resolution	7.26	0.00

 

Linearity

Linearity of the method was studied by injecting five standard solutions

With the concentration in the range 40-200µg/ml of metformin hydrochloride and 1-5µg/ml of empagliflozin. The peak areas were plotted against the corresponding concentration curves. Correlation coefficient value (R2) were found to be 0.9991 and 0.9988 for metformin hydrochloride and empagliflozin. Results were shown in table no.6

 

Table No.6 Data of linearity for Metformin  HCl and Empagliflozin

Conc. of Metformin HCl (µg/ml)	Area	Conc. Of Empagliflozin (µg/ml)	Area
40	1650279	1	86044
80	3053549	2	261448
120	4804020	3	442984
160	6369390	4	647232
200	7907324	5	849178

 

 

 

Fig.No.7 Linearity graph for Metformin HCl

 

 

Fig. No. 8  Linearity graph for Empagliflozin

 

 

Accuracy

Accuracy was determined by calculating recovery of metformin hydrochloride  and empagliflozin by the standard addition  method .Three different levels (50%,100%,150%) of standards were added to formulation containing metformin hydrochloride and empagliflozin . Each solution was injected in triplicate and the recovery was calculated by measuring peak area. The % recovery was found to be 100.4% and 100.06% for metformin hydrochloride and empagliflozin respectively. Results obtained are shown in table no.7 and 8.

 

 

Table.No.7 Accuracy data for Metformin HCl

Conc(%)	Sample amount (ppm)	Amount added(ppm)	Amount recovered (ppm)	% Recovery	% Mean  recovery
50%	80	40	120.16	100.13	100.o6
	80	40	119.94	99.95
	80	40	120.12	100.10
100%	80	80	159.88	99.92	100.01
	80	80	160.20	100.12
	80	80	160	100
150%	80	120	200.29	100.14	100.06
	80	120	199.94	99.97
	80	120	200.15	100.07

 

 

Table NO.8. Accuracy data for Empagliflozin

Conc(%)	Sample amount (ppm)	Amount added(ppm)	Amount recovered (ppm)	% Recovery	% Mean  recovery
50%	2	1	3.00	100.04	99.85
	2	1	2.99	99.78
	2	1	2.99	99.75
100%	2	2	4.002	100.06	100.00
	2	2	3.99	99.95
	2	2	4.00	100
150%	2	3	5.00	100.14	100.33
	2	3	5.01	100.36
	2	3	5.02	100.49

 

 

Precision

The precision of the method was interday and intraday variations

 

Inter-day precision

Interday precision was determined by analyzing day to day variability was assessed using the three different concentrations(40,80,120µg/ml) of metformin hydrochloride,(1,2,3µg/ml)of empagliflozin respectively, analysed on three different days over period of one week. The relative standard deviation for interday was found to be less than 2%.The %RSD of interday was 0.15%for metformin hydrochloride and 0.47 % for empagliflozin .The result shown in table no.9

 

 

Table NO.9 Inter day  variability for metformin HCl and Empagliflozin

Metformin HCL		Empagliflozin
Day1	Area	Day1	Area
	4804020		442984
	4801736		442047
	4809025		441919
Day2	4790180	Day2	444787
	4794858		440195
	4805694		438895
Mean	4805694	Mean	438895
%RSD	0.15%	%RSD	0.47%

 

 

 

Intra -day precision

The intra day precision was determined by analyzing , three different concentration (40,80,120µg/ml) of metformin hydrochloride ,(1,2,3µg/ml) empagliflozin respectively, for three times in the same day. The relative standard deviation for intra day was found to be less than 2%. The % RSD of intra day was 0.11% for metformin hydrochloride and 0.33% for empagliflozin. The result shown in table no. 10

 

 

 

Table NO.10 Intra day variability for Metformin HCl and Empagliflozin

Metformin HCl		Empagliflozin
Morning	Area	Morning	Area
	4804020		442984
	4801736		442047
	4809025		441919
Evening	4796404	Evening	446517
	4806508		443153
	4810061		444669
Mean	4804626	Mean	443398
%RSD	0.11%	%RSD	0.33%

 

Robustness

Robustness test is a measure of the capacity of the chromatographic method to remain unaffected by small deliberated variations in procedural parameters. The robustness was investigated under a variety  of conditions including changes  of flow rate, change in wavelength .This deliberate change in the method has no affect on the peak tailing, peak area and theoretical plate and finally the method was found to be satisfactory within the range. % RSD of change  in flow rate was found 0.81136 for metformin hydrochloride and 0.72708 for eampagliflozin .% RSD of change in wavelength was found 0.73342 for metformin hydrochloride  and 0.5492 for empagliflozin. The result shown in table no.11 and 12

 

Table NO. 11 Robustness data for Metformin HCL and Empagliflozin            (At different Flow Rate)

Drug sample	Flow rate (ml/min)	Area	Mean	SD	% RSD
Metformin HCL	0.8	3053549	3041881	24680.7	0.81136
	0.7	3013530
	0.9	3058565
Empagliflozin	0.8	261448	261343	1900.19	0.72708
	0.7	259392
	0.9	263188

 

Table No. 12 (At different wavelength)

Drug sample	Wavelength (nm)	Area	Mean	SD	% RSD
Metformin HCL	227	3053549	3039951	22295.5	0.73342
	229	3052083
	225	3014220
Empagliflozin	227	261448	260675	1431.66	0.5492
	229	261554
	225	259023

 

Ruggedness

The measurement of ruggedness was evaluated by analyzing different concentration of the standard solution of  metformin hydrochloride  and empagliflozin  .concentration range 40-200µg /ml and 1-5µg/ml for metformin hydrochloride and empagliflozin .The regression coefficient was found to be 0.999 of metformin hydrochloride and 0.998 of empagliflozin  .The equation was found to be y=39568x+17980(MET) and y=19167x-11652(EMPA) as shown in Fig 6

 

Table No. 13 Ruggedness data for Metformin HCL and Empagliflozin

Conc. of Metformin HCl (µg/ml)	Area	Conc. of Empagliflozin (µg/ml)	Area
40	1656592	1	86149
80	3072209	2	262826
120	4806770	3	444171
160	6377570	4	646909
200	7917487	5	852491

 

 

Fig. No. 9 Ruggedness graph for Metformin HCl

 

 

 

Fig .No 10 Ruggedness graph for empagliflozin

 

Limit of Detection (LOD) and Limit of Quantitation(LOQ)

LOD is the lowest concentration of an analyte in a sample that can be detected. Limit of detection was found to be 0.247µg/ml for Metformin HCL and 0.051µg/ml for empagliflozin .

LOQ is the lowest concentration of an analyte in a sample that can be quantitized. Limit of quantitation was found to be 0.751µg/ml for Metformin HCL and 0.157µg/ml for empagliflozin.

 

Table no.14 . LOD and LOQ data for Metformin hydrochloride and Empagliflozin

Sr. No.	Drug	LOD(µg/ml)	LOQ(µg/ml)
1	Metformin Hydrochloride	0.247	0.051
2	Empagliflozin	0.751	0.0157

                                                                                                                              

Assay

The assay performed by the marketed product (25mg/1000mg of EMPA &MET). The prepared sample and standard solution were injected  into HPLC and peak areas were recorded .finally percentage assay of drug was calculated concentration and purity was determined from linearity equation. The result was found to be Metformin HCL 100.108% &empagliflozin 100.192%.as shown in table no.15

 

 

 

Time	Conc	Area	Resolution	T. Plate. No	Asymmetry
5.243	120	4809206	6.29	9538	1.21
6.594	3	443836	0.00	9238	1.07

Fig .no.11 Chromatogram for tablet sample solution MET HCl and EMPA

 

Table No.15  Data for assay of sample of metformin hydrochloride and empagliflozin

Drug Name	Composition in ppm	Area of Std.	Area of Sample	% Assay
Metformin HCl	120	4804020	4809206	100.108%
Empagliflozin	3	442984	443836	100.192%

 

Force degradation studies

Force degradation is the process whereby the natural degradation rate of a product is increased by the application of an additional stress. It shows the chemical behavior of the molecule which in turn helps in the development of formulation. Force degradation studies of drug ,in combination, were performed under different stress condition[ as  mentioned in ICH guideline Q1A(R2)] like acid, alkali, effect by oxidation, photolytic, thermal degradation.

 

Acid Degradation

Acid degradation studies were performed by 1 ml of stock solution transferred into 10 ml of volumetric flask. 1 ml of 0.1N HCL was added and refluxed it for 1 h at 60⁰C in round bottom flask. After time period it was cooled at room temperature. Then the resultant solution was diluted to obtained 200µg/ml for metformin HCl and 5µg/ml for empagliflozin  Then 20µg/ml solution were injected into the system and the chromatogram of acid degradation were recorded. As shown in Fig.no.12

 

 

Time	Conc	Area	Resolution	T. Plate. No	Asymmetry
5.269	200	7586030	3.65	7957	1.05
6.622	5	760325	0.00	7776	1.03

Fig no.12 Acid degradation of MET HCl  and EMPA

Base degradation

Base degradation studies were performed by 1 ml of stock solution transferred into 10 ml of volumetric  flask. 1 ml of 0.1N NaOH was added and  refluxed it for 1 h at 60⁰C in round bottom flask. After time period it was cooled at room temperature. Then the resultant solution was  diluted to obtained 200µg/ml for metformin HCl and 5µg/ml for empagliflozin  Then 20µg/ml solution were injected into the system and the chromatogram of acid degradation were recorded. As shown in Fig.no.13.

 

 

Time	Conc	Area	Resolution	T. Plate. No	Asymmetry
5.323	200	7662447	3.40	9017	1.05
6.668	5	776641	0.00	8114	1.03

Fig no.13 Base degradation of MET HCl  and EMPA

 

Oxidative degradation

Oxidative  degradation studies were performed by 1 ml of stock solution transferred into 10 ml of volumetric  flask. 1 ml of 3%H2O2 solution was added and mixed well and put for 24 h at room temperature  in round bottom flask. After time period it was cooled at room temperature. Then the resultant solution was  diluted to obtained 200µg/ml for metformin HCl and 5µg/ml for empagliflozin  Then 20µg/ml solution were injected into the system and the chromatogram of acid degradation were recorded. As shown in Fig.no.14

 

Time	Conc	Area	Resolution	T. Plate. No	Asymmetry
5.307	200	7826694	6.12	9532	1.04
6.656	5	840113	0.00	9722	1.02

Fig no.14 Oxidative degradation of MET HCl  and EMPA

 

Photolytic degradation

Photolytic degradation studies were performed by 1 ml of stock solution transferred into 10 ml of volumetric  flask. The volumetric flask was kept in room temperature for24 h .Then the resultant solution was  diluted to obtained 200µg/ml for metformin HCL and 5µg/ml for empagliflozin  Then 20µg/ml solution were injected into the system and the chromatogram of acid degradation were recorded. As shown in Fig.no.15

 

 

 

Time	Conc	Area	Resolution	T. Plate. No	Asymmetry
5.160	200	7898336	4.16	6425	1.18
6.343	5	847842	0.00	7051	1.12

Fig .No. 15 Photolytic degradation of Metformin HCl and Empagliflozin

 

Thermal degradation

Thermal degradation studies were performed by 1 ml of stock solution transferred into 10 ml of volumetric  flask. The volumetric flask was kept in room temperature for24 h .Then the resultant solution was  diluted to obtained 200µg/ml for metformin HCl and 5µg/ml for empagliflozin  Then 20µg/ml solution were injected into the system and the chromatogram of acid degradation were recorded. As shown in Fig.no.16

 

Time	Conc	Area	Resolution	T. Plate. No	Asymmetry
5.138	200	7840751	4.11	6378	1.19
6.299	5	819690	0.00	7117	1.12

Fig .No. 16 Thermal degradation of Metformin HCL and Empagliflozin

 

 

Table NO. 16 Degradation data for Metformin HCl

Degradation	Area of std.	Area of sample	Degraded up to%	Actual degradation
Acidic (0.1N HCl)	7907324	7586030	95.93	4.06
Alkaline (0.1 NaOH)	7907324	7662447	96.90	3.09
Oxidation (3%H2O2)	7907324	7826694	98.98	1.01
Photolytic	7907324	7898336	99.88	0.11
Thermal	7907324	7840751	99.15	0.84

 

Table No.17 Degradation data for Empagliflozin

Degradation	Area of std.	Area of sample	Degraded up to%	Actual degradation
Acidic (0.1N HCL)	849178	760325	89.53	10.46
Alkaline (0.1 NaOH)	849178	776641	91.45	8.54
Oxidation (3%H2O2)	849178	840113	98.93	1.06
Photolytic	849178	847842	99.84	0.15
Thermal	849178	819690	96.52	3.47

 

CONCLUSION:

The proposed simultaneous estimation, validation and force degradation method was found to be simple, precise, accurate and rapid  for the determination  of Metformin HCL and Empagliflozin. The coefficient of correlation was obtained in acceptable range. The percentage recovery obtained in acceptable range .variation in flow rate, wavelength, does not have any effect on the % RSD of standard and assay value. The relative standard deviation of main peak area, tailing factor and theoretical plate is well within the acceptable range. Hence the precision of given method is confirmed. Thus from the above result of the individual method is conclude that the analytical method is validated and found to be satisfactory.

 

ACKNOWLEDGMENT:

The authors express their gratitude to the Pravara Rural College of Pharmacy, Loni  and a division of Aurobindo Pharma Limited, Hyderabad for providing the gift sample of Metformin hydrochloride and Empagliflozin.

 

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Received on 05.05.2019       Modified on 08.05.2019 Accepted on 12.05.2019      ©A&V Publications All right reserved Research J. Science and Tech. 2019; 11(2):135-147. DOI: 10.5958/2349-2988.2019.00021.4