INTRODUCTION:

Metformin hydrochloride (MET)

It is chemically known as (3-(diamino methylidene)-1, 1-dimethylguanidine; hydrochloride (shown in figure1). It has a molecular formula of C₄H₁₂ClN₅ and a molecular weight of 165.62g./mol. It is an oral antihyperglycemic agent (type 2 diabetes) that belongs to a class of biguanides and is useful for treating non-insulin-dependent diabetes mellitus¹. It is official in European Pharmacopoeia², United States Pharmacopoeia³ and Indian Pharmacopoeia⁴.

Remogliflozin etabonate (REM):

It is chemically known as ethyl [(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-[5-methyl-1-propan-2-yl-4-[(4-propan-2-yloxyphenyl) methyl] pyrazol-3-yl] oxyoxan-2-yl] methyl carbonate (shown in figure 2).

It has a molecular formula of C₂₆H₃₈N₂O₉ and a molecular weight of 522.6g/mol. It belongs to sodium-glucose co-transporter-2 inhibitors class⁵.

Figure 1: Chemical structure of Metformin hydrochloride (MET)

Figure 2: Chemical structure of Remogliflozin etabonate (REM)

The literature survey reveals that few analytical methods were reported like RP-HPLC methods, Spectrophotometric methods, UFLC, UPLC, HPTLC, Ion pair and LC/MS/MS in single or in combination with other drugs in bulk and pharmaceutical dosage forms^6-30. However, there was only one stability-indicating method reported for these drugs' combination in bulk and pharmaceutical dosage form and hence the present study aimed to develop a new better stability-indicating reverse phase liquid chromatographic method for the simultaneous determination of MET and REM in bulk and combined tablet dosage forms suitable for routine quality control analysis.

MATERIALS AND METHODS:

Materials:

Gift samples were obtained from Elikem Private Limited, Ahmedabad and Glenmark Pharmaceuticals Limited for MET and REM respectively. HPLC-grade water, methanol and acetonitrile were purchased from E. Merck. Chem. Limited, Mumbai. All other chemicals used were of analytical reagent grade (CYNOR Pharma Private Limited).

Instrumentation:

A High-performance liquid chromatograph Agilent 1200 infinity II LC chromatographic system equipped with 1260 Quat Pump VL and UV detector was used. Samples were injected at 20μL volume. Hypersil BDS C₁₈ (250mm x 4.6mm, 5µm) column was utilized to develop an analytical method. Data acquisition and integration were performed using EZ Chrom software.

Selection of UV wavelength:

An acceptable response of two drugs was obtained at 226nm.

Mobile Phase selection and optimization:

Based on different trials, the mixture of Phosphate Buffer (pH 4.0): Acetonitrile (60:40% v/v) at 1.0 mL/min flow rate had proved to be better than the other mixtures of mobile phases in terms of peak shape, theoretical plate and asymmetry. HPLC Chromatogram of MET and REM at optimized condition was shown in Figure 3.

Figure 3: HPLC Chromatogram of MET (40µg./mL, Rt-.4.560 min) and REM (8µg/mL, Rt-7.793 min) using Phosphate buffer pH 4.0: Acetonitrile (60:40% v/v)

Procedure:

1) Preparation of Mobile phase- Phosphate buffer pH 4.0 was prepared by using 5.04g disodium hydrogen phosphate and 3.01g of potassium dihydrogen phosphate in 1000mL. pH 4.0 was adjusted with glacial acetic acid. It was mixed with acetonitrile and prepared mobile phase containing Phosphate buffer pH 4.0: acetonitrile in the ratio of 60:40 % v/v into a mobile phase bottle.

2) Preparation of standard stock solutions- 100mg of MET and 20 mg of REM were dissolved separately with 100mL methanol to prepare a 1000μg/mL MET and 200 µg/mL REM stock solution. 0.4mL from these standard stock solutions of MET and REM were taken respectively and transferred to the same 10mL volumetric flask and volume was made up to the mark with methanol to prepare a 40μg/mL MET and 8 µg/mL REM stock solution.

3) Preparation of solutions for the construction of the calibration curve

The linearity for MET and REM were assessed by analysis of combined standard solution in a range of 20-60μg/mL and 4-12μg/mL respectively.

4) Preparation of sample solution of MET (40 µg/mL) and REM (8µg/mL) -Weighed accurately 20 tablets and calculated the average weight. Tablet powder equivalent to 100mg of MET and 20mg of REM was transferred to a 100mL volumetric flask. 60mL methanol was added and shaken for 15 mins and made up the volume up to the mark with methanol. The solution was filtered through Whatman filter paper no. 41. 0.4mL was taken from this solution and transferred to a 10ml volumetric flask and made up the volume up to the mark with the methanol. 20μL volume of the above solution was injected for assay analysis.

Forced degradation study:

Stress conditions of acid (0.1 N HCl- 2mL for 2 hr at room temperature), base (0.1 N NaOH- 2mL for 30 mins at room temperature), oxidation (3% hydrogen peroxide- 2mL for 3 hrs at room temperature), photolytic stress (1.2million lux hr followed by 200 Watt-hours for 24 hrs), heat (exposed at 80°C for 5 hrs) were applied on sample stock solution containing 40µg/mL MET and 8 µg/mL REM. After completion of degradation, solutions were injected and monitored the chromatograms under optimized conditions and % degradation was calculated.

Method validation:

1) System suitability - Tailing factor, number of theoretical plates and resolution between MET and REM peaks were assessed by injecting a blank mobile phase followed by six replicates of MET (40 µg/mL) and REM (8 µg/mL) mixture.

2) Precision-Inter-day, intra-day precision and repeatability were assessed by injecting 6 independent sample solutions containing MET (40 µg/mL) and REM (8 µg/mL).

3) Specificity - Retention time was compared between the same concentration of standard and sample solution. Chromatograms were shown in figure-9.

4) Linearity and range - The linear correlation was obtained between peak area and concentration of MET and REM in the concentration range of 20-60 μg/mL and 4-12 μg/mL respectively.

5) Accuracy - 40 µg/mL MET and 8 µg/mL REM solution were taken in three different flasks and 80%, 100% and 120% of the respective standard solution was spiked in it to carry out a triplicate recovery study and % recovery was calculated.

6) LOD and LOQ - LOD and LOQ for MET and REM were calculated by using the formula LOD = 3.3*SD/S and LOQ = 10*SD/S, Where SD: the standard deviation of the intercept, S: the slope of the calibration curve.

7) Robustness - It was studied by variations in method parameters like changes in flow rate, mobile phase ratio and pH of the mobile phase.

Assay of marketed sample

Tablet formulation (Zucator M 500 manufactured by Glenmark Pharmaceuticals) containing 500 mg of MET and 100 mg REM was analyzed using the proposed method.

RESULT:

Results of Forced degradation studies in tablet formulation:

MET and REM were undergoing degradation in tablet formulation to different extents under different stress conditions. Obtained chromatograms were given in Figures 4-8. The results of forced degradation studies are shown in Table 1.

Figure 4: Acid degradation in 2 hrs

Figure 5: Base degradation in 30 mins

Figure 6: Oxidation in 3 hrs

Figure 7: Photo degradation in 24 hrs

Figure 8: Thermal degradation in 5 hrs

Table 1: Results of forced degradation study

		% Degradation
Type of degradation	Conditions of Degradation	MET	REM
Acid	0.1 N HCl for 2 hrs at room temperature	20.89	21.39
Alkali	0.1 N NaOH for 30 mins at room temperature	22.45	18.43
Oxidative	3 % H₂O₂ for 3 hrs at room temperature	19.49	18.35
Thermal	80℃ for 5 hrs.	12.69	12.46
Photolytic	UV chamber for 24 hrs	13.31	11.25

Results of method validation:

1) System suitability - System suitability parameters were studied to verify the optimum conditions. The results obtained are summarised in Table 2.

Table 2: System suitability parameters

System suitability parameters, (n=3)	MET	REM	Standard limits
Asymmetry ± RSD	1.414± 0.114	1.260 ± 0.162	A_s of ≤ 2
Theoretical Plates ± RSD	7211 ± 0.140	7849 ± 0.240	>2000
Retention time ± RSD	4.560 mins ± 0.004	7.793 mins ± 0.001	-
Resolution ± RSD	11.303 ± 0.140		>2

2) Specificity- The difference between the retention time of the test and standard was found to be ±0.007 min for MET and ±0.013min for REM.

3) Intraday, Interday Precision and Repeatability- Results were reported as %RSD. The results found are shown in Tables 3 and 4.

Table 3: Data of intermediate precision(n=3)

Drug	Conc. (µg/mL)	Interday precision		Intraday precision
Drug	Conc. (µg/mL)	Peak area±S.D.	% RSD	Peak area ± S.D.	% RSD
MET	20	2523.52 ± 2.36	0.094	2521.43 ± 4.23	0.168
	40	5026.96 ± 2.49	0.050	5032.55 ± 11.01	0.219
	60	7584.04 ± 11.74	0.155	7569.56 ± 4.23	0.056
REM	4	2054.60 ± 1.18	0.058	2054.61 ± 2.37	0.115
	8	4097.72 ± 3.86	0.094	4096.58 ± 8.76	0.214
	12	6169.38 ± 7.48	0.121	6163.96 ± 8.59	0.140

Table 4: Repeatability study (n=6)

MET (40 µg/mL)			REM (8 µg/mL)
Peak Area	Mean ± S.D.	% RSD	Peak Area	Mean ± S.D.	% RSD
5049.494	5037.92 ± 19.05	0.378	4106.537	4105.35 ± 5.93	0.145
5038.521			4096.106
5056.739			4108.054
5035.902			4101.340
5044.703			4106.745
5002.184			4113.359

Linearity and range:

Linear correlation were obtained between peak area and concentration of MET and REM in the concentration range of 20-60μg/mL and 4-12μg/mL respectively. Linearity data was shown in Table 5.

Figure 9: Chromatogram of linearity of MET (20-60μg/mL) and REM ( 4-12μg/mL) standard solution

Table 5: Data of linearity of MET and REM

	MET		REM
Sr. No.	Conc (µg/mL)	Area (mean, n=3) ± S.D.	Conc (µg/mL )	Area (mean, n=3) ± S.D.
1	20	2526.39 ± 7.54	4	2055.47 ± 5.01
2	30	3788.83 ± 5.59	6	3084.09 ± 4.03
3	40	5054.02 ±10.22	8	4111.12 ± 3.97
4	50	6310.79 ± 5.24	10	5137.95 ± 5.12
5	60	7581.25± 6.50	12	6169.65 ± 14.13

4) Accuracy study and recovery-

5) % Recoveries were found in a range of 99.69-100.28% for MET and 99.52-101.26% for REM as shown in Table 6.

Table 6: Recovery data for MET and REM

	MET		REM
Sr. No.	Amount Added (μg/mL)	% Mean (n=3) Recovery ± S.D.	Amount Added (μg/mL)	% Mean (n=3) Recovery ± S.D.
1	32	100.28 ± 0.54	6.4	99.52 ± 0.81
2	40	99.69 ± 0.40	8.0	101.26 ± 0.64
3	48	100.17 ± 0.48	9.6	100.57 ± 1.08

6) LOD and LOQ- LOD and LOQ were calculated in Table 7.

Table 7: LOD and LOQ data

	MET	REM
LOD	0.085 µg/ mL	0.010 µg/mL
LOQ	0.258 µg/mL	0.030 µg/ mL

7) Robustness- The robustness of the method was studied by deliberate variation in method parameters like changes in flow rate, mobile phase ratio and pH of the mobile phase. %RSD was calculated. The mean %RSD was found to be less than 2. The results found are shown in Table 8.

8) Assay of marketed sample:

Table 9: Assay of marketed sample

Tablet	Zucator M 500
Label claim	MET (500 mg)	REM (100 mg)
Assay (% of label claim) Mean ± S. D.	100.082 ± 0.114	100.128 ±0.162

Table 10: Summary of validation parameters

Sr. No	Parameters	MET	REM
1	Linearity and Range	20 - 60 µg/mL	4 - 12 µg/mL
2	Accuracy	99.69-100.28 %	99.52- 101.26 %
3	Regression Equation (y = mx + c)	y = 126.32x -0.4126	y = 514.11x – 1.2354
4	Slope (m)	126.32	514.11
5	Intercept (c)	- 0.4126	- 1.2354
6	Correlation coefficient (R²)	0.998	0.997
7	LOD	0.085 µg/mL	0.010 µg/mL
8	LOQ	0.258 µg/mL	0.030 µg/mL
9	Asymmetry ± RSD	1.414± 0.114	1.260 ± 0.162
10	Theoretical Plates ± RSD	7211 ± 0.140	7849 ± 0.240
11	Retention time ± RSD	4.560 min ± 0.004	7.793 min ± 0.001
12	Assay (% of label claim of tablet) Mean ± S. D.	100.082 ± 0.114	100.128 ±0.162

DISCUSSION:

A specific, selective, sensitive and simple forced degradation RP-HPLC method was developed which is suitable for the determination of MET and REM in the presence of its degradation products in drug formulation. As per ICH guidelines, this method is robust, sensitive, accurate, selective and precise. The developed method is less time-consuming as well as cost-effective. It can be routinely applied for simultaneous estimation of VIL, MET and REM.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 06.09.2023 Modified on 13.11.2023

Research J. Pharm. and Tech 2024; 17(5):2025-2030.

DOI: 10.52711/0974-360X.2024.00320

%R.S.D.
Drug Name	Flow rate (0.8 mL/min)	Flow rate (1.2 mL/ min)	pH (3.8)	pH (4.2)	Mobile phase (58:42% v/ v)	Mobile phase (62:38% v/v)
MET	0.058	0.925	0.289	0.080	0.126	0.128
REM	0.051	0.938	0.064	0.033	0.124	0.067