Method Development and Validation for simultaneous Quantification of Remogliflozin and Metformin in Bulk and Tablets by RP-HPLC

 

Amit Chaudhary¹, Bhuvnesh Kumar Singh^1,2*

¹School of Pharmacy, Abhilashi University, Mandi, Himachal Pradesh, India.

²Teerthanker Mahaveer University, Moradabad, Uttar Pradesh, India.

 

ABSTRACT:

In this study, a novel technique was developed and validated for the simultaneous estimation of Remogliflozin (REMO) and Metformin (MET) in bulk and formulation. The separation was achieved using ODS, C18 (250mm × 4.6mm, i.d.2.5µm) column with isocratic elution. A mixture of acetonitrile: 20 mM ammonium formate buffer (pH 3.5) at the ratio of (60:40) as mobile phase with a flow rate of 1.0ml min^-1 was used for the separation of analytes. The separated effluents were monitored with diode array detector at 243nm. The results showed good linearity over a range of 2.5–25μg ml^-1 and 12.5–125μg ml^-1 for REMO and MET respectively. Furthermore, the average percentage assay of marketed formulations of REMO and MET was found to be 100.30% and 100.52%, respectively. REMO limit of detection (LOD) and limit of quantitation (LOQ) were 0.42 and 1.28μg ml^-1 while LOD and LOQ for MET were 1.97 and 5.96μg ml^-1 respectively. The proposed method was simple, rapid and precise for the study of new formulations which has been approved for the cure of diabetes mellitus and applicable for the analysis of both drugs in pharmaceutical industries effectively.

 

 

INTRODUCTION:

Remogliflozin etabonate (REMO, Figure1, A), a prodrug of remogliflozin, is a selective antagonist of sodium-dependent glucose transporter-2 located in renal proximal tubules. It depletes glucose level in diabetes type 2 by preventing reabsorption of renal glucose. This mechanism work independently of insulin so that, these drugs could be an effective for the treatment of type 1 diabetes as an oral formulation¹.sodium-dependent glucose transporter-2 inhibitors have many advantageous effects that include reduction in systolic blood pressure, loss of body weight, and lowering haemoglobin (HbA1c)². It is chemically “(5-Methyl-4-[4-(1-methylethoxy)benzyl]-1-(1-methylethyl)-1H-pyrazol-3-yl6-O-(ethoxycarbonyl)-β-D glucopy-ranoside)”³.

 

Metformin (MET, Figure 1, B) chemically represents biguanides group and is effective in the treatment of Type 2 diabetes. It is chemically 1,1-dimethyl biguanide hydrochloride^4-11.

 

It decreases the blood glucose level by reducing the gluconeogenesis (production of hepatic glucose), thus decreasing the glucose absorption in the intestine and results in increased uptake and utilization of peripheral glucose by increasing insulin sensitivity. Several HPLC methods have been developed for the determination of MET alone or with other drugs like dapagliflozin, empagliflozin, canagliflozin, gliclazide, rosiglitazone, linagliptinglimepiride, pioglitazonein and other biological products^12-17. Simultaneous estimation of REMO and MET by isocratic elution technique has limited literature so optimised method was established

and validated as per ICH Q2 A and ICH Q2 B^18-25.

 

Figure 1: Chemical structure of (A) Remogliflozin etabonate and (B) Metformin HCl

 

MATERIAL AND METHODS:

Analytes and Reagents:

REMO and MET as reference drugs were gifted by AKUMS drugs pharmaceutical Ltd, Haridwar, Uttrakhand and tablets were purchased from the local market of Moradabad, U.P. Ammonium formate buffer, (Rankem Laboratory Chemicals) acetonitrile (Merck® India Ltd, Mumbai, India) and water HPLC grade were procured by Teerthanker Mahaveer University, U.P. All the chemicals used in the study were of AR grade. HPLC grade water was produced in the laboratory using Milli Q water purification technique.

 

Chromatographic conditions:

The Chromatographic separation of REMO and MET was attained by hypersil “ODS-C18 (250mm × 4.6mm, i.d.2.5µm)” columns having two LC-20AD pumps, adaptable wavelength programmable photodiode array detector (Model SPD-M20A) with system controller (Model CBM-20A) and LC solution software (Model LC solution) of Shimadzu^® Corporation, Japan. The analytes eluted by a diode array detector at 243nm. The injection volume was set at 20µl. The flow rate of an isocratic mobile phase was found to be 1 ml ml^-1 which consisted of acetonitrile: 20mM ammonium formate (pH adjusted to 3.5 with formic acid) in a ratio of 40:60% (v/v) and the filtration was done with 0.45µm membrane filter and also degassed the system for removing the entrapped gas molecules^26-27.

 

Preparation of Mobile Phase:

The mobile phase used in this study was composed of acetonitrile: ammonium formate buffer (pH 3.5) at the ratio of 40:60% v/v, with isocratic elution. Filtration was done by 0.45-micron nylon membrane (Millipore) filter and an ultrasonic bath was used to degas to prevent clogging of the column due to small particles. Standard & sample preparations were achieved with methanol and acetonitrile as diluent. The mobile phase was screened based on system suitability.

 

Preparation of buffer solution:

Buffer solution was prepared by dissolving ammonium formate (1.26gm) in a volumetric flask (1 litre) and the volume was adjusted with HPLC grade water to make 20mM buffer solution.  Final pH of buffer solution was adjusted to 3.5 by using formic acid. This buffer solution was filtered through the membrane filter of 0.45µm porosity and degassed the mobile phase with the help of sonicator⁹.

 

Preparation of Standard Solution:

The primary standard stock solution was prepared by adding 10mg REMO and 50mg MET in 80ml of diluent and then made the final volume upto 100ml using same diluents. The solution was sonicated for 10 min with the help of sonicator to make the drugs completely soluble and after sonication, volume was made up 100ml with the same solvent. Dilutions with different concentration range of primary standard stock solution (0.25, 0.5, 1, 1.25, 1.5, and 2.5) were prepared and their volume was made up to 10ml using the same diluent. The final concentration of REMO and MET was found to be 2.5 to 25µg ml^-1and 12.5 to 125µg ml^-1, respectively for linearity and other analytical methods. The final solutions were filtered through a 0.45μm Millipore membrane filter.

 

Preparation of Sample Solution:

20 tablets of different brands of REMO and MET were accurately weighed and then crushed to form the powder equivalent to 10mg REMO and MET 50mg and then transferred into a cleaned and dried 100ml volumetric flask. The powder was dissolved into the mobile phase using sonication, and the volume was made up to 100ml with the same solvent and filtered with a membrane filter resulting in the concentrations 10 and 50μg ml^-1 respectively. The working sample was repeatedly injected with 20μl solution by using the flow rate of 1 ml min^-1. The optimum wavelength for detection was set at 243nm.

 

RESULTS ANDDISCUSSION:

Method Development and optimization:

The main objective of this study was to develop and validate an optimized method by changing different parameters like column temperature, wavelength, pH, flow rate and mobile phase composition for simultaneous detection of REMO and MET with sharp peaks, maximum theoretical plates, less tailing factors and short analysis time as per International Conference of Harmonisation (ICH) guidelines A typical chromatogram found by the same condition has been illustrated in Figure 2.

 

Figure 2: Chromatograms of Blank, remogliflozin, metformin, remogliflozin and metformin mixture

 

System suitability:

The system suitability test is of importance and integral part of the chromatographic method for validation of the analytical techniques. All-important parameters like the theoretical plate’s values, retention time and symmetry factors of both drugs were calculated, and the results are as reported in Table 1.  The method was found to be within the acceptance criteria.

 

Method Validation:

Specificity:

Good resolution and no interference from blank and excipients were observed indicating that this method is specific for simultaneous estimation of both drugs. The explanatory chromatogram did not show any other peak confirming to the specificity of this method.

 

Accuracy:

The recovery was estimated at 80 %, 100 % & 120 % of the selected concentrations. Three samples were prepared for each recovery level and mean recovery data of REMO and MET were100.73 and 100.23% (Table 2), respectively demonstrated that the method is accurate within the desired range as described in Figure 3.

 

 

Table 1: System suitability parameters

Injection Number	Retention Time Remogliflozin	Peak Area of Remogliflozin	Retention Time Metformin	Peak Area of Metformin
1	3.164	353872	4.956	1593926
2	3.178	352635	4.888	1587382
3	3.084	347327	4.894	1613925
4	3.146	347328	4.934	1583926
5	3.156	354754	4.952	1618395
6	3.214	354737	4.954	1603936
Mean	3.157	351775.5	4.930	1600248.33
SD	0.043	3531.159	0.031	14155.455
%RSD	1.358	1.003	0.629	0.884
USP Tailing Factor	1.28		0.95
USP Plate Count	5245		6547

 

Table 2: Accuracy and recovery data (n=6)

Name of the drug	% Level Spiking	Amount of drug (Tablet) µg	Amount of drug (Standard) µg	Total Drug (µg)	Total Found (µg) Mean ± SD	%RSD	% Recovery
Remogliflozin	80%	5	4	9	9.132 ± 0.107	1.17	101.47
	100%	5	5	10	9.996 ± 0.190	1.90	99.96
	120%	5	6	11	11.08 ± 0.103	0.93	100.76
Metformin	80%	25	20	45	45.05 ± 0.161	0.36	100.11
	100%	25	25	50	50.20 ± 0.152	0.30	100.40
	120%	25	30	55	55.10 ± 0.214	0.39	100.18

 

 

Figure 3: Accuracy study of remogliflozin and metformin at 80% (A), 100% (B) and 120% (C)

 

Precision:

For System and method precision parameter the %RSD of REMO was found to be 1.51 and 0.72 and for the MET it was found to be 0.61 and 1.02. These results were summarized in Table 3.

 

Table 3: System and Method precision study data.

	System Precision		Method Precision
Injection Number	Peak areas of Remogliflozin*	Peak areas of Metformin*	% Assay of Remogliflozin*	% Assay of Metformin*
1	354742	1599372	99.23	99.98
2	351832	1587283	100.45	99.27
3	347281	1586237	100.34	99.45
4	346261	1578289	101.02	101.4
5	356271	1595360	99.99	99.56
6	342637	1573724	101.23	101.59
Mean	349837	1586711	100.38	100.21
SD (±)	5302.76	9749.36	0.72	1.02
RSD (%)	1.51	0.61	0.72	1.02
Acceptance criteria	% RSD value should be ≤ 2

*Concentration of Remogliflozin (10µg/ml) and Metformin (50µg/ml)

 

 

Intermediate precision (Ruggedness):

Intraday precision was done by taking relative standard deviation for 6 replicates on the same day. In intraday precision, the %RSD values for REMO and MET were recorded as 0.73 and 0.28 at 10µg/ml and 50µg/ml, respectively. For interday precision was assessed by 6 samples on three different days and %RSD values were found to be 1.29 and 1.02 for RMEO and MET, respectively. Intraday and interday precisions were found to be significant and summarized in Table 4.

 

Table 4: Intraday and Interday precision study data.

	Intraday Precision		Interday Precision (Day 1,2,3)
Conc.	Peak areas of Remogliflozin	Peak areas of Metformin	Peak areas of Remogliflozin	Peak areas of Metformin
Remogliflozin 8 (µg/ml) & Metformin 40 (µg/ml)	292832	1278402	298594	1293354
	289480	1284943	289480	1284943
	288894	1284382	294834	1294832
Mean	290402	1282576	294302.70	1291043
SD	2124.74	3625.37	4580.17	5334.19
%RSD	0.73	0.28	1.56	0.41
Remogliflozin 10 (µg/ml) & Metformin 50 (µg/ml)	351134	1589323	342678	1583873
	349986	1583810	349986	1583930
	349282	1583930	341899	1612094
Mean	350134	1585688	344854.3	1593299
SD	934.83	3148.86	4461.19	16276.97
%RSD	0.27	0.19	1.29	1.02
Remogliflozin 12 (µg/ml) & Metformin 60 (µg/ml)	422891	1998294	439292	1983824
	419382	1992372	428548	1972389
	428548	1972389	428738	1983832
Mean	423607	1987685	432192.7	1980015
SD	4624.76	13573.62	6148.94	6604.31
%RSD	1.09	0.68	1.42	0.33

 

Linearity and range:

The method was linear between the range from 2.5-25µg ml^-1and 12.5-125µg ml^-1for REMO and MET respectively. The value of correlation coefficients calculated for REMO (R² =0.999, y = 35223x + 4824.5) and MET (R² = 0.9999, y = 31458x - 22.591) were shown in the figure 4.

 

 

Figure 4: Calibration curve of (A) remogliflozin, (B) metformin

 

Robustness:

No significant changes were found in the analytical method for validation when changing of flow rate, pH, wavelength and mobile phase composition. In all cases of REMO and MET, peaks were symmetric with tailing factor less than two and well separated. The robustness data were presented in Table 5.

 

Table 5: Robustness data.

	Remogliflozin			Metformin
Parameter	Modification/Level	% RSD	Tailing Factor	% Recovery	% RSD	Tailing Factor	% Recovery
Flow rate (ml min-1)	0.8	0.67	1.11	99.23	0.17	1.01	99.89
	1.0	0.56	1.03	100.12	0.34	1.23	99.45
	1.2	0.91	1.34	99.11	0.45	1.09	100.45
	3.0	0.78	1.23	100.11	0.28	1.11	99.78
pH	3.5	0.08	1.02	99.78	0.56	1.23	99.29
	4.0	0.69	1.45	99.66	0.48	1.18	98.99
Wavelength (nm)	241	0.67	1.21	99.23	0.89	1.11	99.11
	243	0.08	1.03	99.46	0.58	1.09	99.78
	245	0.56	1.34	100.78	0.99	1.24	99.45
Mobile Phase ratio (ACN: Buffer)	30:70	0.98	1.35	101.44	0.19	1.08	101.23
	40:60	0.46	1.02	99.34	0.89	1.09	100.79
	50:50	0.88	1.04	99.44	0.56	1.21	100.76

 

Detection and quantitation limits:

The calculated LOD and LOQ were obtained by using the signal to noise ratio and it found to be 0.42µg ml^-1,1.28µg ml^-1 for REMO and 1.97µg ml^-1, 5.96µg ml^-1for ETOR, respectively.

 

Assay of marketed formulation:

Percentage purity of three different brands (ZUCATOR M^TM, REMO M^TM, RENIVAM^TM) of REMO and MET tablets were calculated by using simultaneous quantification. The mean contents of the formulations were between the acceptable range of 90 to 110 % of the label amount. The results (Table 6) indicate that the new method is accurate and precise for quantification of fixed-dose combination of REMO and MET by HPLC technique. 

 

Table: 6 Assay data for marketed formulation.

Marketed Formulation		Retention Time (min)	Peak Area	% Assay
ZUCATOR M	Remogliflozin 100 mg	3.34	348576	99.09
	Metformin 500 mg	5.10	1598328	99.88
REMO M	Remogliflozin 100 mg	3.37	352739	100.27
	Metformin 500 mg	5.12	1608246	100.5
RENIVA M	Remogliflozin 100 mg	3.27	357272	101.56
	Metformin 500 mg	4.98	1619353	101.19

 

CONCLUSION:

A newly developed and validated RP-HPLC method is simple, precise, fast and accurate for the simultaneous detection REMO and MET in combined dosage form (tablets). The %RSD for all parameters was found to be within the prescribed limits thus the proposed method used in pharmaceutical industries for the routine analysis, quality control and stability studies of REMO and MET combined dosage form.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGEMENTS:

The authors would like to thank AKUMS drugs pharmaceutical Ltd, Haridwar, Uttarakhand, India for providing the gift samples.

 

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Accepted on 20.12.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(10):4709-4714.