STABILITY INDICATING UPLC METHOD FOR SIMULTANEOUS ESTIMATION OF REMOGLIFLOZIN ETABONATE AND TENELIGLIPTIN IN PHARMACEUTICAL DOSAGE FORM

Abstract

The present invention provides an easy, sensitive, specific and precise method for the simultaneous estimation of Remogliflozin Etabonate and Teneligliptin in pharmaceutical dosage form using UPLC, comprising: dissolving Remogliflozin Etabonate and Teneligliptin using 40:60% v/v ratio of Ethanol: 0.1% TFA buffer pH-2.2 mobile phase; running chromatogram through column C18, 50mm x 2.1mm, 1.7µm mm using mobile phase; optimizing conditions of column at flow rate 0.2ml/min, detecting wavelength at 236nm with 5µl injection volume and 2.00 min run time at ambient temperature; running the sample and recording chromatogram from the chromatograph for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin. The retention time of Remogliflozin Etabonate and Teneligliptin are 0.412 and 0.794 min respectively, %RSD of the Repeatability precision is ≤ 2.0; LOD values obtained from regression equation of Remogliflozin Etabonate and Teneligliptin are 0.6µg/ml, 2µg/ml, LOQ values of Remogliflozin Etabonate and Teneligliptin 0.06µg/ml, 0.2µg/ml; Regression equation of Remogliflozin Etabonate and Teneligliptin are y=30104x+11443 and y=30423x+1299.3 respectively and %Recovery ranges between 99.7% and 100.0%. The present invention provides a conduction of forced degradation studies of Remogliflozin Etabonate and Teneligliptin under different stress conditions. The developed method is simple and economical and can be used in regular Quality control test in Industries as routine analysis of Remogliflozin Etabonate and Teneligliptin.

Specification

Description:Technical Field of the Invention
The present invention relates to Ultraperformance Liquid Chromatography (UPLC) method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin. This invention is more specifically related to UPLC method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin in bulk and pharmaceutical dosage form. The present invention is used to develop an environment friendly, green, simple, rapid, accurate, precise, and specific, analytical technique for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin in tablet dosage form.
Background of the Invention
Remogliflozin etabonate is known to exhibit a strong inhibitory action for SGLT2 and is proposed for treatment of diabetic patients. Remogliflozin Etabonate is a pro-drug of Remogliflozin, the active entity that inhibits the sodium glucose transport proteins, which are responsible for glucose reabsorption in the kidney. Blocking of sodium glucose transport proteins helps blood glucose to eliminate through the urine. The glucagon-like peptide-1 is secreted from alimentary canal in response to food intake and that promotes insulin secretion from pancreas and regulates blood sugar after food by controlling secretion of glucagon. Teneligliptin exhibits a hypoglycemic effect by controlling the degradation of glucagon-like peptide-1 by inhibiting activity and thereby increasing blood concentration of active glucagon-like peptide-1[Patel, G., Maradia, R. and Meshram, D., A CRITICAL REVIEW ON ANALYTICAL METHODS FOR NEWLY APPROVED DRUGS: REMOGLIFLOZIN ETABONATE AND TENELIGLIPTIN HYDROBROMIDE HYDRATE; Attimarad, Mahesh, Katharigatta Narayanaswamy Venugopala, Anroop Balachandran Nair, Nagaraja Sreeharsha, Effren II Plaza Molina, Ramling Bhagavantrao Kotnal, Christophe Tratrat, Abdulrahman Ibrahim Altaysan, Abdulmalek Ahmed Balgoname, and Pran Kishore Deb. "Environmental sustainable mathematically processed UV spectroscopic methods for quality control analysis of remogliflozin and teneligliptin: Evaluation of greenness and whiteness." Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 278 (2022): 121303]. Many methods have been proposed on the quantitative and qualitative estimation of simultaneous estimation of Remogliflozin Etabonate and Teneligliptin in bulk and its pharmaceutical formulation [Menda, J., Chintala, V., Kanuparthy, P.R., Katari, N.K., Kowtharapu, L.P. and Jonnalagadda, S.B., 2024. Quality by Design Tool Assessed Ultraperformance Liquid Chromatography Method for the Analysis of Remogliflozin and Teneligliptin in Oral Dosage Form. ACS omega, 9(11), pp.12553-12563; Patel, R. and Kotadiya, R., 2024. Stability-indicating green HPLC method for fixed-dose tablets containing remogliflozin etabonate and teneligliptin: an AQbD approach. Drug Development and Industrial Pharmacy, pp.1-13; Muruganathan, G., Thangavel, M. and Kochupapy, R.T., 2024. Stability-Indicating HPTLC Method for Determination of Remogliflozin Etabonate and Vildagliptin in Tablets. Journal of Chromatographic Science, 62(1), pp.27-34]. However, little work has been done on UPLC method for simultaneous estimation of remogliflozin etabonate and teneligliptin in pharmaceutical dosage form and stability studies. Therefore, an accurate, precise, selective and rapid UPLC method for simultaneous estimation of Remogliflozin Etabonate and teneligliptin in pharmaceutical dosage form.
Objects of the Invention
The main object of the present invention is to provide a UPLC method for simultaneous estimation of remogliflozin etabonate and teneligliptin in bulk and pharmaceutical dosage form.
Another object of the present invention is to develop an environment friendly, green, simple, rapid, accurate, precise, and specific, analytical technique for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin.
Yet another object of the present invention is to provide a conduction of forced degradation studies of Remogliflozin Etabonate and Teneligliptin under different stress conditions.
Still another object of the present invention is to develop a simple and economical and method that can be used in regular Quality control test in Industries as a routine analysis of Remogliflozin Etabonate and Teneligliptin.
Summary of the Invention
The present invention provides a simple, sensitive, specific and precise method for the estimation of Remogliflozin Etabonate and Teneligliptin in pharmaceutical dosage form. In an embodiment of the present invention, the method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin in pharmaceutical dosage form using UPLC, comprising: a) dissolving Remogliflozin Etabonate and Teneligliptin using 40:60 % v/v ratio of Ethanol: 0.1% TFA buffer pH-2.2 mobile phase; b) running chromatogram through column C18, 50 mm x 2.1 mm, 1.7 µm mm using mobile phase of step a); c) optimizing conditions of column of step b) at flow rate 0.2ml/min, detecting wavelength at 236 nm with 5µl injection volume and 2.00 min run time at ambient temperature; d) running the sample of step c) and recording chromatogram from the chromatograph for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin. In an embodiment of the present invention, the method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin, wherein the concentration of sample solution Remogliflozin Etabonate 100µg/ml and Teneligliptin10µg/ml. In an embodiment of the present invention, the method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin, wherein the Retention time of Remogliflozin Etabonate and Teneligliptin are 0.412 and 0.794 min respectively, %RSD of the Repeatability precision of Remogliflozin Etabonate and Teneligliptin is ≤ 2.0%. In an embodiment of the present invention, the method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin, wherein the LOD value obtained from regression equation of Remogliflozin Etabonate and Teneligliptin are 0.6 µg/ml, 2 µg/ml, LOQ value obtained from regression equation of Remogliflozin Etabonate and Teneligliptin is 0.06 µg/ml, 0.2 µg/ml; Regression equation of Remogliflozin Etabonate and Teneligliptin are y = 30104x + 11443 and y = 30423x + 1299.3 respectively and %Recovery of Remogliflozin Etabonate and Teneligliptin ranges between 99.7% and 100.0%. In an embodiment of the present invention, the method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin, wherein the invention provides a conduction of forced degradation studies of Remogliflozin Etabonate and Teneligliptin under different stress conditions. The present invention provides a process for the preparation of sample stock solutions. In an embodiment of the present invention, the process for the preparation of sample stock solutions, comprising: a) dissolving 238mg tablet powder representing a corresponding weight of 100mg Remogliflozin etabonate and 10mg Teneligliptin using diluent in 100-ml volumetric flask; b) sonicating for 30 minutes and then centrifuging for 30minutes to obtain clear solution; c) adjusting the volume with the diluent and filtering using 0.45-μ membrane filter to obtain sample stock solution. The present invention provides a process for the preparation of sample working solution. In an embodiment of the present invention, the process for the preparation of sample working solution, comprising: a) transferring 5ml of filtered sample stock solution to 50ml volumetric flask; and b) making up volume with diluent to obtain sample working solution of 100µg/ml Remogliflozin Etabonate and 10µg/ml Teneligliptin. sample working solution the method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin, wherein the process for the preparation of standard stock solution comprising: a) weighing accurately 100mg Remogliflozin etabonate and 10mg Teneligliptin and transferring to 100ml volumetric flask; b) adding diluent to the flask and sonicating and making up the flask with diluents to obtain Standard stock solution of Remogliflozin Etabonate and Teneligliptin. sample working solution the method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin, wherein the process for the preparation of standard working solution comprising of preparing Standard working solutions of 100% solution by pipetting out 5ml from stock solution and taking into a 50ml volumetric flask and making up with diluent to obtain 100µg/ml Remogliflozin Etabonate and 10µg/ml Teneligliptin. sample working solution the method for estimation of Remogliflozin Etabonate and Teneligliptin, wherein the developed method is simple and economical and can be used in regular Quality control test in Industries as a routine analysis of Remogliflozin Etabonate and Teneligliptin.
Brief Description of drawings
In the drawings accompanying the specification, Figure 1 shows calibration curves for Remogliflozin Etabonate and Teneligliptin; Optimized UPLC chromatogram of Remogliflozin Etabonate and Teneligliptin.
In the drawings accompanying the specification, Figure 2 shows Representative chromatogram for Remogliflozin Etabonate and Teneligliptin system suitability test; UPLC chromatograms of a) blank, b) placebo & c) Remogliflozin Etabonate and Teneligliptin sample.
In the drawings accompanying the specification, Figure 3 shows LOD, LOQ of Remogliflozin Etabonate and Teneligliptin and Representative chromatogram for Remogliflozin Etabonate and Teneligliptin Assay.
In the drawings accompanying the specification, Figure 4 shows Acid, Alkali and Peroxide degradation chromatogram for Remogliflozin Etabonate and Teneligliptin.
In the drawings accompanying the specification, Figure 5 shows Reduction, Hydrolysis, Thermal and Photolytic degradation chromatogram for Remogliflozin Etabonate and Teneligliptin.
Detailed description of the Invention
The present invention provides a method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin in pharmaceutical dosage form using UPLC, comprising: a) dissolving Remogliflozin Etabonate and Teneligliptin using 40:60 % v/v ratio of Ethanol: 0.1% TFA buffer pH-2.2 mobile phase; b) running chromatogram through column C18, 50 mm x 2.1 mm, 1.7 µm mm using mobile phase; c) optimizing conditions of column at flow rate 0.2ml/min, detecting wavelength at 236 nm with 5µl injection volume and 2.00 min run time at ambient temperature; d) running the sample and recording chromatogram from the chromatograph for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin. The present invention provides a method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin, wherein the concentration of sample solution Remogliflozin Etabonate 100µg/ml and Teneligliptin10µg/ml. The present invention provides a method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin, wherein the Retention time of Remogliflozin Etabonate and Teneligliptin are 0.412 and 0.794 min respectively, %RSD of the Repeatability precision of Remogliflozin Etabonate and Teneligliptin is ≤ 2.0%. The present invention provides a method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin, wherein the LOD value obtained from regression equation of Remogliflozin Etabonate and Teneligliptin are 0.6 µg/ml, 2 µg/ml, LOQ value obtained from regression equation of Remogliflozin Etabonate and Teneligliptin is 0.06 µg/ml, 0.2 µg/ml; Regression equation of Remogliflozin Etabonate and Teneligliptin are y = 30104x + 11443 and y = 30423x + 1299.3 respectively and %Recovery of Remogliflozin Etabonate and Teneligliptin ranges between 99.7% and 100.0%. The present invention provides a conduction of forced degradation studies of Remogliflozin Etabonate and Teneligliptin under different stress conditions. The present invention provides a process for the preparation of sample stock solutions. The process for the preparation of sample stock solutions, comprising: a) dissolving 238mg tablet powder representing a corresponding weight of 100mg Remogliflozin etabonate and 10mg Teneligliptin using diluent in 100-ml volumetric flask; b) sonicating for 30 minutes and then centrifuging for 30minutes to obtain clear solution; c) adjusting the volume with the diluent and filtering using 0.45-μ membrane filter to obtain sample stock solution. The present invention provides a process for the preparation of sample working solution. The process for the preparation of sample working solution, comprising: a) transferring 5ml of filtered sample stock solution to 50ml volumetric flask; and b) making up volume with diluent to obtain sample working solution of 100µg/ml Remogliflozin Etabonate and 10µg/ml Teneligliptin. sample working solution the method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin, wherein the process for the preparation of standard stock solution comprising: a) weighing accurately 100mg Remogliflozin etabonate and 10mg Teneligliptin and transferring to 100ml volumetric flask; b) adding diluent to the flask and sonicating and making up the flask with diluents to obtain Standard stock solution of Remogliflozin Etabonate and Teneligliptin. sample working solution the method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin, wherein the process for the preparation of standard working solution comprising of preparing Standard working solutions of 100% solution by pipetting out 5ml from stock solution and taking into a 50ml volumetric flask and making up with diluent to obtain 100µg/ml Remogliflozin Etabonate and 10µg/ml Teneligliptin. sample working solution the method for estimation of Remogliflozin Etabonate and Teneligliptin, wherein the developed method is simple and economical and can be used in regular Quality control test in Industries as a routine analysis of Remogliflozin Etabonate and Teneligliptin.
MATERIALS AND METHODS
Instrumentation
Acquity UPLC coupled with quarternary pump, Empower2 software, A Eutech pH metre, a weight scale manufactured by Sartouris Pvt.Ltd., Chennai, a Shimadzu UV-1700 UV-Vis-spectrophotometer, and a Nichrome ultrasonicator, model UCA 701 are used in the study.
Composition of solutions
Buffer: 1ml TFA was topped upto 1000ml with Milli-Q water and passed through a 0.45µ nylon filter. 2.2 was the observed pH of the buffer solution.
Mobile Phase (Diluent): 40 volumes Ethanol along with 60 volumes 0.1% TFA buffer (pH-2.2) were mixed to be utilized as mobile phase. 0.22μ membrane filter was utilized for the filtration of mobile phase.
Standard solution: Remogliflozin etabonate (100 milligram) & Teneligliptin (10mg) were accurately measured then taken in a 100ml volumetric flask. Contents of the flask were sonicated along with diluent and the volume was made up with the same (Stock solution). Further, diluent along with 5 ml of the stock solution was sonicated in a 50 ml volumetric flask. Diluent was utilized to top off the solution (100µg/ml Remogliflozin etabonate, 10µg/ml Teneligliptin).
Sample Solution: Twenty Zita® plus-R tablets were weighed and individual tablet weight was calculated. 238mg tablet powder representing a corresponding weight of 100mg Remogliflozin etabonate and 10mg Teneligliptin was taken precisely and moved to a 100ml volumetric flask. Contents of flask were sonicated along with diluent for 30 min and then centrifuged for 30min. Diluent was utilized to top off the solution and was filtered by 0.45µ injection filter (Sample Stock). 5ml of sample stock was taken into 50ml volumetric flask and the solution was top off with diluent. (Remogliflozin etabonate 100µg/ml and Teneligliptin 10µg/ml).
Placebo: Composition of placebo (150mg) - lactose (135mg), magnesium stearate, talc, starch (5mg each). It was weighed equivalent to sample weight and was further processed as sample preparation.
Determination of wavelength (λmax)
The wavelength of maximum absorption of the Remogliflozin etabonate and Teneligliptin solution in diluent was scanned utilizing a photodiode detector in 200-400 nm wavelength range with diluent as blank.
Optimization of chromatographic conditions
To attain acceptable plate count, tolerable tailing factor and satisfactory resolution peaks, trials were executed during the development of analytical procedure by altering the mobile phase composition and stationary phase.
Method Validation Ensuing ICH Q2R1, the progressed technique parameters viz., system suitability, specificity, accuracy, precision, linearity, robustness, limit of detection (LOD) and limit of quantification (LOQ) were validated.
System suitability: System suitability was performed to confirm that the measuring system and the analytical approach were appropriate for the suggested analysis. Six indistinguishable samples were assessed.
Specificity: Capability to determine the analyte in the existence of anticipated contaminants and degradants by any nosiness noted at Remogliflozin etabonate and Teneligliptin retention times. Assessed by evaluating the outcomes of blank, placebo and sample.
Accuracy: Degree of agreement amid the actual value and the discovered value is stated as accuracy, also known as trueness. It was calculated as a percentage recovery by adding three different concentrations of recognized standards to samples that had already undergone analysis.
Repeatability- Precision over a brief period with the same operating conditions. Six analyses of the sample solution were utilized for evaluation.
Intermediate precision- The variability within laboratories, such as different analysts, instruments, days etc. Often referred to as method precision. Six different determinations of sample made on six different days were utilized for evaluation.
Linearity
Ability of a technique to analyze and produce findings that are proportionate to the sample's analyte concentration within a certain range. The calibration curve's regression equation, created from six linear standard concentrations was considered to estimate linearity.
Forced Degradation Studies
Forced degradation reagents:
1N HCl: 8.5 ml of hydrochloric acid diluted to 100 ml, with milli-Q water.
1N NaOH: 0.4gm of NaOH dissolved in 100 ml of milli-Q water.
3% H2O2: 1ml of 30% hydrogen peroxide was added with 9 ml milli-Q water.
10% w/v NaHSO4: 1g of NaHSO4 dissolved in 10 ml milli-Q water.
Ensuing ICH Q1A (R2) and Q1B guidelines, forced degradation studies were performed which comprises acid and alkali hydrolysis, thermal, peroxide, reduction, hydrolytic degradations along with photolytic degradation.
Acid Hydrolysis
5ml sample stock was added to a 50ml volumetric flask, followed by 1ml of 1N HCl. The volumetric flask was maintained at 60°C for 1hr before being neutralized with 1N NaOH and diluted to 50ml with diluent.
Alkali Hydrolysis
5ml sample stock was added to a 50ml volumetric flask, followed by 1ml of 1N NaOH. The volumetric flask was maintained at 60ºC for 1hour before being neutralized with 1N HCl and diluted to 50ml with diluent.
Peroxide degradation
5ml sample stock was added to a 50ml volumetric flask, followed by 1ml of 3% hydrogen peroxide solution and the volume was top off using diluent. The volumetric flask contents were maintained at 60ºC for 1hour and was then left for 15 minutes at room temperature.
Reduction degradation
5ml sample stock was added to a 50ml volumetric flask, followed by 1ml of 10% w/v sodium bisulphate and the volume was built up to the required volume with diluent. The volumetric flask was maintained at 60oC for 1 hour. The volumetric flask was left at room temperature for 15 minutes.
Hydrolytic degradation
5ml of the sample stock solution was added to a 50ml volumetric flask, 1ml of milli Q water was added to a flask and the volume was built up to the required volume with diluent. The volumetric flask was then maintained at 60°C for 1 hour and was left for 15 minutes at room temperature.
Thermal degradation
Petri dish added with Remogliflozin etabonate and Teneligliptin sample was placed at 1050C for 24 hours in a hot air oven. Diluent was utilized to dilute this sample.
Photolytic degradation
The Remogliflozin etabonate and Teneligliptin sample was placed in UV light for 24 hours. Diluent was utilized to dilute this sample.
All the above degradation solutions were filtered using 0.22µ syringe filter, diluted and analysed separately using UPLC technique.
RESULTS
Method development and Optimization of chromatographic conditions
The various trials performed and the results are depicted in Table 1 and the chromatograms are shown.
Table 1: Trial conditions and results for Remogliflozin etabonate and Teneligliptin UPLC method development
Condition Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6
Column Phenyl Column,1.7 µ, 2.1 mm x 100 mm Phenyl Column,1.7 µ, 2.1 mm x 100 mm Acquity UPLC BEH C18 Colum, 1.7µ, 2.1 mm x 50 mm Acquity UPLC BEH C18 Colum, 1.7µ, 2.1 mm x 50 mm Acquity UPLC BEH C18 Colum, 1.7µ, 2.1 mm x 50 mm Acquity UPLC BEH C18 Colum, 1.7µ, 2.1 mm x 50 mm
Mobile Phase ACN: 0.1% TFA (70:30) ACN: 0.1% TFA (60:40) ACN: 0.1% TFA (50:50) EtOH: 0.1% TFA (60:40) EtOH: 0.1% TFA (50:50) EtOH: 0.1% TFA (40:60)
Injection vol. 5µl 5µl 5µl 5µl 5µl 5µl
Flow rate 0.2ml /min 0.2ml /min 0.2ml /min 0.2ml /min 0.2ml /min 0.2ml /min
Run time 4.0 min 5.0 min 10.0 min 2.0 min 2.0 min 2.0 min
λmax 200-400 nm 236nm 236nm 236nm 236nm 236nm
Observation Baseline is not good Unknown peak was formed Less resolution between the peaks Second peak tailing is not within the limit Second peak was broad.
Optimized method
Optimized method
Table 2: UPLC Chromatogram for Remogliflozin etabonate and Teneligliptin method development - Trial 1
Name Retention Time Area USP Resolution USP Tailing USP Plate Count
1 1.247 2599031 1.01 10755
2 2.421 834251 12.05 1.05 3164
Table 3: UPLC Chromatogram for Remogliflozin etabonate and Teneligliptin method development - Trial 2
Name Retention Time Area USP Resolution USP Tailing USP Plate Count
1 1.008 440102 1.08 1853
2 2.939 99441 17.54 1.03 8152
Table 4: UPLC Chromatogram for Remogliflozin etabonate and Teneligliptin method development - Trial 3
Name Retention Time Area USP Resolution USP Tailing USP Plate Count
1 1.093 2351044 1.01 3658
2 1.367 485679 1.86 1.08 5428
Table 5: UPLC Chromatogram for Remogliflozin etabonate and Teneligliptin method development - Trial 4
Name Retention Time Area USP Resolution USP Tailing USP Plate Count
1 0.689 2625475 1.73 4526
2 0.973 725462 5.47 2.09 2845
Table 6: UPLC Chromatogram for Remogliflozin etabonate and Teneligliptin method development - Trial5
Name Retention Time Area USP Resolution USP Tailing USP Plate Count
1 0.573 1066766 1.17 2317
2 0.746 190664 3.60 1.05 2969
Optimized technique
The idyllic isocratic UPLC conditions for Remogliflozin etabonate and Teneligliptin determination were acquired to remain with Acquıty UPLC BEH C18 (50 mm x 2.1 mm, 1.7 µm) column and 40:60 % v/v ratio of Ethanol: 0.1% TFA buffer (pH-2.2) thrust with 0.2ml/min flow rate using PDA detection at 236 nm wavelength with 5µl injection volume and 2.00 min run time at ambient temperature. Remogliflozin etabonate and Teneligliptin were separated at 0.412 and 0.794 min of retention times respectively. The procedure was considered to be optimized as the plate count, tailing factor and peak resolution were observed to remain in the tolerable ranges.
Table 7. Optimized technique
S.No Name Retention Time Area USP Resolution USP Tailing USP Plate Count
1 Remogliflozin 0.418 3052467 1.17 13671
2 Teneligliptin 0.786 304851 6.41 1.13 5578
Figure 2 shows optimized UPLC chromatogram of Remogliflozin Etabonate and Teneligliptin.
Method Validation
System suitability
Overall, the parameters of system suitability were within the range and are acceptable as per ICH guidelines and are presented in Table 8.
Inj RGE TEN
Plate count Tailing factor Plate count Tailing factor Resolution
1 13671 1.16 5578 1.13 6.41
2 13684 1.13 5563 1.12 6.63
3 13632 1.15 5542 1.09 6.43
4 13695 1.16 5587 1.1 6.6
5 13664 1.12 5554 1.09 6.53
6 13675 1.17 5579 1.1 6.56
Mean 13670.2 1.1 5567.2 1.1 6.5
SD 21.57 0.02 17.17 0.02 0.09
% RSD 0.16 1.69 0.31 1.49 1.37
Specificity
0.412min and 0.794 min respectively were observed retention times of Remogliflozin etabonate & Teneligliptin in the proposed technique. Interfering peaks were not observed in blank, placebo and sample at the retention times of the analytes.
Accuracy
50%, 100% and 150% concentrations were considered for accuracy and the mean percent recovery was 99.7% & 100.0% for Remogliflozin etabonate & Teneligliptin respectively which was satisfactory and manifest the trueness of the technique.
Table 9: accuracy data for Remogliflozin etabonate & Teneligliptin
Remogliflozin etabonate Teneligliptin
%Level API Added (mg) Actual API (mg) Peak Area Amt recovered
(μg/ml) % Recovery Level Mean % Recovery API Added (mg) Actual API (mg) Peak Area Amt recovered
(μg/ml) % Recovery Level Mean % Recovery
50 % 5 5.00 1502436 4.95 99.0 99.3 0.5 0.50 152285 0.499 99.8 100.6
5 5.00 1512407 4.98 99.6 0.5 0.50 155186 0.51 102.0
5 5.00 1508562 4.97 99.4 0.5 0.50 153494 0.5 100.0
100% 10.00 10.00 3025685 9.96 99.6 100.1 1.00 1.00 305184 0.999 99.9 100.0
10.00 10.00 3011256 9.91 99.1 1.00 1.00 303769 0.99 99.0
10.00 10.00 3085647 10.16 101.6 1.00 1.00 307113 1.01 101.0
150 % 15 15.00 4534521 14.93 99.5 99.7 1.5 1.50 451948 1.48 98.7 99.3
15 15.00 4575826 15.06 100.4 1.5 1.50 453785 1.49 99.3
15 15.00 4523568 14.89 99.3 1.5 1.50 457743 1.5 100.0
Mean % Recovery 99.7 Mean % Recovery 100.0
Precision
Precision is the degree to which a set of measurements taken under predetermined conditions using repeated samples of the different homogeneous material agree closely. It is reported as %RSD. Repeatability and intermediate precision were considered and the % RSD was measured to be within the range. Table 10: precision data for Remogliflozin etabonate & Teneligliptin UPLC method
Peak Area RGE TEN
Injection Repeatability Intermediate precision Repeatability Intermediate precision
1 3052467 3024157 304851 305714
2 3042658 3036958 305174 302855
3 3014572 3010258 303849 307133
4 3036259 3021457 307718 304915
5 3032014 3015697 306491 308487
6 3047125 3021052 304033 305948
Mean 3037516 3021597 305352.7 305842
SD 13420.8 9013.5 1495.1 1922.1
% RSD 0.44 0.30 0.49 0.63
Linearity
The technique's linearity was recognized by plotting a calibration curve for the individual drug's concentration level and their corresponding peak area. It was perceived on the concentration scale of 25-150µg/ml of Remogliflozin etabonate and 2.5-15µg/ml of Teneligliptin. The coefficient of determination, R2 was noticed to be more than 0.999% and therefore the technique is linear.
Table 11: linearity data for Remogliflozin etabonate & Teneligliptin UPLC method
Linearity data RGE TEN
Inj. Conc. μg/mL Peak Area Conc. μg/mL Peak Area
1 25.00 785496 2.5 76741
2 50.00 1526387 5 153485
3 75.00 2236524 7.5 230226
4 100.00 3007458 10 305468
5 125.00 3754816 12.5 382209
6 150.00 4562389 15 456854
Slope 30104 30423
Intercept 11443 1299.3
R2 0.9997 1
Figure 1 shows Calibration curves for Remogliflozin etabonate and Teneligliptin.
Robustness
Regarding deliberate changes in method parameters, it is the method's validity which was carried out by altering the mobile phase composition and flow rate. The technique's robustness was measured by varying the mobile phase and the flow rate at the method's proposed wavelength. The values obtained were briefed and remained to be satisfactory signifying the technique remains robust.
Table 12: Robustness data for Remogliflozin etabonate & Teneligliptin UPLC method
Area RGE TEN
Inj . FR (-) FR (+) MP(-) MP (+) FR (-) FR (+) MP(-) MP (+)
1 2852641 3356982 2794138 3534138 287881 326594 292166 313898
2 2866214 3332658 2766214 3566214 282465 328083 295850 318155
3 2896325 3378512 2776325 3576325 285669 325196 291543 315749
Mean 2871726.7 3356050.7 2778892.3 3558892.3 285338.3 326624.3 293186.3 315934.0
SD 22357.7 22941.2 14137.9 22025.9 2723.1 1443.7 2327.7 2134.5
% RSD 0.78 0.68 0.51 0.62 0.95 0.44 0.79 0.68
LOD and LOQ
Detection and quantification limits were assessed for Remogliflozin etabonate and Teneligliptin at 3:1 and 10:1 S/N ratio respectively, by analyzing series of known concentration dilutions. The observed values of LOD were 0.6µg/ml, 0.06µg/ml respectively and the LOQ were 2µg/ml and 0.2µg/ml respectively for the Remogliflozin etabonate & Teneligliptin by considering S/N ratio.
Forced degradation Studies
Inj Condition Remogliflozin etabonate Teneligliptin
Peak Area % Recover % Drug Degradation Peak purity Peak Area % Recover % Drug Degradation Peak purity
Purity Angle Purity Threshold Pass / Fail Purity Angle Purity Threshold Pass / Fail
1 Control 3035730 100 0 0.424 4.053 Pass 305193 99.9 0.1 0.936 1.252 Pass
2 Acid 2645128 87.1 12.9 0.478 4.024 Pass 268044 87.7 12.3 0.924 1.243 Pass
3 Alkali 2680214 88.3 11.7 0.423 4.039 Pass 271541 88.9 11 0.927 1.258 Pass
4 Peroxide 2598647 85.6 14.4 0.415 4.071 Pass 265113 86.7 13.2 0.938 1.234 Pass
5 Reduction 2935641 96.7 3.3 0.424 4.054 Pass 275184 90 9.9 0.956 1.222 Pass
6 Hydrolysis 2985642 98.4 1.6 0.476 4.032 Pass 299412 98 1.9 0.958 1.231 Pass
7 Thermal 2758421 90.9 9.1 0.483 4.028 Pass 291436 95.4 4.5 0.939 1.257 Pass
8 Photolytic 2958456 97.5 2.5 0.447 4.063 Pass 295164 96.6 3.3 0.951 1.248 Pass
Stress studies were carried out under various degradation conditions. Amid all observations, the minimum was hydrolytic degradation while the maximum degradation was for peroxide. Results are shown in Table 7.
Table 13: Forced degradation studies data for Remogliflozin etabonate & Teneligliptin UPLC method

S NO Degradation Condition RGE TEN
% Recovery % Drug degradation % Recovery % Drug degradation
1 Control 100 0 99.9 0.1
2 Acid
(2N HCl, 600C, 30min) 87.1 12.9 87.7 12.3
3 Alkali
(2N NaOH, 600C, 30min) 88.3 11.7 88.9 11
4 Peroxide
(20% H2O2, 600C, 30min) 85.6 14.4 86.7 13.2
5 Reduction
(10%w/v NaHSO4, 600C, 1hr ) 96.7 3.3 90 9.9
6 Hydrolysis
(water reflux, 600C, 1hr) 98.4 1.6 98 1.9
7 Thermal
(1050C, 1hr in oven) 90.9 9.1 95.4 4.5
8 Photolytic
(UV chamber, 24hrs) 97.5 2.5 96.6 3.3
Figure 3 shows Representative chromatogram for Remogliflozin Etabonate and Teneligliptin system suitability test; UPLC chromatograms of a) blank, b) placebo & c) Remogliflozin Etabonate and Teneligliptin sample.
Figure 4 shows LOD, LOQ of Remogliflozin Etabonate and Teneligliptin and Representative chromatogram for Remogliflozin Etabonate and Teneligliptin Assay.
Figure 5 shows Acid, Alkali and Peroxide degradation chromatogram for Remogliflozin Etabonate and Teneligliptin.
Figure 6 shows Reduction, Hydrolysis, Thermal and Photolytic degradation chromatogram for Remogliflozin Etabonate and Teneligliptin.
Assay
Validated UPLC technique was successfully implied to analyse the sample solution under the same chromatographic conditions as linearity. Two separate assays were performed to calculate the mean. The % assay of Remogliflozin etabonate and Teneligliptin were assessed from the calibration curve. 99.3% and 99.5% were the calculated mean % assays of RGE and TEN respectively which were satisfactory and in good agreement with the % label claim for RGE and TEN.
Table 14: Assay data for Remogliflozin etabonate & Teneligliptin UPLC method
Peak area Remogliflozin etabonate Teneligliptin
INJ. NO. STD SAMPLE STD SAMPLE
1 3035594 3024157 305617 305143
2 3005698 302894
Mean 3014928 304019
% Assay 99.3 99.5
Greenness assessment of the developed procedure by AGREE tool
For the evaluation of the environmental impact & greenness assessment of analytical procedure, AGREE® tool was employed. The 12 elements of Green analytical chemistry are the basis for a Pictogram provided by AGREE. The compatibility of proposed analytical procedure with green analytical chemistry concept is graded on a red-yellow-green colour scale, where each principle is represented on a separate subdivision. The central area of the AGREE pictogram has a numerical between 0 and 1 and color which represents the overall rating. Reagent toxicity, waste generation, energy consumption, intensity of labour, degree of automation and integration were few factors which are calculated based on their impact on environment and operator's safety. The eco-friendliness of the approach, was calculated by assigning various weights & scores to the parameters utilized in the proposed method and was reflected with a final score (0.88).
CONCLUSION
Considering the results of specificity, accuracy, precision, linearity, robustness and recovery the proposed stability indicating UPLC technique is considered to be ideal for qualitative and quantitative determination of Remogliflozin etabonate & Teneligliptin. Additionally, the solvents and mobile phase are non toxic, economical, simple to compose and resulting in decent resolution. Moreover, neither the degradation products nor any of the excipients interfered with the outcome of the technique. The proposed method is the initial reported UPLC method for the estimation of Remogliflozin etabonate & Teneligliptin. The eco-friendliness of the current method was evaluated utilizing AGREE calculator. Therefore, the Green technique can be employed for rapid & systematic analysis in repetitive laboratories evaluation aimed at the simultaneous assessment of RGE & TEN in the drug substance and pharmaceutical dosage form for quality control purposes.
Acknowledgements: The authors gratefully acknowledge that the funding for this publication was provided by the Pradhan Mantri Uchchatar Shiksha Abhiyan (PM-USHA), under the Multi-Disciplinary Education and Research Universities (MERU) Grant sanctioned to Sri Padmavati Mahila Visvavidyalayam, Tirupati.
References
1. Patel, G., Maradia, R. and Meshram, D., A CRITICAL REVIEW ON ANALYTICAL METHODS FOR NEWLY APPROVED DRUGS: REMOGLIFLOZIN ETABONATE AND TENELIGLIPTIN HYDROBROMIDE HYDRATE.
2. Attimarad, Mahesh, Katharigatta Narayanaswamy Venugopala, Anroop Balachandran Nair, Nagaraja Sreeharsha, Effren II Plaza Molina, Ramling Bhagavantrao Kotnal, Christophe Tratrat, Abdulrahman Ibrahim Altaysan, Abdulmalek Ahmed Balgoname, and Pran Kishore Deb. "Environmental sustainable mathematically processed UV spectroscopic methods for quality control analysis of remogliflozin and teneligliptin: Evaluation of greenness and whiteness." Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 278 (2022): 121303.
3. Menda, J., Chintala, V., Kanuparthy, P.R., Katari, N.K., Kowtharapu, L.P. and Jonnalagadda, S.B., 2024. Quality by Design Tool Assessed Ultraperformance Liquid Chromatography Method for the Analysis of Remogliflozin and Teneligliptin in Oral Dosage Form. ACS omega, 9(11), pp.12553-12563.
4. Patel, R. and Kotadiya, R., 2024. Stability-indicating green HPLC method for fixed-dose tablets containing remogliflozin etabonate and teneligliptin: an AQbD approach. Drug Development and Industrial Pharmacy, pp.1-13.
5. Muruganathan, G., Thangavel, M. and Kochupapy, R.T., 2024. Stability-Indicating HPTLC Method for Determination of Remogliflozin Etabonate and Vildagliptin in Tablets. Journal of Chromatographic Science, 62(1), pp.27-34.
, Claims:We claim:
1. A method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin in pharmaceutical dosage form using UPLC, comprising:
a) dissolving Remogliflozin Etabonate and Teneligliptin using 40:60 % v/v ratio of Ethanol: 0.1% TFA buffer pH-2.2 mobile phase;
b) running chromatogram through column C18, 50 mm x 2.1 mm, 1.7 µm mm using mobile phase of step a);
c) optimizing conditions of column of step b) at flow rate 0.2ml/min, detecting wavelength at 236 nm with 5µl injection volume and 2.00 min run time at ambient temperature;
d) running the sample of step c) and recording chromatogram from the chromatograph for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin.
2. The method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin as claimed in claim 1, wherein the concentration of sample solution Remogliflozin Etabonate 100µg/ml and Teneligliptin10µg/ml.
3. The method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin as claimed in claim 1, wherein the Retention time of Remogliflozin Etabonate and Teneligliptin are 0.412 and 0.794 min respectively, %RSD of the Repeatability precision of Remogliflozin Etabonate and Teneligliptin is ≤ 2.0%.
4. The method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin as claimed in claim 1, wherein the LOD value of Remogliflozin Etabonate and Teneligliptin are 0.6 µg/ml, 2 µg/ml, LOQ value of Remogliflozin Etabonate and Teneligliptin is 0.06 µg/ml, 0.2 µg/ml; Regression equation of Remogliflozin Etabonate and Teneligliptin y = 30104x + 11443 and y = 30423x + 1299.3 respectively and %Recovery of Remogliflozin Etabonate and Teneligliptin ranges between 99.7% and 100.0%.
5. The method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin as claimed in claim 1, wherein the forced degradation studies of Remogliflozin Etabonate and Teneligliptin under different stress conditions.
6. A process for the preparation of sample stock solutions, comprising:
a) dissolving 238mg tablet powder representing a corresponding weight of 100mg Remogliflozin etabonate and 10mg Teneligliptin using 100-ml diluent;
b) sonicating for 30 minutes and then centrifuging for 30minutes to obtain clear solution;
c) adjusting the volume with the diluent and filtering using 0.45μ membrane filter to obtain sample stock solution.
7. A process for the preparation of sample working solution, comprising:
a) transferring 5ml of filtered sample stock solution to 50ml volumetric flask; and b) making up volume with diluent to obtain sample working solution of 100µg/ml Remogliflozin Etabonate and 10µg/ml Teneligliptin.
8. The method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin as claimed in claim 1, wherein the process for the preparation of standard stock solution comprising:
a) weighing accurately 100mg Remogliflozin etabonate and 10mg Teneligliptin and transferring to 100ml volumetric flask;
b) adding diluent to the flask and sonicating and making up the flask with diluents to obtain Standard stock solution of Remogliflozin Etabonate and Teneligliptin.
9. The method for simultaneous estimation of Remogliflozin Etabonate and Teneligliptin as claimed in claim 1, wherein process for the preparation of standard working solution comprising of preparing Standard working solutions of 100% solution by taking 5ml from stock solution and and making up with diluent to obtain 100µg/ml Remogliflozin Etabonate and 10µg/ml Teneligliptin.
10. The method for estimation of Remogliflozin Etabonate and Teneligliptin as claimed in claim 1, wherein the developed method is simple, economical and can be used in regular Quality control test in Industries as a routine analysis of Remogliflozin Etabonate and Teneligliptin.

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