Remogliflozin Enabonate (REMO) has IUPAC nomecature ethyl[(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-v 6-[5-methyl-1-propan-2yl-4-[(4-propan-2- yloxyphenyl)methyl]pyrazol-3- yl]oxyoxan-2-yl]methylcarbonate (Figure 2) with antimineralocorticoid activity [1-2]. Comprehensive literature survey reveals that several chromatographic methods have been official and reported for the estimation of Remogliflozin Enabonate (REMO) and with other drug combination which includes RP-HPLC [4-7]. Metformin Hydrochloride(MET) 1,1-dimethylbiguanidehydrochloride (Figure 1) act by reducing the hepatic glucose production and improving the insulin sensitivity of the tissue. [2-3]. Comprehensive literature survey reveals that several spectophotometric and Chromatographic methods have been official and reported for the estimation of Metformin Hydrochloride(MET) alone and with other drug combination which includes RP-HPLC [10-18], UPLC [30],UV Spectrophotometry [8-9], and HPTLC [19-29]. The analytical method for simultaneous determination of MET and REMO has been reported are HPLC [31], but there is no HTPLC method is available. The present work HPTLC method for simultaneous determination of these drugs in tablet.
INSTRUMENT AND REAGENTS:
Chromatographic separation was carried out on HPTLC instrument equipped with 100 microliter micro syringe (Linomat Syringe 659.0014, Hamilton-Bonaduz Schweiz, Camag Linomat V sample applicator Switzerland), twin trough glass chamber (Camag, Switzerland),Camag TLC scanner IV with WinCATS software (V 1.4.6.2002, camag), the source of radiation was deuterium lamp. MET and REMO were obtained as a gift sample by Alembic Pharmaceuticals. AR grade Methanol, Ethanol, Chloroform, Acetonitrile, Ethyl Acetate, Acetic Acid, Triethylamine and Water (RANKEM) was used.
PREPARATION OF MOBILE PHASE:
optimized mobile phase was Methanol: Ethyl acetate: Acetic acid in 6: 3.5: 0.5 v/v. The mobile phase was sonicated for 15 min and then it was filtered through 0.45μm membrane filter paper.
PREPARATION OF STOCK SOLUTION: (MET 500 μg/ml and REMO 100 μg/ml) :
10 mg of REMO is dissolved in mobile phase and made up to 10 ml to get 1000 μg/ml. 1 ml of this solution diluted up 10 ml in mobile phase to get 100 μg/ml solution
50 mg of MET is dissolved in mobile phase and made up to 10 ml to get 5000 μg/ml. 1 ml of this solution diluted up to 10 ml in mobile phase to get 500 μg/ml solution.
SAMPLE SOLUTIONS:
Twenty tablets were weighed accurately and powdered. Powder equivalent to 500 mg of MET and 100 mg REMO was weighed and transferred into 100 ml volumetric flasks and dissolved in mobile phase. The flask was shaken and sonicated for 15 min and then volume was made up to mark with mobile phase to get 5000 μg/ml MET and 1000 μg/ml of REMO. 1 ml of resulting solution was taken in 10 ml flask and made upto the mark with mobile phase to get final concentration of MET (500 μg/ml) and REMO (100μg/ml).
CHROMATOGRAPHIC CONDITION:
The samples were spotted in the form of band having band width 8mm with a microliter micro syringe (Linomat syringe 659.0014, Hamilton-Bonaduz Schweiz, Camag, Switzerland ) on precoated silica gel aluminium plate 60F254, (20×10) 100μm thickness; (E. Merck, Darmstadt, Germany) using a camag Linomat V sample applicator (Switzerland). Linear ascending development was carried out in 20×10cm twin trough glass chamber (Camag, Switzerland). The optimized chamber saturation time before chromatographic development was 20 min at room temperature (25ºC ± 2). The length of chromatographic run was 8cm which looks average 15min to develop. Subsequent to the development; TLC plates were dried in a current of air with the help of air dryer. Densitometry scanning was performed using Camag TLC scanner IV with WinCATS software (V 1.4.6.2002, Camag). All instruments were made in the reflectance – absorbance mode at 234nm, slit dimension (6.00× 0.30mm), scanning speed 20mm/s, data resolution 100μm/step, optical filter, filter factor. The source of radiation was deuterium lamp emitting a continuous UV spectrum between 190nm to 400nm.