HPTLC instruments
CAMAG (Muttenz, Switzerland) HPTLC instrument containing Linomat V sample applicator was used for the study (Hamilton, Bonadauz, Switzerland). Aluminum HPTLC plates (10 cm × 10 cm), precoated with silica gel F254 were used as stationary phase (E. Merck, Darmstadt, Germany; supplied by Anchrom Technologists, Mumbai, India). Densitometric scanning of developed densitograms were performed using Camag TLC scanner. Shimadzu electronic balance was used for weighing drugs and chemicals (AX 200, Shimadzu Corp., Japan).
Chemicals and reagents
Analytically pure REM (99.23% w/w as per the certificate of analysis provided by company) was obtained as gift sample from Reputed Pharmaceutical company. Methanol (HPLC grade) was procured from Avantor Performance Materials India Ltd.; Ethyl acetate (AR grade) and toluene (HPLC grade) were procured from Loba Chemie Pvt. Ltd., and Ammonia solution (25%) was purchased from Aatur Instru Chem. Pvt. Ltd., India. Marketed formulation with a brand name Remo-Zen (each film-coated tablet contains Remogliflozin etabonate 100 mg by Glenmark Pharmaceuticals Ltd., Mumbai, India) was procured from the local pharmacist.
Chromatographic system
Sample application
Standard and formulation samples of REM were applied on stationary phase (precoated plates containing silica gel F254 ) in the form of narrow band (6 mm) with the help of sample applicator and distance of 9 mm was kept between two bands. The bands were applied using continuous drying stream of nitrogen gas.
Mobile phase and development
Linear ascending chromatographic development was performed using mobile phase methanol: ethyl acetate: toluene: NH3 (2:4:4:0.1, v/v/v). The development was carried out in twin-trough glass chamber previously equilibrated with the mobile phase for 30 min. The mobile phase was allowed to migrate a distance of 80 mm, and after development, plates were removed and dried.
Densitometric analysis
The developed TLC plates were scanned in the reflectance mode using winCATS software. The light source used was deuterium lamp, and bands were scanned at 229 nm. The dimension of the slit was 5 mm in length and 0.45 mm in width. Peak area and peak height data were obtained for each developed band, and regression equation was developed by plotting peak areas versus concentration.
Preparation of standard stock solution
REM (10 mg) was weighed accurately and transferred to 10-mL volumetric flasks and dissolved in a few milliliter of methanol and sonicated for 5 min. Solution was diluted up to the mark with methanol which gave a concentration of 1000 μg/mL. The above solution was further diluted in another volumetric flask to obtain a working standard of 500 μg/mL REM.
Validation
International Conference on Harmonization (ICH) published a guideline Q2 (R1) for the validation of analytical method [9]. The developed method was validated with respect to linearity, accuracy, precision, specificity, and robustness.
Linearity of calibration curves
Linearity of the method was studied by plotting calibration curves over a range of 500–8000 ng/band. Peak area versus concentration data were used to plot calibration curve (n = 6).
Accuracy
The accuracy is the closeness of test result to the true value. To perform accuracy, recovery studies were carried out. Known amount of REM was spiked at three concentration levels (50, 100, and 150%) to a pre quantified formulation. The solutions were diluted and analyzed by developing densitograms using optimized chromatographic conditions. The peak area was noted, and the amount of REM was estimated with the help of regression equation.
Intermediate precision
Intermediate precision study was performed using intraday and interday precisions. Intraday precision was carried out by estimating drug solutions of REM (500, 4000, 8000 ng/band) at three levels covering low, medium, and high concentrations of the calibration curve. The study was performed three times on the same day. Inter-day precision was determined by estimating drug solutions of REM (500, 4000, 8000 ng/band) at three levels covering low, medium, and high concentrations, and the study was conducted for 3 days. Developed densitograms were analyzed, peak area was obtained, and variability of data was calculated in terms of percentage of relative standard deviation (%RSD).
Repeatability
Repeatability of the method was assessed by applying band of REM (4000 ng/band) six times on an HPTLC plate. The plates were developed, and peak areas were determined. To study the scanner repeatability, the same spot was scanned six times, peak area was determined, and variability in the result was analyzed.
Specificity
To check the interference of impurities, degradants, and matrix in the estimation of drug, specificity study was performed by preparing synthetic mixture. Synthetic mixture was prepared using formula REM (30%), Talc 10%, microcrystalline cellulose 20%, Starch 20%, and Lactose 20% [10,11,12]. Synthetic mixture was analyzed for the drug using the optimized chromatographic method, and interference due to exipients were noted.
Sensitivity
The lowest amount of analyte that can be detected in a method is called limit of detection (LOD), and the lowest amount of anlayte that can be quantified is called limit of quantification (LOQ). LOD and LOQ were calculated using the following equation as per ICH guidelines.
$$ \mathrm{LOD}=3.3\times \sigma /S $$
$$ \mathrm{LOQ}=10\times \sigma /S $$
where σ is the standard deviation of y-intercepts of regression lines, and S is the slope of the calibration curve.
Robustness
Small deliberate changes were introduced in the method to assess the robustness of the method. Changes in mobile phase ratio and chamber saturation time were introduced, and the effects on densitogram were analyzed. The study was performed triplicate and %RSD was calculated.
Forced degradation study
To find out the intrinsic stability of the drug molecule and possible degradation pathway, forced degradation studies were performed as per ICH guideline Q1A (R2) and Q1B using different stress conditions [13, 14]. Stress studies were performed using acid and base hydrolysis, oxidative hydrolysis, thermal degradation, and UV light exposure conditions.
Alkali hydrolysis
To perform base hydrolysis, 2 mL stock solution (500 μg/mL) of REM was taken in 10 mL volumetric flasks, and 2.5 mL of 0.1N NaOH was added to it. Solution was kept at room temperature for 30 min, neutralized and diluted with methanol up to the mark. The solution was analyzed and developed; densitogram showed complete degradation of REM. Hence, the same study was repeated by keeping solution for 5 min. The solution was neutralized and diluted up to the mark with methanol.
Acid hydrolysis
To perform acid hydrolysis, 2 mL of stock solution of REM (500 μg/mL) was taken in 10-mL volumetric flasks, and 2.5 mL of 0.1N HCl was added. Then the mixture was heated in a water bath at 70 °C for 30 min and allowed to cool to room temperature. The solution was neutralized with 0.1 N NaOH, and it was diluted up to the mark with methanol.
Oxidative stress degradation
To perform oxidative stress degradation, 2 mL stock solution (500 μg/mL) of REM was taken in 10-mL volumetric flasks, and 2.5 mL of 3% hydrogen peroxide was added. Then the mixture was heated in a water bath at 70 °C for 30 min and allowed to cool to room temperature and diluted up to the mark with methanol.
Thermal degradation
To perform dry heat degradation study, pure drug samples of REM was exposed in the oven at 70 °C for 2 h. It was cooled, and drug was weighed (10 mg) and transferred to 10-mL volumetric flask. It was dissolved and diluted up to the mark using methanol.
Photo degradation
Analytically pure samples of REM were exposed to UV light for 24 h. A 10 mg of drug was weighed and transferred to a 10-mL volumetric flask. It was dissolved and diluted up to the mark using methanol.
All the reaction solutions were applied using applicator microliter syringe on TLC plates. Plates were developed using optimized chromatographic conditions, and densitograms were recorded.
Solution stability
The stock solutions of REM was stored at room temperature for 24 h and analyzed at 0, 4-, 8-, and 24-h intervals.
Analysis of marketed formulation
Twenty tablets were weighed and powdered. The powder equivalent to the 25 mg of REM was weighed and transferred to the 50-mL volumetric flask. A small amount of (10 mL) methanol was added to the above volumetric flask and sonicated for 10 min. The solution was filtered using Whatman filter paper No. 45, and the volume was made up to the mark with methanol (500 μg/mL).
Using a sample applicator, 8 μl of sample was applied on the stationary phase which gave 4000 ng/band concentration of REM. The stationary phase plates were developed as per optimized chromatographic conditions and scanned. The areas were determined, and quantification was carried out by keeping this value in regression equation.