INTRODUCTION:
Remogliflozin
Etabonate:
Chemically
Remogliflozin Etabonate is a 5-methyl-4-[4-1(1-methylethoxy) benzyl]-1(methylethyl)-1H-pyrazol-3-yl-6-0-(ethoxycarbonyl)-β-D-glucopyranoside.
It is also be spelled as "Remo”.¹-⁷ It is available in the Indian
market as tablet dosage form in the brand names Remo and Remozen.⁸
It
is used therapeutically in non-alcoholic steatohepatitis and type II diabetes.
It is Highly soluble in ethanol, DMSO, DMF, sparingly soluble in aqueous
buffers.⁹ Mode of action Remogliflozin Etabonate is a prodrug of drug of
the gliflozin. Literature survey reveals that only HPTLC method has been
developed for estimation of Remogliflozin Etabonate so there is no
spectroscopic method available to estimate either from bulk drug or from the
pharmaceutical dosage forms. The present investigation was proposed to estimate
Remogliflozin Etabonate in bulk and pharmaceutical dosage form by spectroscopic
method which is rather simple, sensitive, specific, precise, accurate method.
The present work was to develop and validate as per ICH guidelines so the
method was developed validated according to ICH guidelines for accuracy,
precision, reproductivity, repeatability and robustness.¹⁰
MATERIALS
AND METHODS:
Materials
and reagents:
A
gift sample of Remogliflozin Etabonate
from Glenmark Pharmaceuticals, Mumbai used as a standard drug. Glenmark (Remo
100mg) film coated tablet dosage form bought from the local market. Other
chemicals like Ethanol were bought from SD fine chemicals, Mumbai, India.
Apparatus
and Equipment Required:
1.
UV-Vis double beam spectrophotometer (Model; Shimadzu: 1700S, Japan), Electric
Sonicator, Volumetric flasks (10ml, 50ml, 100ml), Calibrated analytical
pipettes, Electronic digital balance (Techno, Mumbai)
Preparation
of concentration range (Beer’s limit):
Determination
of concentration range which obeys the Lambert and Beer’s law is necessary for
accuracy and reproducibility in Spectrophotometric analysis for quantitative
determination of any drug. For this; Remogliflozin Etabonate stock solution
(100µg/ml) was prepared using pure drug in Ethanol. Further dilutions were made
using 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, 1.2ml, 1.4ml, 1.6ml and 1.8ml of above
solution was transferred to a series of 10ml volumetric flasks this gave a
series of concentrations ranging from 2 to 18 µg/ml of Remogliflozin Etabonate.
The final volume was made up to 10ml mark using Ethanol, sonicated for 5
minutes. The resultant solutions were measured using a double beam
uv-vis-spectrophotometer at wave length of 239.8nm against a reagent blank. A
calibration curve was plotted with concentration against absorbance. From the
graph it was clear that Beer’s law was obeyed in concentration range of
2-14µg/ml and deviation was observed above these concentrations.
Preparation
of standard calibration curve:
100mg
of pure Remogliflozin Etabonate drug was dissolved in little quantity of
Ethanol in a 100ml volumetric flask, the volume was made up to the mark using
Ethanol. The solution was sonicated for 10 minutes. This gave a Remogliflozin
Etabonate solution with concentration of 1mg/ml (1000µg/ml). 10ml of this
solution was further diluted 100ml in volumetric flask using Ethanol to obtain
a concentration of 100µg/ml. Further dilutions were made using 0.2, 0.4, 0.6,
0.8, 1.0, 1.2, 1.4, 1.6 and 1.8ml solution was transferred to a series of 10ml
volumetric flasks (to Obtain a series of concentrations ranging from 2-14µg/ml
of Remogliflozin Etabonate). The final volume was made up to 10ml mark using
Ethanol, sonicated for 5 minutes, the absorbances were measured at of 239.8nm
against a reagent blank. A calibration curve was plotted with concentration
against absorbance.
Estimation
of Remogliflozin Etabonate in tablet dosage forms:
Twenty
tablets were weighed accurately and powdered. The tablet powder equivalent to
100mg of Remogliflozin Etabonate was transferred into a 100mL volumetric flask
and dissolved in little quantity of Ethanol. Then the solution was sonicated
for 30 minutes and filtered using whatman filter paper No#41. The filtrate so
obtained was diluted with Ethanol to produce 100ml. Further dilutions were made
with Ethanol to get required concentrations within Beer’s - Lambert limits. The
resultant solutions were measured at wave length of 239.8nm against a reagent
blank. The concentration of drug was calculated with the help of standard
calibration curve.
ANALYTICAL
METHOD VALIDATION:
Validation
of an analytical procedure is the process by which it is established, by
laboratory studies, that the performance characteristics of the procedure meet
the requirements for its intended use. All analytical methods that are intended
to be used for analyzing any clinical samples will need to be validated.
Validation of analytical methods is an essential but time consuming activity
for most analytical development laboratories. It is therefore important to wave
length. Understand the requirements of method validation in more detail and the
options that are available to allow for optimal utilization of analytical
resources in a development laboratory.
RESULTS
AND DISCUSSION:
Results
of Determination of Beer's Limit:
Table:1.
Beer’s range for Remogliflozin Etabonate:
|
Sl. No.
|
Concentration in µg/ml
|
**Absorbance at λmax
239.8nm
|
|
1.
|
0.0
|
0.000
|
|
2.
|
2
|
0.180
|
|
3.
|
4
|
0.350
|
|
4.
|
6
|
0.535
|
|
5.
|
8
|
0.740
|
|
6.
|
10
|
0.920
|
|
7.
|
12
|
1.101
|
|
8.
|
14
|
1.300
|
|
9.
|
16
|
1.420
|
|
10.
|
18
|
1.860
|
(**
Average of three determinations)
Figure:
1. Beer’s limit for Remogliflozin Etabonate pure drug:
(Figure:1
showing Beer's limit for Remogliflozin Etabonate at 239.8nm)
Standard
Calibration Curve of Remogliflozin Etabonate:
Table:
2. Absorbance of Remogliflozin Etabonate at various concentrations:
|
Sl. No.
|
Concentration in µg/ml
|
Absorbance at λmax
239.8nm
|
|
1.
|
0.0
|
0.000
|
|
2.
|
2
|
0.180
|
|
3.
|
4
|
0.350
|
|
4.
|
6
|
0.535
|
|
5.
|
8
|
0.740
|
|
6.
|
10
|
0.920
|
|
7.
|
12
|
1.101
|
|
8.
|
14
|
1.300
|
Figure:
2. Standard calibration curve for Remogliflozin Etabonate
Table:9
showing calibration data for Remogliflozin Etabonate at 239.8nm:
|
 Parameters
|
Calibration data at
239.8nm
|
|
λmax
|
239.8nm
|
|
Beer’s law limit (µg/ ml)
|
2 -14µg/ml
|
|
Molar Absorptivity
|
1.1156Lmol-1cm-1
|
|
Regression Equation(Y=a+bc)
|
Y= 0.050X+0.011
|
|
Slope (b)
|
0.01421 to 0.01467
|
|
Intercept(a)
|
- 0.004515 to 0.0016752
|
|
Correlation Coefficient (R2)
|
0.997
|
|
Limit of detection (LOD)
|
1.220µg/ml
|
|
Limit of quantitation (LOQ)
|
5.220µg/ml
|
Determination
of Beer’s limit:
The
Beer’s limit felled in the range of 2-14µg/ml under given experimental
conditions.
Determination
of absorption maxima:
100µg/ml
stock solution of Remogliflozin Etabonate was prepared and absorbances were
measured from 200nm to 340nm. The optimum wave length was found to be 239.8nm
Assay:
Marketed
tablets contained Remogliflozin Etabonate were used for the assay. After
extraction, proper dilution, measurement, the concentration was determined
using standard calibration curve. The amount of drug found in the range of
99.80 – 100.70%w/w
Method
validation:
The
proposed method was validated in accordance to ICH guidelines.
a)
Accuracy:
Percentage
of recoveries of Remogliflozin Etabonate in tablets was found in the range of
99.94 – 100.30% w/w
b)
precision:
The
percent coefficient of variations (% C.V) was between 0.34-0.60 for intra-day
and 0.23-0.62 for inter-day absorbances.
c)
Repeatability:
Repeat
ability was determined by analyzing the sample at the given concentration
wavelength for at least six times and it was found that the variability in the
results was not more than 0.5%.
d)
Reproducibility:
The
standard solution of Remogliflozin Etabonate by analyst-I and analyst-II
separately. The values obtained were evaluated using F-test and t-test to
verify their reproducibility. Calculated value for t-test was found to be less
than the tabulated (standard) value it can calculated that no significant
difference was observed in the result of analysis.
e)
Ruggedness:
Ruggedness
of the developed method was determined by changing the analytical tools such as
laboratory, instruments, analyst and chemicals. The result (in terms of %RSD)
of six determinations indicated that there were no significant variations in
the data.
f)
Robustness:
Robustness
of the method was established by slightly changing the temperature and PH of
the reaction mixture. The data so obtained showed no significant variation in
the absorption pattern.
g)
Limit of detection and limit of quantitation:
Were
determined from the standard deviation of y – intercepts of six calibration
curves and average slope of six calibration curves. LOD and LOQ of
Remogliflozin Etabonate was found to be 1.220µg/ml and 5.220µg/ml respectively.
CONCLUSION:
A
new spectrophotometric method was developed to estimate Remogliflozin Etabonate
in pure and tablet dosage forms. Ethanolic solutions of Remogliflozin Etabonate
was estimated by using UV-spectrophotometer (Shimadzu 1700S, Japan) with
matched 1cm quartz cell. It showed maximum absorption at the range of 2-14µg/ml
at 239.8nm with coefficient of correlation (R2) of 0.997
The
method so developed was validated according to ICH guide lines for accuracy,
precision (inter and intra-day precisions), repeatability, reproducibility,
ruggedness, robustness etc.
The
proposed method was found to be simple, accurate, sensitive, precise,
reproducible and rapid.
This
method can be successfully employed for routine quantitative analysis of
Remogliflozin Etabonate in bulk and tablet dosage form.
ACKNOWLEDGMENT:
The
authors are thankful to Head of the Department of Chemistry.
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