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Research Paper
Stability-indicating Analytical Method Development
using Quality by Design Approach for Simultaneous
Estimation of Ezetimibe and Glimepiride
U. SHAH*, KUNTI SHAH AND RUPAL PATEL
Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology, CHARUSAT Campus, Changa,
Anand-388 421, India
Shah et al.: Stability-indicating Analytical Method for Ezetimibe and Glimepiride
Stability-indicating reversed-phase high-performance liquid chromatography method was developed and
validated for the simultaneous estimation of ezetimibe and glimepiride. Chromatographic separation was
achieved with a Shimadzu’s high performance liquid chromatograph and PhenomenexL1 analytical C18
column with a mobile phase of acetonitrile:ammonium acetate buffer (30 mM):methanol (50:45:5 %, v/v/v).
The flow rate was set at 1.5 ml/min and the detection wavelength was 232 nm. Quality by design approach
was employed for optimization of method parameters like proportion of mobile phase, concentration of
buffer and a model highlighting the design space was generated. This developed chromatographic method
gave well resolved symmetric peaks. Ezetimibe and glimepiride were eluted at 6.7 and 4.4 min, respectively.
This method was validated according to International Conference on Harmonisation Q2(R1) guideline. The
method was linear in range of 50-400 µg/ml for ezetimibe and 5-40 µg/ml for glimepiride with r2=0.9999 and
0.9996, respectively. The sample recoveries were in good agreement with the respective label claim, which
suggested non-interference from formulation additives in the estimation. Forced degradation studies were
carried out and the stressed samples were analysed using the developed methods.
Key words: Quality by design, stability-indicating, ezetimibe, glimepiride, RP-HPLC
Quality by design (QbD) approach has been instability may affect the bioavailability and can further
introduced by the Food and Drug Administration for [2,3]
lead to toxic effects .
the pharmaceutical development to ensure predefined Ezetimibe (EZE; fig.1a), chemically (3R,4S)-1-(4-
quality attributes of the product. Simultaneously, fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxy
the application of the QbD concept to the analytical propyl]-4-(4-hydroxyphenyl)azetidin-2-one, is plasma
method development leads to a more robust method. cholesterol lowering agent that acts by decreasing
As per the International Conference on Harmonisation cholesterol absorption in the small intestine. It is
(ICH) Q8(R2) guidelines, QbD is defined as [4,5]
“a systematic approach to development that begins soluble in methanol . EZE is official in Indian
[6]
with predefined objectives and emphasizes product Pharmacopoeia .
and process control, based on sound science and Glimepiride (GLM; fig.1b), chemically 3-ethyl-4-
quality risk management”. Thus in this approach, the methyl-N-{2-[4-({[(4-methylcyclohexyl) carbamoyl]
variables that overall contribute to the quality of the amino}sulfonyl)phenyl]ethyl}-2-Oxo-2,5-dihydro-
method are identified, their interactions are studied and 1H-pyrrole-1-carboxamide, is a sulfonylurea
finally a method with optimum values of the variables
[1]
is developed . Stability studies are an integral part This is an open access article distributed under the terms of the Creative
of the drug development program. The need for the Commons Attribution-NonCommercial-ShareAlike 3.0 License, which
stability studies on a drug candidate arises from the allows others to remix, tweak, and build upon the work non-commercially,
fact that the chemical integrity of the drug substance as long as the author is credited and the new creations are licensed under
the identical terms
should be maintained until the compound is delivered Accepted 06 February 2019
to the intended site of action. Any form of chemical Revised 08 September 2018
*Address for correspondence Received 02 March 2018
E-mail: umangshah.ph@gmail.com Indian J Pharm Sci 2019;81(2):273-281
March-April 2019 Indian Journal of Pharmaceutical Sciences 273
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Fig. 1: Structure of (a) ezetimibe and (b) glimepiride
antidiabetic agent that works by causing the pancreas phase (RP)-HPLC method for simultaneous estimation
to release insulin, which helps to lower blood sugar. of EZE and GLM in a combined dosage form within
[7,8]
It is soluble in methanol and is official in Indian the QbD framework.
[9]
Pharmacopoeia . MATERIALS AND METHODS
Literature survey revealed that several analytical
methods have been reported, such as ultraviolet The GLM reference standard was procured from
[10] Baroque Pharma, Sokhda, Khambhat and EZE
spectrophotometry , high-performance liquid
[11,12] reference standard was procured from Ranbaxy
chromatography (HPLC) , liquid chromatography-
[13] [14] Pharmaceuticals Ltd., Gurgaon, India. The commercial
mass spectrometry (LC-MS) and stability study
for the estimation of GLM either individually and formulation Eziwa tablets containing 10 mg of EZE
in combination with other drugs and similarly for and 1 mg of GLM was procured from a local pharmacy.
[15-17] HPLC grade methanol, acetonitrile, and ammonium
EZE . Few HPLC methods were reported for the
simultaneous estimation of EZE and GLM in literature. acetate were procured from Loba Chemicals, India.
Pavani et al. reported the separation of EZE and GLM The HPLC system (LC-2010C HT, Shimadzu
from bulk and tablet dosage form by an HPLC method Corporation, Kyoto, Japan) equipped with a SPD M20A
using 0.01 N of potassium di hydrogen ortho phosphate photodiode array detector and LC solutions software
buffer solution and acetonitrile as a mobile phase in was used for chromatographic separation. Separation
[18]
the ratio of 30/70 (v/v) . Sudheer et al. developed was carried out on a Phenomenex L1 HPLC analytical
and validated an HPLC method for simultaneous C 100 Aº (250×4.6 mm, 5 μ) column. Isocratic
estimation of EZE and GLM using phosphate buffer 18
(pH 3.6) and acetonitrile in the ratio of 45:55 (v/v) as a condition with mobile phase of acetonitrile:ammonium
[19] acetate buffer (30 mM):methanol (50:45:5 %, v/v/v)
mobile phase . and 1.5 ml/min flow rate was used for analysis.
All reported liquid chromatographic methods for the Ammonium acetate buffer was filtered through 0.45 µ
simultaneous estimation of the EZE and GLM have filter. Detection wavelength selected for the estimation
used phosphate buffer as one of the components of the of the two drugs was 232 nm.
mobile phase, which can adversely affect the lifespan Preparation of standard solution:
of the column. The phosphate buffer at an intermediate
and high pH values complexes with the silica surface About 100 mg of EZE and 10 mg of GLM were
and weakens the surface silica-siloxane bonds. Thus, accurately weighed and transferred into a 100 ml
a need for a method arises, for estimating the drugs clean volumetric flask and diluted up to the mark
in combination that comprises a mobile phase, which with methanol. From the above stock solution, 2 ml
has such solvents and buffers that do not interfere was pipetted out into a 10 ml volumetric flask and the
with silica of the chromatographic column as well volume was made with methanol to make concentration
as contain volatile components that can be used with 200 µg/ml EZE and 20 µg/ml GLM, respectively.
[20]
MS detectors. The ICH Q1A (R2) guideline entitled Method development:
“Stability testing of new drug substances and products”
requires stress testing to be carried out to elucidate Optimal chromatographic conditions were determined
the inherent stability characteristics of the active after studying various parameters affecting the
substance. An ideal stability-indicating method is the chromatographic separation of a mixture including
one that resolves the drug and its degradation products the column, mobile phase ratio, buffer concentration,
efficiently. So the aim of the present work was to column temperature and flow rate to achieve maximal
[21]
develop and validate a stability-indicating reversed- separation of the drugs and better peak shape. Various
274 Indian Journal of Pharmaceutical Sciences March-April 2019
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combinations of organic solvents in different ratios compounds in sample matrices, and is used particularly
were tried to obtain a well-resolved chromatogram for the determination of impurities and/or degradation
of EZE and GLM. The concentration and proportion products. The limit of detection (LOD) and limit of
of ammonium acetate buffer was varied in the mobile quantitation (LOQ) were separately determined at a
phase to obtain good peak shape. The QbD approach signal to noise ratio (S/N) of 3 and 10.
was applied to get better resolution between the two Specificity is the ability to assess unequivocally the
drugs and optimization of such robust method to get analyte in the presence of components, which may be
good peak shape. expected to be present. Typically these might include
Method validation, linearity: impurities, degradants, matrix. Thus solution of the
formulation of EZE and GLM was injected and peak
Standard solutions containing 50-400 µg/ml of EZE purity was determined for both the peaks. Further stress
and 5-40 µg/ml of GLM were prepared. Peak areas for studies were performed for EZE and GLM to provide
the two drugs were measured at 232 nm. A calibration an indication of the stability indicating property and
curve was plotted for peak areas vs. concentration. specificity of the proposed method.
Regression equation, correlation coefficient, slope and To assess system suitability of the method, the retention
intercept were calculated. time, peak areas, tailing factor, theoretical plates,
Precision: and resolution of six replicate injections of standard
Six sample solutions of the same concentration solution of EZE and GLM having the concentration
(300 µg/ml EZE and 30 µg/ml GLM) were prepared of 300 and 30 µg/ml respectively, was used and the
and injected into the HPLC system as per test % RSD values were calculated in each case.
procedure. The peak areas for both the drugs in all the Robustness study was performed to establish the ability
sample solutions were determined and percent relative of method to remain unaffected for slight variations in
standard deviation (% RSD) was calculated. The the method conditions like mobile phase ratio, flow
intraday and interday precision of the proposed method rate and detection wavelength. No substantial effect
was determined by analysing the corresponding was observed on system suitability parameters.
responses three times on the same day and on three Solution stability:
different days of the three standard solution mixtures The solution stability of EZE and GLM was carried
of 100, 300, and 400 μg/ml of EZE and 10, 30, and 40 out leaving the standard solution in tightly capped
μg/ml GLM, respectively. The result was reported in volumetric flask for 48 h at room temperature. The
terms of % RSD. peak areas for both the drugs were measured initially,
Accuracy: after 24 and 48 h.
Accuracy was determined by calculating % recovery Forced degradation studies:
by standard addition method. Known amount of The studies were carried out for API as well as
standard solution of (50, 100 and 150 µg/ml of EZE formulation. Stress studies were performed to provide
and 5, 10 and 15 µg/ml of GLM) were added in pre- an indication of the stability indicating property of
analysed sample solution having EZE (100 µg/ml) and the proposed method. Forced degradation conditions
GLM (10 µg/ml). Peak area of each solution at 232 nm applied to the drug mixture and formulation was
was taken in triplicates and recovery was calculated by acid hydrolysis (0.1 M HCl/50°/15 min), base
using the regression equation. hydrolysis (0.1 M NaOH/50°/15 min), oxidation
The detection limit of an individual analytical (3 % H2O2/50°/15 min), photolysis (sunlight/ 1 h) and
procedure is the lowest amount of analyte in a sample thermal degradation (60°/1 h). The stressed samples
that can be detected but not necessarily quantitated as were further diluted in methanol and further studied to
an exact value. The quantitation limit of an individual evaluate the ability of the proposed method to separate
analytical procedure is the lowest amount of analyte EZE and GLM from their degradation products. Peak
in a sample that can be quantitatively determined with purity test was carried out of EZE and GLM by using
suitable precision and accuracy. The quantitation limit PDA detector. Assay studies were carried out of stress
is a parameter of quantitative assays for low levels of samples.
March-April 2019 Indian Journal of Pharmaceutical Sciences 275
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Applicability of developed method: good peak shape and resolution of the chromatographic
Twenty tablets were weighed accurately and the peaks. EZE showed two wavelength maxima at 232
average weight was determined. The tablets were and 250 nm while the wavelength maxima of GLM was
further crushed; powder equivalent to 100 mg of 225 nm. The detection wavelength used for the method
EZE and 10 mg of GLM was transferred to a 100 ml development for simultaneous estimation of both
volumetric flask and dissolved in methanol. An aliquot drugs was 232 nm. The optimized chromatographic
of 2 ml was withdrawn from the above solution into condition for the separation and quantification of EZE
10 ml volumetric flask and the final solution was filtered and GLM was acetonitrile:ammonium acetate buffer
by using 0.45 μ membrane filter to get concentration of (30 mM):methanol in the ratio of 50:45:5 (v/v/v) as
200 and 20 μg/ml for EZE and GLM, respectively. the mobile phase with the flow rate of 1.5 ml/min. The
optimized chromatogram is shown in fig. 2. From the
RESULTS AND DISCUSSION Table 2, the p-values for the studied factors are noted.
The factors and levels considered in the experimental For the analysis of overall effect of all critical factors,
design are shown in Table 1. Evaluation of the selected 3D response surface plots were generated that shows
critical quality attributes (CQAs) was carried out by simultaneous effect of critical factors on selected
ANOVA statistics for experimental design (Table 2). responses (fig. 3) and an overlay plot of all responses
Various proportions of acetonitrile and methanol in the is shown in fig. 4. The overlay plot shows that the
mobile phase were explored for the chromatographic entire range of the chromatographic conditions having
[22] the proportion of acetonitrile from 45 to 55 % v/v and
separation of EZE and GLM. The pKa of EZE is 9.73 buffer concentration ranging from 25 to 35 mM with
[23]
and that of GLM is 6.2 . the other previously mentioned chromatographic can
Ammonium acetate buffer was selected to maintain a be applied for the chromatographic separation of EZE
lower pH of the mobile phase in order to have both the and GLM and that any chromatographic condition used
drugs in unionised form for efficient elution through within the optimised range will not affect the CQAs.
the stationary phase. The organic content of the mobile Data for predicted value vs. actual value obtained by
phase and the concentration of buffer were optimised for cross validation is shown in Table 3.
TABLE 1: DoE SUMMARY, CRITICAL FACTORS Linear correlation was found between peak areas
AND CRITICAL RESPONSES versus concentration of EZE and GLM in the range
Critical factors of 50-400 and 5-40 µg/ml, respectively. The results
Factors Levels Value
-1 45:50:5
Mobile phase ratio 0 50:45:5
1 55:40:5
Concentration of -1 25
buffer 0 30
1 35
Responses
Peak area
Tailing factor
Resolution
Theoretical plates Fig. 2: Chromatograms of EZE and GLM
2
TABLE 2: ANOVA FOR 3 FACTORIAL DESIGNS
Independent p-value (probability at 95 % confidence interval)
Factors Area Area Tailing factor Tailing factor Resolution Theoretical Theoretical
EZE GLM EZE GLM plate EZE plate GLM
X 0.0385 0.0940 0.3812 <0.0001 0.0015 0.0021 0.0048
1
X 0.0071 0.3759 0.8845 0.0804 0.0083 0.9107 0.6503
2
X X 0.4835 0.6441 0.7628 0.5552 0.0245 0.2853 0.6617
1 2
X 2 0.3441 0.9203 0.3927 <0.0001 0.1832 0.4322 0.0463
1
X 2 0.1875 0.6037 0.0078 0.0475 0.0553 0.0228 0.4033
2
X : mobile phase ratio
1 *p>0.05 (insignificant)
X : conc. of buffer
2
X X : interaction *p<0.05 (significant)
1 2
276 Indian Journal of Pharmaceutical Sciences March-April 2019
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