Development and Validation Of Analytical Methods

For The Simultaneous Estimation Of

Epalrestat And Pregabalin
In Tablet Dosage Form By RP-HPLC Method
 Introduction

 Literature Review
 Drug Profile
 Aim and Objective
 Validation Parameters
 Degradation Studies
 Bibliography

Overview..
 High Performance Liquid
Chromatography is one mode of
chromatography; the most
widely used analytical
technique.

 HPLC is a separation technique.

 HPLC utilizes a liquid mobile

phase to separate the
components of a mixture.

 The principle of separation is

adsorption.

 Types of Techniques:
◦ Reverse phase chromatography
◦ Normal phase chromatography
◦ Isocratic separation
◦ Gradient separation

Introduction : HPLC
METHOD DEVELOPMENT PROCEDURE

 Step 1 - Selection of the HPLC method and initial system

Sample preparation
Types of chromatography
Column dimensions
Detectors
 Step 2 - Selection of initial conditions
Mobile phase solvent strength
 Step 3 - Selectivity optimization
 Step 4 - System optimization
 Step 5 - Method validation.

Introduction : Method Development

 • Kasawar GB et al..
•Developed and Validated RP-HPLC For The Estimation Of pregabalin
2010 in capsule dosage form by using photodiode array detector.
•The mobile phase consisted of Phosphate buffer pH
6.9:Acetonitrile(95:5)

• P. Janaki Pathi et al..

• Developed and Validated RP-HPLC For The Estimation Of

2012 Epalrestat in tablet dosage form.
• The mobile phase consisted of 0.03M Potassium
Dihydrogen phosphate in water at pH 3.2 with ortho-
phosphoric acid) and acetonitrile in the gradient program

• Thejo Moorthy Karvadi et al..

2014 • Studied the most suitable bioanalytical method based on

solid phase extraction has been developed and validated for
quantification of pregabalin in rat plasma

Literature Review
 • Keval L Chaudhari et al..

• Developed and Validated RP-HPLC For The Estimation Of

2014 Epalrestat and Methylcobalamin.
• The mobile phase consisted of ACN: 50Mm Ammonium Acetate
buffer with pH 4.0 by glacial acetic acid(50:50).

•Mili Patel et al..

•Developed and Validated RP-HPLC For The Estimation Of

2015 Epalrestat and Methylcobalaminin tablet formulation.
•The mobile phase consisted of Acetonitrile: 0.05 M Potassium
Dihydrogen Phosphate buffer with pH 4.0 by ortho phosphoric
acid(60:40)

•S.M.Malipatil, Bharath

2017 • Developed a new stability- indicating RP-HPLC for estimation of

Epalrestat and Pregabalin in bulk and tablet dosage form
• Mobile phase containing a mixture of Ammonium acetate buffer
(pH 10): ACN (70:30 )

Literature Review
 To develop and validate reversed phase high
performance liquid chromatography (RP-HPLC)
method for the Epalrestat and Pregabalin in
pharmaceutical dosage forms.

 To develop & validate a method that to ensure a

suitable strategy for a particular analyte which is
more specific, accurate and precise.

Aim & Objective

 Epalrestat Pregabalin
IUPAC Name : (3S)-
3-(aminomethyl)-5-methylhexanoic acid
IUPAC Name : 2-[(5Z)- Molecular formula :
5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4- C8H17NO2
oxo-2- Molecular weight :
sulfanylidene-1, 3-thiazolidin-3-yl] acetic acid 159.2261
Molecular formula : Physical State : Solid
C15H13NO3S2 Solubility : Freely
Molecular weight : 319.393 soluble in both basic and acidic solutions

Molecular Structure of Epalrestat Molecular Structure of Pregabalin

Drug Profile of Epalrestat and Pregabalin
 Trail-1
Mobile phase : Water : Acetonitrile (50:50)
Flow rate : 1 ml/min
Column : Discovery C18 (4.6 x 250mm, 5µm)
Detector wave length : 274nm
Column temperature : 25°C
Injection volume : 10L
Run time : 10 min
Diluent : Water and Acetonitrile in the ratio 50:50
Results : Pregabalin and Epalrestat were eluted with
good retention times but Epalrestat shows fronting.

Trail-2
Mobile phase : Water : Acetonitrile (40:60)
Flow rate : 1 ml/min
Column : BDS C8 (4.6 x 150mm, 5µm)
Detector wave length : 274nm
Column temperature : 25°C
Injection volume : 10L
Run time : 10 min
Diluent : Water and Acetonitrile in the ratio 50:50
Results
Trail-3
Mobile phase : OPAbuffer : Acetonitrile (50:50)
Flow rate : 1 ml/min
Column : BDS C8 (4.6 x 150mm, 5µm)
Detector wave length : 274nm
Column temperature : 30°C
Injection volume : 10L
Run time : 7 min
Diluent : Water and Acetonitrile in the ratio 50:50
Results : Epalrestat and Pregabalin both peak are
eluted but Resolution were
less .

Trails-HPLC
 Optimized Method
 Mode of operation : Isocratic
 Mobile Phase : 0.01N KH2PO4 (4.8PH):Acetonitrile (45:55)
 Column temperature : 25°C
 Column : Discovery C8 (4.6 x 250mm, 5µm)
 Detector wavelength : 274 nm
 Injection volume : 10 μL
 Flow rate : 1.0ml/min
 Run time : 10min

Observation: Epalrestat and Pregabalin were eluted at 2.373min and

2.967min respectively with good resolution. Plate count and tailing factor
was very satisfactory, so this method was optimized and to be validated

Chromatographic - Conditions
 Linearity Table Data
S. No. Epalrestat Pregabalin
Conc(µg/ml) Area Conc(µg/ml) Area
1 37.5 1097916 18.75 445789
2 75 2021248 37.5 966841
3 112.5 2970983 56.25 1414804
4 150 4008313 75 1913792
5 187.5 5135687 93.75 2348127
6 225 6087067 112.5 2776280

Calibration curve of Epalrestat Calibration curve of Pregabalin

Validation – Linearity
 Accuracy 50% Chromatogram of Epalrestat and Pregabalin

Accuracy 100% Chromatogram of Epalrestat and Pregabalin

Accuracy 150% Chromatogram of Epalrestat and Pregabalin

Validation Parameter-Accuracy
Validation data for Accuracy of Epalrestat and Pregabalin
Amount Spiked Amount recovered
% Level % Recovery Mean %Recovery
(μg/mL) (μg/mL)

75 75.09 100.12
50% 75 74.03 98.70
75 75.46 100.62
150 150.30 100.20
100% 150 150.35 100.23 99.85%
150 150.52 100.35
225 224.68 99.86
150% 225 223.21 99.21
225 223.59 99.38

Amount Spiked Amount recovered

% Level % Recovery Mean %Recovery
(μg/mL) (μg/mL)
37.5 36.94 98.51
50% 37.5 36.98 98.61
37.5 36.90 98.40
75 74.16 98.87
100% 75 73.67 98.22 99.42%
75 74.69 99.58
112.5 113.47 100.86
150% 112.5 113.39 100.79
112.5 113.53 100.91

Validation Parameter-Accuracy
 Precision
Chromatograms of System Precision

Validation Parameter-System Precision

 Validation data of System Precision

S. No Area of Epalrestat Area of Pregabalin

1. 4030625 1933592
2. 4013874 1929075
3. 4021592 1934777
4. 4033815 1931264
5. 4034095 1924751
6. 4015891 1925878
Mean 4024982 1929890
S.D 9060.2 4065.6
%RSD 0.2 0.2

Validation Parameter-System Precision

 LOD and LOQ

Molecule LOD LOQ

Epalrestat 0.22 0.71
Pregabalin 0.03 0.08

LOD Chromatogram of Standard LOQ Chromatogram of Standard

Validation Parameter-LOD,LOQ
 Chromatograms of Robustness
Flow Plus Chromatogram of Epalrestat and Pregabalin
Flow minus Chromatogram of Epalrestat and Pregabalin

Mobile Phase Plus Chromatogram of Epalrestat and Pregabalin

Mobile Phase minus Chromatogram of Epalrestat and Pregabalin

Temperature minus Chromatogram of Epalrestat and Pregabalin Temperature Plus Chromatogram of Epalrestat and Pregabalin

Validation Parameter
 Validation data of Robustness
S. No Condition %RSD of %RSD of
Epalrestat Pregabalin

1 Flow rate (-) 1.1ml/min 0.1 0.3

2 Flow rate (+) 1.3ml/min 0.3 0.8

3 Mobile phase (-) 35B:65A 0.1 0.1

4 Mobile phase (+) 45B:55A 0.1 0.5

5 Temperature (-) 25°C 0.1 0.2

6 Temperature (+) 35°C 0.2 0.2

Validation Parameter-Robustness
 Standard Chromatograms of Assay

Assay-Standard
 Sample Chromatograms of Assay

Assay-Sample
 Assay data of Epalrestat and Pregabalin
S. No Standard Area Sample area % Assay
1 4030625 4025748 99.82
2 4013874 4027767 99.87
3 4021592 4018426 99.64
4 4033815 4029788 99.92
5 4034095 4025517 99.81
6 4015891 4022579 99.74
Avg 4024982 4024971 99.80
Stdev 9060.2 4012.0 0.099
%RSD 0.3 0.2 0.2
S. No Standard Area Sample area % Assay
1 1933592 1918227 99.20
2 1929075 1915135 99.04
3 1934777 1910180 98.78
4 1931264 1929603 99.79
5 1924751 1919061 99.24
6 1925878 1921673 99.38
Avg 1929890 1918980 99.24

Assay
Stdev 4065.6 6525.3 0.3374
%RSD 0.2 0.3 0.3
 Acid chromatogram of Epalrestat and Pregabalin Base chromatogram of Epalrestat and Pregabalin

Peroxide chromatogram of Epalrestat and Pregabalin Base chromatogram of Epalrestat and Pregabalin

UV chromatogram of Epalrestat and Pregabalin Water chromatogram of Epalrestat and Pregabalin

Degradation Studies
 Degradation Data of Epalrestat
S.NO Degradation % Drug Purity Angle Purity Threshold
Condition Degraded
1 Acid 4.67 0.186 0.298
2 Alkali 2.77 0.169 0.297
3 Oxidation 2.00 0.165 0.289
4 Thermal 0.95 0.186 0.298
5 UV 0.94 0.169 0.297
6 Water 0.94 0.165 0.289
Degradation Data of Pregabalin
S.NO Degradation % Drug Purity Angle Purity Threshold
Condition Degraded
1 Acid 4.83 0.148 0.296
2 Alkali 2.61 0.150 0.299
3 Oxidation 1.82 0.124 0.294
4 Thermal 0.69 0.148 0.296
5 UV 0.54 0.150 0.299
6 Water 0.63 0.124 0.294

Degradation Studies
 Parameters
Epalrestat Pregabalin LIMIT
Linearity
37.5-18.75µg/ml 18.75-112.5 µg/ml
Range(µg/ml)
Regression coefficient 0.999 0.999
Slope(m) 26994 24913
R< 1
Intercept(c) 13337 7779
Regression equation
y = 26994x + 13337 y = 24913x + 7779
(Y= mx+c)
Assay (% mean assay) 99.80% 99.24% 90-110%

Specificity Specific Specific No interference of any peak

Parameters
Epalrestat Pregabalin LIMIT
System precision %RSD 0.2 0.2 NMT 2.0%
Method precision
0.1 0.3 NMT 2.0%
%RSD
Accuracy % recovery 99.85% 99.42% 98-102%
LOD 0.24 0.02 NMT 3
LOQ 0.73 0.07 NMT 10
FM 0.1 0.3
FP 0.3 0.8
MM 0.1 0.1
%RSD NMT
Robustness MP 0.1 0.5
2.0
TM 0.1 0.2
TP 0.2 0.2

Summary
 The proposed HPLC method was found to be simple,
specific, precise, accurate, rapid and economical for
simultaneous estimation of Epalrestat and Pregabalin in
pharmaceutical dosage form.

 The developed method was validated in terms of accuracy,

precision, linearity, robustness and ruggedness, and results
will be validated statistically according to ICH guidelines.

 Hence the suggested RP-HPLC method can be used for

routine analysis of Epalrestat and Pregabalinin API and
Pharmaceutical dosage form.

Conclusions
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