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WORLD JOURNAL OF PHARMACEUTICAL
Prasanthi et la. World Journal of Pharmaceutical and Medical Research
ISSN 2455-3301
AND MEDICAL RESEARCH
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ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF RP-HPLC FOR

ESTIMATION OF PREGABALIN AND EPALRESTAT IN PURE AND
PHARMACEUTICAL DOSAGE FORM

D. Prasanthi*, Ch. M.M. Prasada Rao and D. Dhachinamoorthi

Department of Pharm. Analysis and Quality assurance, QIS College of Pharmacy, Ongole-523272.

*Corresponding Author: D. Prasanthi

Department of Pharm. Analysis and Quality assurance, QIS College of Pharmacy, Ongole-523272.

Article Received on 19/07/2019 Article Revised on 07/08/2019 Article Accepted on 28/08/2019

ABSTRACT
A simple, Accurate, precise method was developed for the simultaneous estimation of the Pregabalin and
Epalrestat in Tablet dosage form. Chromatogram was run through Std Azilent 150 x 4.6 mm, 5. Mobile phase
containing Buffer: Acetonitrile taken in the ratio 45:55 was pumped through column at a flow rate of 1.0 ml/min.
Buffer used in this method was 0.1% OPA buffer. Temperature was maintained at 30°C. Optimized wavelength
selected was 241 nm. Retention time of Pregabalin and Epalrestatwere found to be 2.930 min and 2.179 min.
%RSD of the Epalrestat and Pregabalin were and found to be 0.4and 0.2 respectively. % Recovery was obtained as
98.98% and 99.32% for Epalrestat and Pregabalin respectively. LOD, LOQ values obtained from regression
equations of Epalrestat and Pregabalin were 0.02, 0.06 and 0.26, 0.77 respectively. Regression equation of
Epalrestat is y = 20545x + 16173, and y = 18476x + 10803of Pregabalin. Retention times were decreased and run
time was decreased, so the method developed was simple and economical that can be adopted in regular Quality
control test in Industries.

KEYWORDS: Epalrestat, Pregabalin, RP-HPLC.

INTRODUCTION technique and is today called as High Performance

Liquid Chromatography (HPLC).
Liquid chromatography (3) is an analytical
chromatographic technique that is useful for separating
Epalrestat is a carboxylic acid derivative and a
ions or molecules that are dissolved in a solvent. If the
noncompetitive and reversible used for the treatment
sample solution is in contact with a second solid or liquid
of which is one of the most common long-term
phase to differing degrees due to differences in
complications in patients with. It reduces the
adsorption, ion exchange, partitioning or size. These
accumulation of intracellular sorbitol which is believed
differences will allow the mixture components to be
to be the cause of diabetic neuropathy, retinopathy and
separated from each other by using these differences to
nephropathy It is well tolerated, with the most
determine the time of the solutes through a column.
commonly reported adverse effects being gastrointestinal
During 1970’s, most chemical separations were carried
issues such as nausea and vomiting, as well as increases
out using a variety of techniques including open-column
in certain liver enzymes. Chemically, epalrestat is
chromatography, paper chromatography and thin layer
unusual in that it is a drug that contains a group. Aldose
chromatography (TLC). However, these
reductase is the key enzyme in the polyol pathway whose
chromatographic techniques were inadequate for
enhanced activity is the basis of diabetic neuropathy.
quantification of compounds and resolution between
Aldose reductase inhibitors (ARI) target this enzyme.
similar compounds. During this time pressure liquid
Out of the many ARIs developed, ranirestat and
chromatography began to be used to decreased flow
fidarestat are in the trial stage. Others have been
through time, thus reducing separation time of
discarded due to unacceptable adverse effects or weak
compounds being isolated by column chromatography.
efficacy. Epalrestat is the only ARI commercially
However, flow rates were inconsistent, and the question
available. It is easily absorbed into the neural tissue and
of whether it was better to have constant flow rate or
inhibits the enzyme with minimum side effects.
constant pressure debated. High pressure liquid
chromatography quickly improved with the development
of column packing materials. Additional convenience of
on-line detectors became rapidly a powerful separation

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Prasanthi et la. World Journal of Pharmaceutical and Medical Research

Structure Preparation of Standard working solutions (100%

solution): 1ml from each stock solution was pipetted out
and taken into a 10ml volumetric flask and made up with
diluent. (150 µg/ml of EPAL and 150µg/ml of PREGA).

Preparation of Sample stock solutions: 5 tablets were

weighed and the average weight of each tablet was
calculated, then the weight equivalent to 1 tablet was
transferred into a 100 ml volumetric flask, 50ml of
PREGABALIN diluents was added and sonicated for 25 min, further the
Description: Pregabalin is an anticonvulsant drug used volume was made up with diluent and filtered by HPLC
for neuropathic pain, as an adjunct therapy for partial filters. (1500 µg/ml of EPAL and 1500 µg/ml of
seizures, and in generalized anxiety disorder. It was PREGA).
designed as a more potent successor to gabapentin.
Pregabalin is marketed by Pfizer under the trade name Preparation of Sample working solutions (100%
Lyrica. It is considered to have a dependence liability if solution): 1ml of filtered sample stock solution was
misused, and is classified as a Schedule V drug in the transferred to 10ml volumetric flask and made up with
U.S. diluent. (150µg/ml of EPAL and 150µg/ml of PREGA).
Structure Preparation of buffer
0.01N KH2PO4 Buffer: Accurately weighed 1.36gm of
Potassium dihyrogen Ortho phosphate in a 1000ml of
Volumetric flask add about 900ml of milli-Q water
added and degas to sonicate and finally make up the
volume with water then PH adjusted to 5.4 with dil.
Orthophosphoric acid solution.

0.1%OPA Buffer: 1ml of Ortho phosphoric acid was

diluted to 1000ml with HPLC grade water.

VALIDATION
System suitability parameters
The system suitability parameters were determined by
preparing standard solutions of Epalrestat (150ppm) and
Pregabalin (150ppm) and the solutions were injected six
times and the parameters like peak tailing, resolution and
USP plate count were determined.
EXPERIMENTAL WORK The % RSD for the area of six standard injections results
MATERIALS should not be more than 2%.
Epalrestat and Pregabalin pure drugs (API), Combination
Epalrestat and Pregabalin tablets (PREALDONIL Specificity: Checking of the interference in the
150MG TABLET) received from spectrum lab Distilled optimized method. We should not find interfering peaks
water, Acetonitrile, Phosphate buffer, Methanol, in blank and placebo at retention times of these drugs in
Potassium dihydrogen ortho phosphate buffer, Ortho- this method. So this method was said to be specific.
phosphoric acid. All the above chemicals and solvents
are from Rankem Ltd. PRECISION
Preparation of Standard stock solutions: Accurately
METHODOLOGY weighed 75 mg of Epalrestat, 75 mg of Pregabalin and
Diluent: Based up on the solubility of the drugs, diluent transferred to 50ml&50ml volumetric flasks. 3/4 th of
was selected, Acetonitrile and Water taken in the ratio of diluents was added and solicited for 10 minutes. Flasks
50:50. were made up with diluents and labeled as Standard
stock solution 1and 2.(1500µg/ml EPAL of and
Preparation of Standard stock solutions: Accurately 1500µg/ml of PREGA)
weighed 75 mg of Epalrestat, 75 mg of Pregabalin and
transferred to 50ml&50ml volumetric flasks. 3/4 th of Preparation of Standard working solutions (100%
diluents was added and solicited for 10 minutes. Flasks solution): 1ml from each stock solution was pipetted out
were made up with diluents and labeled as Standard and taken into a 10ml volumetric flask and made up with
stock solution 1and 2.(1500µg/ml EPAL of and diluent. (150 µg/ml of EPAL and 150µg/ml of PREGA).
1500µg/ml of PREGA).
Preparation of Sample stock solutions: 5 tablets were

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Prasanthi et la. World Journal of Pharmaceutical and Medical Research

weighed and the average weight of each tablet was Acceptance Criteria
calculated, then the weight equivalent to 1 tablet was The % Recovery for each level should be between 98.0
transferred into a 100 ml volumetric flask, 50ml of to 102
diluents was added and sonicated for 25 min, further the
volume was made up with diluent and filtered by HPLC Robustness: Small deliberate changes in method like
filters.(1500 µg/ml of EPAL and 1500 µg/ml of PREGA) Flow rate, mobile phase ratio, and temperature are made
but there were no recognized change in the result and are
Preparation of Sample working solutions (100% within range as per ICH Guide lines.
solution): 1ml of filtered sample stock solution was
transferred to 10ml volumetric flask and made up with Robustness conditions like Flow minus (0.9ml/min),
diluent. (150µg/ml of EPAL and 150µg/ml of PREGA) Flow plus (1.1ml/min), mobile phase minus, mobile
phase plus, temperature minus (25°C) and temperature
LINEARITY plus(35°C) was maintained and samples were injected in
duplicate manner. System suitability parameters were not
Preparation of Standard stock solutions: Accurately
much effected and all the parameters were passed.
weighed 75 mg of Epalrestat, 75 mg of Pregabalin and
%RSD was within the limit.
transferred to 50ml&50ml volumetric flasks. 3/4 th of
diluents was added and solicited for 10 minutes. Flasks
LOD sample Preparation: 0.25ml each from two
were made up with diluents and labeled as Standard
standard stock solutions was pipetted out and transferred
stock solution 1and 2.(1500µg/ml EPAL of and
to two separate 10ml volumetric flasks and made up with
1500µg/ml of PREGA)
diluents. From the above solutions 0.1ml each of
25% Standard solution: 0.25ml each from two standard
Epalrestat, Pregabalin, solutions respectively were
stock solutions was pipetted out and made up to 10ml.
transferred to 10ml volumetric flasks and made up with
(37.5µg/ml of EPAL, and 37.5 µg/ml of PREGA)
the same diluents
50% Standard solution: 0.5ml each from two standard
stock solutions was pipetted out and made up to 10ml.
LOQ sample Preparation: 0.25ml each from two
(75µg/ml of EPAL, and 75 µg/ml of PREGA)
standard stock solutions was pipetted out and transferred
75% Standard solution: 0.75ml each from two standard
to two separate 10ml volumetric flask and made up with
stock solutions was pipetted out and made up to 10ml.
diluent. From the above solutions 0.3ml each of
(112.5µg/ml of EPAL, and 112.5µg/ml of PREGA)
Epalrestat, Pregabalin, solutions respectively were
100% Standard solution: 1.0ml each from two standard
transferred to 10ml volumetric flasks and made up with
stock solutions was pipetted out and made up to 10ml.
the same diluent.
(150µg/ml of EPAL, and 150µg/ml of PREGA)
125% Standard solution: 1.25ml each from two
DEGRADATION STUDIES
standard stock solutions was pipetted out and made up to
Oxidation
10ml. (187.5µg/ml of EPAL and 187.5µg/ml of PREGA)
To 1 ml of stock solution of Epalrestat and Pregabalin, 1
150% Standard solution: 1.5ml each from two standard
ml of 20% hydrogen peroxide (H2O2) was added
stock solutions was pipettede out and made up to 10ml.
separately. The solutions were kept for 30 min at 60 0c.
(225µg/ml of EPAL and 225µg/ml of PREGA)
For HPLC study, the resultant solution was diluted to
obtain 150µg/ml&150µg/ml solution and 10 µl were
Accuracy
injected into the system and the chromatograms were
Preparation of Standard stock solutions: Accurately
recorded to assess the stability of sample.
weighed 75 mg of Epalrestat, 75 mg of Pregabalin and
transferred to 50ml&50ml volumetric flasks. 3/4 th of
Acid Degradation Studies
diluents was added and solicited for 10 minutes. Flasks
To 1 ml of stock s solution Epalrestat and Pregabalin,
were made up with diluents and labeled as Standard
1ml of 2N Hydrochloric acid was added and refluxed for
stock solution 1and 2.(1500µg/ml EPAL of and
30mins at 600c. The resultant solution was diluted to
1500µg/ml of PREGA)
obtain 150µg/ml&150µg/ml solution and 10 µl solutions
Preparation of 50% Spiked Solution: 0.5ml of sample
were injected into the system and the chromatograms
stock solution was taken into a 10ml volumetric flask, to
were recorded to assess the stability of sample.
that 1.0ml from each standard stock solution was
pipetted out, and made up to the mark with diluent.
Alkali Degradation Studies
Preparation of 100% Spiked Solution: 1.0ml of
To 1 ml of stock solution Epalrestat and Pregabalin, 1 ml
sample stock solution was taken into a 10ml volumetric
of 2N sodium hydroxide was added and refluxed for
flask, to that 1.0ml from each standard stock solution
30mins at 600c. The resultant solution was diluted to
was pipetted out, and made up to the mark with diluent.
obtain 150µg/ml&150µg/ml solution and 10 µl were
Preparation of 150% Spiked Solution: 1.5ml of
injected into the system and the chromatograms were
sample stock solution was taken into a 10ml volumetric
recorded to assess the stability of sample.
flask, to that 1.0ml from each standard stock solution
was pipetted out, and made up to the mark with diluent.

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Dry Heat Degradation Studies to 150µg/ml&150µg/ml solution and 10 µl were injected

The standard drug solution was placed in oven at 105°C into the system and the chromatograms were recorded to
for 6 h to study dry heat degradation. For HPLC study, assess the stability of the sample.
the resultant solution was diluted to
150µg/ml&150µg/ml solution and10µl were injected into RESULTS AND DISCUSSION
the system and the chromatograms were recorded to
Optimized method
assess the stability of the sample.
Chromatographic conditions
Mobile phase : 40% OPA (0.1%): 60%
Photo Stability studies
Acetonitrile
The photochemical stability of the drug was also studied
Flow rate : 1 ml/min
by exposing the 1500µg/ml&1500µg/ml solution to UV
Column : Azilent C18 (4.6 x 150mm,
Light by keeping the beaker in UV Chamber for 7days or
5µm)
200 Watt hours/m2 in photo stability chamber. For HPLC
Detector wave length : 241nm
study, the resultant solution was diluted to obtain
Column temperature : 30°C
150µg/ml&150µg/ml solutions and 10 µl were injected
into the system and the chromatograms were recorded to Injection volume : 10L
assess the stability of sample. Run time : 7 min
Diluent : Water and Acetonitrile in
the ratio 50:50
Neutral Degradation Studies
Results : Both peaks have good
Stress testing under neutral conditions was studied by
resolution, tailing factor, theoretical plate count and
refluxing the drug in water for 6hrs at a temperature of
60º. For HPLC study, the resultant solution was diluted resolution.

Fig 6.8 Optimized Chromatogram.

Table: 6.1 System suitability parameters for Epalrestat and Pregabalin.

S no Pregabalin Epalrestat
Inj RT(min) USP Plate Count Tailing RT(min) USP Plate Count Tailing Resolution
1 2.149 3226 1.15 2.901 3845 1.08 4.4
2 2.154 3380 1.18 2.915 3846 1.08 4.3
3 2.159 3315 1.21 2.910 3832 1.10 4.3
4 2.166 3292 1.19 2.916 3946 1.10 4.4
5 2.169 3314 1.18 2.920 3981 1.08 4.4
6 2.179 2515 1.29 2.930 3054 1.22 3.7

Fig 6.11: System suitability Chromatogram.

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Prasanthi et la. World Journal of Pharmaceutical and Medical Research

Discussion: According to ICH guidelines plate count system suitable parameters were passed and were within
should be more than 2000, tailing factor should be less the limits.
than 2 and resolution must be more than 2. All the

Validation: Specificity

Figure No. 6.12. Chromatogram of blank.

Figure No. 6.13 Chromatogram of placebo.

Linearity
Table 6.2 Linearity table for Epalrestat and Pregabalin.
Epalrestat Pregabalin
Conc (μg/mL) Peak area Conc (μg/mL) Peak area
0 0 0 0
37.5 793975 18.75 392023
75 1538713 37.5 696010
112.5 2335643 56.25 1033486
150 3160302 75 1385625
187.5 3852671 93.75 1736003
225 4611087 112.5 2107476

Fig No. Calibration curve of Epalrestat.

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Prasanthi et la. World Journal of Pharmaceutical and Medical Research

Fig No. Calibration curve of Pregabalin.

Discussion: Six linear concentrations of Epalrestat (37.5- Repeatability

225µg/ml) and Pregabalin (18.75-112.5µg/ml) were Table 6.4 Repeatability table of Epalrestat and
injected in a duplicate manner. Average areas were Pregabalin.
mentioned above and linearity equations obtained for Area of Area of
S. No
Epalrestat was y =20545.x + 16173 and of Pregabalin Epalrestat Pregabalin
was y = 18476x + 10803 Correlation coefficient obtained 1. 3115267 1310152
was 0.999 for the two drugs. 2. 3145891 1311046
3. 3152013 1310287
PRECISION 4. 3140181 1312589
System Precision 5. 3142258 1309030
Table 6.3: System precision table of Epalrestat and 6. 3140106 1304812
Pregabalin. Mean 3139286 1309653
S. No Area of Epalrestat Area of Pregabalin S.D 12592.2 2647.1
1. 3105736 1300458 %RSD 0.4 0.2
2. 3146003 1316596
3. 3145407 1320473 Intermediate precision (Day_ Day Precision)
4. 3159858 1314421 Table 6.5 Intermediate precision table of Epalrestat
5. 3146567 1321248 and Pregabalin.
6. 3145825 1322787 Area Area of
Mean 3141566 1315997 S. No
of Epalrestat Pregabalin
S.D 18417.3 8222.1 1. 3252623 1241050
%RSD 0.6 0.6 2. 3279229 1230421
3. 3296583 1211693
4. 3230020 1226513
5. 3238091 1240509
6. 3251171 1224768
Mean 3257953 1229159
S.D 25274.9 10983.2
%RSD 0.8 0.9

Accuracy
Table Accuracy table of Epalrestat.
Amount Spiked Amount recovered
% Level % Recovery Mean %Recovery
(μg/mL) (μg/mL)
75 74.08 98.77
50% 75 74.39 99.18
75 74.62 99.49
150 150.49 100.33
100% 150 147.25 98.17 98.98%
150 148.32 98.88
225 223.30 99.25
150% 225 221.78 98.57
225 220.86 98.16

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Prasanthi et la. World Journal of Pharmaceutical and Medical Research

Accuracy table of Pregabalin.

Amount Spiked Amount recovered Mean
% Level % Recovery
(μg/mL) (μg/mL) %Recovery
37.5 37.04 98.78
50% 37.5 37.54 100.10
37.5 37.34 99.56
75 73.67 98.22
100% 75 74.06 98.74 99.35%
75 75.16 100.22
112.5 111.51 99.12
150% 112.5 112.15 99.69
112.5 112.23 99.76

Sensitivity
Table: Sensitivity table of Epalrestat and Pregabalin.
Molecule LOD LOQ
Epalrestat 0.20 0.62
Pregabalin 0.18 0.56

Robustness
Table 6.8: Robustness data for Epalrestat and Pregabalin.
%RSD of %RSD of
S.no Condition
Epalrestat Pregabalin
1 Flow rate (-) 0.7ml/min 1.3 1.3
2 Flow rate (+) 0.9ml/min 1.3 1.3
3 Mobile phase (-) 50B:50A 0.4 0.2
4 Mobile phase (+) 40B:60A 0.7 0.8
5 Temperature (-) 25°C 1.8 1.8
6 Temperature (+) 35°C 0.3 0.6

Assay

Fig.: Chromatogram of working standard solution.

Fig. No: Chromatogram of working sample solution.

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Prasanthi et la. World Journal of Pharmaceutical and Medical Research

DEGRADATION
Epalrestat Pregabalin
Standard Sample % Standard Sample
S.no % Assay
Area area Assay Area area
1 3105736 3115267 98.96 1300458 1310152 99.36
2 3146003 3145891 99.94 1316596 1311046 99.42
3 3145407 3152013 100.13 1320473 1310287 99.37
4 3159858 3140181 99.76 1314421 1312589 99.54
5 3146567 3142258 99.82 1321248 1309030 99.27
6 3145825 3140106 99.75 1322787 1304812 98.95
Avg 3141566 3139286 99.73 1315997 1309653 99.32
Stdev 18417.3 12592.2 0.4 8222.1 2647.1 0.2
%RSD 0.6 0.4 0.4 0.6 0.2 0.2

Degradation Studies: Degradation studies were performed with the formulation and the degraded samples were
injected. Assay of the injected samples was calculated and all the samples passed the limits of degradation.

Table: Degradation Data of Epalrestat.

Degradation % Drug Purity Purity
S.NO
Condition Degraded Angle Threshold
1 Acid 4.46 0.155 0.373
2 Alkali 2.88 0.140 0.366
3 Oxidation 1.44 0.105 0.374
4 Thermal 0.78 0.120 0.367
5 UV 0.72 0.128 0.361
6 Water 0.74 0.143 0.371

Table Degradation Data of Pregabalin.

Degradation % Drug Purity Purity
S.NO
Condition Degraded Angle Threshold
1 Acid 4.58 0.605 1.474
2 Alkali 2.48 0.988 1.445
3 Oxidation 1.52 0.172 0.976
4 Thermal 0.81 0.158 0.415
5 UV 0.55 0.132 0.454
6 Water 0.49 0.346 0.504

SUMMARY AND CONCLUSION

Parameters Epalrestat Pregabalin LIMIT
Linearity
37.5-225µg/ml 18.75-112.5µg/ml
Range (µg/ml)
Regressioncoefficient 0.999 0.999
R< 1
Slope(m) 20545 18476
Intercept(c) 16173 10803
Regression equation (Y=mx+c) y = 20545x + 16173 y = 18476x + 10803
Assay(% mean assay) 99.73% 99.32% 90-110%
Specificity Specific Specific No interference of any peak
System precision %RSD 0.6 0.6 NMT 2.0%
Method precision %RSD 0.4 0.2 NMT 2.0%
Accuracy %recovery 98.98% 99.32% 98-102%
LOD 0.20 0.18 NMT 3
LOQ 0.62 0.56 NMT 10
FM 1.3 1.3
FP 1.3 1.3
MM 0.4 0.2
Robustness %RSD NMT 2.0
MP 0.7 0.8
TM 1.8 1.8
TP 0.3 0.6

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Prasanthi et la. World Journal of Pharmaceutical and Medical Research

CONCLUSION Pharmaceutica Sciencia, Jul-Sep, 2012; 2(3).

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