DEVELOPMENT AND VALIDATION OF HIGH PERFORMANCE
LIQUID CHROMATOGRAPHIC METHOD FOR SIMULTANEOUS
ESTIMATION OF EPALRESTAT AND METHYLCOBALAMIN
IN COMBINED DOSAGE FORM
MADHURI A. HINGE*, NIRAJ K. PATEL, RAJVI J. MAHIDA
*
Department of Quality Assurance, Rofel Shri G.M.Bilakhia College of Pharmacy, Vapi
email id: madhuri_shreyal@yahoo.co.in
ABSTRACT
A simple, rapid and sensitive isocratic reversed-phase high-performance liquid
chromatography (RP-HPLC) method was developed and validated for the simultaneous
determination of Epalrestat and Methylcobalamin in various pharmaceutical formulations.
Separation of two drugs was achieved with a Aligent SHIMADZU, C18 (250 x 4.6 mm, 5
μm) and a mobile phase consisting of Methanol: Water in the ratio 30: 70, (v/v) adjusted to
pH 3.5 using orthophosphoric acid. Analyses were performed at a flow rate of 1 ml/min and
at a detection wavelength of 438 nm. The validity of the methods was further
confirmed using the standard addition method. The proposed procedures were successfully
applied to the determination of Epalrestat and Methylcobalamin in bulk and tablet form, with
high percentage of recovery, good accuracy and precision.
Keywords
Epalrestat ; Methylcobalamin; HPLC method.
INTRODUCTION
Epalrestat is Aldose reductase inhibitor. Epalrestat inhibited high glucose-mediated
Neutrophil endothelial adhesion molecules not only through inhibition of a PKCdependent pathway,but also through increased endothelial NO production. It is Used in
treatment of Diabetes. Chemically it is described 2-[(5Z)-5-[(E)-3-phenil-2-methylprop-2enylidene]-4-oxo-2-thioxo-3-thiazolidinyl]acetic acid.(Figure 1)
[1-2]
Methylcobalamin is
use as Vitamin supplement. The synthesis of methionine from homocysteine requires a
folate coenzyme as vitamin B12-dependent enzyme. Chemically it is described Coα-[α(5,6-Dimethylbenz-1H-imidazolyl)]-Co
β-Methylcobamid.(Figure
2)
[3-4]
Methylcobalamin is official in Japan Pharmacopoeia and HPLC method is given for its
estimation in Japan pharmacopeia.
[5]
Extensive literature survey revealed that methods
were reported for the estimation of Epalrestat
[6-8]
and Methylcobalamin
combination with other drugs are spectrophotometric methods
[11]
[9-10]
alone and in
, HPTLC method[12-13],
and RP-HPLC methods[14-17] for Methylcobalamin and Epalrestat.
MATERIALS AND METHODS
Equipments used:
HPLC Model was Aligent SHIMADZU, Column C18 (250 x 4.6 mm, 5 μm), Detector: UV
Detector used for analytical work. The analysis was carried by using LC Solutions software.
All the weighing was carried out on the Electronic analytical balance (Mettler Toledo),
Sonication of samples was carried out by sonicator.
Chemicals and reagents:
Pharmaceutical grade of Epalrestat and Methylcobalamin were supplied by Triveni
Chemicals and Pvt. Ltd.,Vapi). Tablet formulation (Alrista Plus) was procured
from local market.The analytical grade methanol was purchased from Fisher Scientific
(India). The distilled water was used for analytical work and rinsing of clean glasswares.
Chromatographic Condition
C18 column, 250mm × 4.6 mm, 5μm was used for the chromatographic separation at a
detection wavelength of 438 nm. Mobile phase of composition M e t h a n o l : W a t e r
( 3 0 : 7 0 ) v / v , p H - 3 . 5 a d j u s t e d w i t h o r t h o p h o s p h o r i c a c i d was
selected for elution and same mixture was used in the preparation of standard and sample
solutions. Flow rate was adjusted to 1.0 ml/min and the injection volume was 10.0 μl.
Typical chromatogram is given in Figure 3.
Standard solutions and calibration graphs for chromatographic procedure (HPLC)
Standard solutions of EPAL and MCB containing concentration range of 50–150 g/ ml and
0.5-2.5 g/ ml and a fixed concentration 500 μg/ ml of EPAL and 100 μg/ ml of MCB were
prepared in the mobile phase. Triplicate 10 l injections were made for each concentration
and the peak height ratio of each concentration to the internal standard were plotted against
the corresponding concentrations to obtain the calibration graph.
Sample preparation
A total of 20 tablets containing EPAL and MCB as the active ingredient were weighed and
newly powdered. A portion of the powder equivalent to 100 mg EPAL were weighed
accurately, transferred to a 100 ml volumetric flask .The flask was placed in ultrasonic
water bath for 15 min before completion to volume with the same solvent. For HPLC
analysis, 1 ml aliquots of the resulting solution were transferred into a 10 ml volumetric flask
and volume was made up to mark with mobile phase. Solution was then filtered through
0.45μ membrane filter. The diluted solution was analyzed under optimized chromatographic
conditions. The areas of resulting peak were measured at 438 nm.
Validation of RP-HPLC Method
System Suitability Study
The typical values for evaluating system suitability of a chromatographic procedure include
the RSD <1%, tailing factor <2 and theoretical plates >2000. The determination of system
suitability of analytical method was accomplished by assaying six samples of the same
strength of EPAL or MCB. The sample concentration of EPAL and MCB used in this
analysis was 50-250 g/ml and 0.5-2.5 g/ml, respectively. The retention time, peak area,
theoretical plates and tailing factor were evaluated for system suitability.Data of system
suitability study is given in Table 1.
Linearity and Range
The sample strengths ranged from 50 to 250 g/ml and 0.5 to 2.5 g/ml for EPAL and MCB
respectively. The regression analysis was accomplished by slope, intercept and correlation
coefficient (r2).The calibration curves for EPAL and MCB were shown in Figure 4 and 5 and
their corresponding linearity parameters were given in Table 2.
Accuracy and precision
The accuracy was determined by percent recovery method. Furthermore, precision (inter-day
variance and intra-day variance) were determined by assaying samples over a period of 1 day
and 3 days, respectively. The standard concentrations of EPAL used in this investigation
were 50, 100 and 150 g/ml. And, the standard strengths of MCB used in this study were
0.5, 1 and 1.5 g/ml. The results were given in Table 3 and 4.
Specificity
Specificity of a method was determined by testing standard substances against potential
interferences. The method was found to be specific when the test solution was injected and no
interferences were found because of the presence of excipients.
Limit of Detection (LOD) and Limit of Quantitation (LOQ).
The LOD and LOQ were calculated from the slope(s) of the calibration plot and the standard
deviation (SD) of the peak areas using the formulae LOD = 3.3 σ/s and LOQ = 10 σ/s. The
results were given in Table 2.
Robustness
The influence of slight deliberate changes in chromatographic conditions such as column
temperature, flow rate of mobile phase and pH of mobile phase on the retention time and
peak area were observed one by one. The test was performed in triplicate for each set of
conditions. The standard concentrations of EPAL and MCB used in this analysis were 100
g/ml and 1 g/ml, respectively. %RSD was reported in Table 5.
Assay of Marketed Formulations
10.0 l of sample solution of concentration of 100 g/ml EPAL and 1 g/ml of MCB was
injected into chromatographic system and the peak responses were measured and shown in
the figure 3. The solution was injected three times in to the column. The amount of drug
present and percentage purity was calculated by comparing the peak areas of the standards
with that of test samples. Results are given in Table 6.
RESULTS AND DISCUSSION
A reversed phase HPLC method was developed to provide a specific procedure suitable
for rapid quality control of EPAL and MCB tablet dosage form. A mobile phase
consisting of methanol and Water in the ratio of (30:70 v/v) and pH 3.5 adjusted with
ortho phosphoric acid, was chosen after several trials with acetonitrile: water and
methanol: water. The apparent pH of the aqueous phase was adjusted to 3.5 using
orthophosphoric acid. The above described chromatographic system allowed an
adequate resolution (Rs 22.98) between EPAL (tr 3.049) and MCB (tr 10.107) in a
reasonable time (Figure 3) (Rs, resolution; tr, retention time). The applied analytical
conditions produced the peaks with suitable peak symmetry (<2).
The typical conditions for system suitability of an analytical method encompass the
relative standard deviation (RSD) < 1%, peak symmetry <2 and theoretical plates
>2000.The results of system suitability of present chromatographic method are
described in Table 1. The peak area, retention time, tailing factor and theoretical plates
were within the recommended limits. Therefore, the method was considered as suitable.
For quantitative determinations a linear calibration graph (Y =12.351x+13.005, r2 =
0.9987; n=3 , for EPAL and Y=783.95x+7.2218,r2=0.9976;n=3,for MCB) was obtained
over the working concentration range of 50-150 g/ml for EPAL and 0.5-1.5 g/ml
for MCB.
The specificity and selectivity of the HPLC system were ascertained by a separate
chromatographic analysis of either the excipient mixtures or sample; no interfering
peaks at the retention times of EPAL and MCB peaks were observed.
The LOD and LOQ in accordance with the ICH guidelines is 3/1 and 10/1, respectively.
LOD and LOQ values for EPAL and MCB for HPLC method are given in Table 2.
For HPLC method, the calibration curve of EPAL was made with 50, 75, 100, 125 and 150
g/ml concentrations ( Figure 4). Similarly, the concentrations used in the formation of
calibration curve of MCB were 0.5,0.75,1,1.25 and 1.5 g/ml ( Figure 5). The regression
analysis is displayed in Table 2. The correlation coefficient (r2) was close to 0.9999 for
both EPAL and MCB.
The results of precision (inter-day variance and intra-day variance) and accuracy are
shown in Table 3 and 4. In HPLC method the recovery values of EPAL and MCB was
99.21% and 100.75 %. For inter-day and intra-day variance assessment %RSD was
calculated. All the samples exhibited RSD values <1% confirming that the analytical method
was precise.
Robustness study perform by making minor modifications in analytical conditions such
as composition of mobile phase, flow rate of mobile phase and pH of mobile phase. No
substantial variances were observed in the retention time and peak area of each
component when the chromatographic conditions were slightly changed one by one.
Moreover, the RSD for each value was <1%. Thus, the proposed method was
considered as robust.
CONCLUSION
A new RP-HPLC method has been developed for simultaneous estimation of EPAL
and MCB in marketed formulation. The method gave good resolution for both the drugs
with a short analysis run time within 10 min. The developed method was validated. It was
found to be novel, simple, accurate, precise, sensitive and cost effective. Hence
the
proposed RP-HPLC method is suitable for routine assay of EPAL and MCB in
pharmaceutical dosage form in quality control laboratories.
Acknowledgment
The authors are thankful to the Triveni Chemicals and Pvt. Ltd., Vapi for providing the gift
sample of EPAL and MCB. The authors are also thankful to the Principle, ROFEL Shri G.
M. Bilakhia College of Pharmacy, VAPI, India, for providing the required facilities to carry
out this research work.
REFERENCES
[1] Drug profile Epalrestat Drug Information
www.medicinenet.com/ Epalrestat /article.html.
[2] Drug profile Epalrestat Drug Information
www.drugbank.ca/drug/DB01165/ Epalrestat /article.html
[3] Drug profile Methylcobalamin Drug Information
www.drugbank.ca/drug/DB0207/zithromycin/article.html.
[4] Drug profile Methylcobalamin Drug Information
www.medicinenet.com/ Methylcobalamin /article.html.
[5] Japan Pharmacopoeia, Volume-I,part- 1, pp 590-591.J.P.XIV
[6] Sharthachandra S, Dhanlakshmi K, Nagarjuna RG. New Derivative
Spectrophotometric Methods for the Determination of Epalrestat. Int. J. Pharma Sci.
Rev. & Res.21.2013 (1), 247-250.
[7] Janakipathi P, AppalaRaju N. Analytical Development and Validation RP HPLC
method for the estimation of Epalrestat in tablet dosage form. Asian Journal
Pharm Analytical.2013, 2(2), 49-51.
[8] Sengamalam R, Ravindran M, Manishan G, Meena S. Analytical method
development and Dissolution profile of Duloxetine and Methylcobalamine by
Vierodt’s mehod. J. Pharm. Res. 2011, 4, 449-451.
[9] Ganesan M, Solairaj P, Rajesh SC, Senthilkumar T, Thangathirupathi A. A
Simple spectrophotometric method for the estimation of Methylcobalamine in
Injections. Int. J. Pharm. and Phrmaceuticla Sci.2012, 4, 559-562.
[10] Saravanan J, Shajan A, Joshi NH, Valliappan K. A simple and validated
RP- HPLC method for estimation of Methylcobalamine in bulk and capsule dosage
form. Int. J. Chem and Pharmaceutical Sci. 2001,1,13-16.
[11] Galande VR, Baheti KG, Dehghan MH .UV-Vis spectrophotometric method
for estimation of Gabapentine and Methylcobalamine in bulk and tablet. Int.
J. Chemtech res. Coden USA.2010, 0974-4290.
[12] Baheti KG, Galande VR. Validated simultaneous estimation of Gabapentine in
the Presence of Methylcobalamine in tablet by HPTLC method.Int. J.
Res. Pharmaceutical & Biomed. Sci.2011 , 2(3),1199- 1202.
[13] Shah DA, Emanual MP, Mori A .Simultaneous estimation of Pregabalin
and Methylcobalamin in Pharmaceutical formulation by HPTLC- Densitometry
method. J Chromat Separation Techniq 2010,4(2), 100169
[14] Poongothai S, Ilavarasan R, Karrunakaran CM. Simultaneous and
Accurate determination of Vitamins B1, B6, B12 and Alpha Lipoic Acid in
Multvitamin capsule by RP-HPLC method. Int. J. Pharma & Pharmaceuti. Sci. 2010,
2, 133-139.
[15] Narmada P, Vijaya Lakshmi G, Nalini G, Gowtham Y , Suhasini B, Jogi KV.
RPHPLC Method development and validation for the determination of
Methylcobalamin and pregabalamin in combined capsule dosage form. Int. J.
Res. Pharmaceutical sci. 2012, 4(1),25-29.
[16] Kanapan N , Nayak SP, Venkatachalam T , Prabhakaran V. Analytical
RP-HPLC method development and validation of pregabalin and methylcobalamin
in combined capsule dosage formulation. J. App. chem.. Res.2010,13,85-89.
[17] Bhatt KK, Emanual Michael ,Patelia & Aswin Mori .RP-HPLC method
development and validation for Simultaneous estimation of pregabalin
and methylcobalamin in capsules. J.Anal. Bioanal. Tech. 2013, 4(1), 103-109.
Table 1: Mean values of system suitability study (n=5)
EPAL
± % RSD
3.053
1.389
7082.8
22.98
Parameters
Retention time
Tailing factor
Theoretical plates
Resolution
MCB
± % RSD
10.147
1.371
7345.2
Table 2: Results for Linearity (n=3)
Parameters
EPAL
MCB
Slope
Y intercept
12.351
13.005
783.95
7.2218
Correlation
coefficient (r2)
0.9980
0.9979
Regression
equation
12.351x+13.005
783.95x+7.2218
50-150
0.5-1.5
1.978
5.998
0.132
0.401
Linearity
range (g/ml)
LOD (g/ml)
LOQ (g/ml)
Table 3: Results of precision (n=6)
Drug
Intraday
Interday
Precision(%RSD)
Precision(%RSD)
EPAL
1.18-1.25
0.86-1.07
MCB
1.04-1.19
1.06-1.43
n=No. of determinants
Table 4: Results for Accuracy (n=3)
Method
Drug
Amt.present
(g/ml)
Amt.
added
(g/ml)
Amt.recovered %
Recovery
(g/ml)
EPAL
100
80
180.01
100.65
100
100
200.10
100.75
100
120
219.90
99.21
1
0.8
1.81
100.12
1
1.0
1.98
99.62
1
1.2
2.18
99.58
HPLC
Method
MCB
n= No. of determinants
Table 5: Results for Robustness
%RSD
Parameters (n=3)
EPAL
MCB
Mobile Phase +2
0.9437
0.9196
Mobile Phase -2
0.9437
0.9196
Flow rate +2
0.6528
0.7488
Flow rate -2
0.8468
0.8797
pH +2
0.8320
0.9251
pH -2
1.1785
1.1564
n= No. of determinants
Table 6 : Analysis of marketed formulation (n=3)
MARKETED
Labeled claimed
Amt.
found
FORMULATION
(mg/tab)
(mg/tab)
EPAL
ALRISTAPLUS
100
MCB
1
EPAL
99.445
Fig 1 Structure of Epalrestat
Fig 2 Structure of Methylcobalamin
%
Label claim
MCB
1.104
EPAL
99.98
MCB
100.41
Fig.3. Typical Chromatogram of EPAL and MCB
Fig. 4 Calibration curve for EPAL
Fig. 5 Calibration curve for MCB