Development and Validation of
new RP-HPLC Method for simultaneous Estimation
of Methylcobalamin, Epalrestat
and Pregabalin in bulk and Pharmaceutical dosage
form
GITAM Institute of Pharmacy, GITAM Deemed to be University, Rushikonda, Visakhapatnam - 5303045,
India.
*Corresponding Author
E-mail: krishnaphanisri@gmail.com
A new rapid, accurate, precise and economical
reverse phase high performance liquid chromatographic method has been developed and validated for
simultaneous estimation of Methylcobalamin, Epalrestat and Pregabalin in bulk and pharmaceutical dosage form. The
separation was accomplished utilizing Agilent C18, 150 x 4.6mm, 5m column at a detection wavelength of
210nm utilizing the mobile phase water and acetonitrile 60: 40 v/v at the flow rate of 0.8ml/min and injection
volume of 10µl. The total run time was 6.0 min. Validation discovered the method was specific, rapid, accurate,
precise, reliable and reproducible. The calibration curve was linear over the concentration range of 0.37 –
2.25μg/ml of Methylcobalamin, 37.5-225μg/ml
of Epalrestat and 37.5-225μg/ml of Pregabalin
respectively with correlation coefficient of 0.999. The accuracy was determined
by recovery studies and was found to
be 99.5-100%. The precision of the
results stated that the %RSD was <2%. The limits of detection for Methylcobalamin, Epalrestat and Pregabalin
were 0.2, 0.9 and 1.2μg/ml, while the limits of quantification were 0.5, 1.5 and 0.9μg/ml respectively.
Forced degradation study was carried out under acidic, alkaline, oxidative, photolytic and thermal conditions to prove
the stability-indicating ability of the developed HPLC method. The high recovery
confirms the suitability of developed method
and can be further used in routine
analysis.
KEYWORDS: High performance liquid chromatography, Methylcobalamin, Epalrestat, Pregabalin, Method development and Method validation.
Analytical chemistry was used to determine the qualitative and quantitative composition of material under study1. Pharmaceutical
analysis is considered as interdisciplinary subject
comprising of chemistry, pharmacy, physics, electronics, biology etc. It is used in identifying drug substances, called
qualitative analysis, determining the concentration or amount of drug substances, called quantitative analysis
and confirming the structures of drug substances2. Methylcobalamin3 was chemically 3-[({2-[(diaminomethylidene) amino]-1, 3-thiazol-4-yl}
methyl) sulfanyl]-N'- sulfamoylpropanimidamide4.
It is one of the two coenzyme forms of vitamin B12 in the enzyme methionine synthase
which functions to transfer methyl
groups for the regeneration of methionine from
homocysteine5. Epalrestat chemically 2-[(5Z)-5-[(E)-2- methyl-3-phenylprop-2-enylidene]-4-oxo-2- sulfanylidene-1, 3-thiazolidin-3-yl] acetic acid is an aldose reductase inhibitor used for the treatment of diabetic neuropathy6,7,8. Epalrestat (EP), a poorly water-
soluble acidic type drug, with β-cyclodextrin (CD) and with water-soluble polymers9. Pregabalin Chemically is (3S)-3-(aminomethyl)-5-methylhexanoic
acid, an anticonvulsant drug used for neuropathic pain, for partial
seizures partial seizures
in adults currently10 and in generalized anxiety disorder11. It is a white crystalline solid, which is soluble in water and in both basic and acidic aqueous
solutions12. Pregabalin13 (PRN)
is a medication used to treat epilepsy,
neuropathic pain, fibromyalgia, and generalized anxiety
disorder14. These triple
combination drugs used for neuropathy, eye- related disorders
and for treatment of Vitamin B deficiency. The chemical structures of Methylcobalamin, Epalrestat and Pregabalin are shown in Fig.1. The extensive
literature survey revealed that several methods were reported for estimation of Methylcobalamin, Epalrestat and Pregabalin
individually or in combination 15-19,
but no chromatographic method was reported for
simultaneous estimation of triple combination in combined tablet dosage form and yet to be reported. Therefore, it was essential to develop
such a method for bulk and tablet
dosage form. Thus, the method described was simple,
sensitive, rapid, accurate,
precise and stability indicating for routine
quality control analysis.
Figure 1(a) Methylcobalamin
Figure 1(b) Epalrestat
Figure 1(c) Pregabalin
Figure 1 Chemical
structure of Methylcobalamin, Epalrestat and Pregabalin
Methylcobalamin,
Epalrestat and pragabalin were procured from spectrum pharma research
solutions (Hyderabad). Water and acetonitrile were purchased from MERCK (India)
Ltd, Worli and Mumbai India.
High purity water was obtained by using Millipore Milli Q plus water purification system and are of analytical grade.
Instrumentation
and chromatographic conditions: The
chromatographic analysis was carried out on Waters
HPLC equipped with Photo diode array detector.
The output signal
was checked and processed using
Empower 2 software.
UV-Visible spectrophotometer PG Instruments T60 with special
bandwidth of 2mm and 10mm matched quartz cells integrated
with UV win 6 Software was used for measuring absorbance of Methylcobalamin, Epalrestat and Pregabalin. Denver Electronic Balance was used for weighing.
The PH meter and Ultrasonicator obtained
from BVK enterprises, India The Agilent
C18, (150 x 4.6mm, particle
size 5mm), column
was used for the study. Various mobile phases
were tried in order to find the best conditions for separation of Methylcobalamin, Epalrestat and Pregabalin. The suitable mobile phase identified was water:
acetonitrile (60:40 v/v). Flow rate of mobile phase was 0.8ml/min; column temperature was maintained at 30°C. The eluted compounds were monitored at a wavelength of 210nm. The sample injection
volume was 10ml. Mobile
phase was degassed by sonicator prior to use. All determinations were performed at ambient temperature.
On the basis of solubility study acetonitrile
was selected as the solvent for dissolving.
The Mobile phase was prepared by mixing water
and acetonitrile in the ratio of 60:40 (v/v). The resultant solutions were mixed thoroughly and
filtered through a poly-tetra-fluoro ethanol (PTFE) filter of 0.45μm pore size by means of vacuum pump and degassed by sonication to expel
the dissolved gases in solvent system.
Standard solution was prepared by transferring an accurately weighed
0.75mg, 75mg and 75mg of Methylcobalamin, Epalrestat and Pregabalin to 50ml of clean dry volumetric flasks.
The compounds were dissolved with water: acetonitrile 60:40 (v/v) and sonicated for 2 min. The solution was diluted up to the volume
with water: acetonitrile 60:40 (v/v).
The developed method was applied for the assay
of this drug combination in the
pharmaceutical formulation. 5 tablets
were weighed and the average weight of tablets
was calculated. Weight equivalent to 1 tablet was transferred to 100ml volumetric flask, diluents was added
and sonicated for 25 minutes. Further the volume was made up with diluents and filtered.
1ml was pipetted from the filtered solution
and transferred to 10ml volumetric flask. Estimation was carried
out at 210nm.
The HPLC system was stabilized for thirty
minutes by passing mobile phase,
detector was set at 210nm, flow rate of 0.8ml/min was maintained to get a stable base line. Six replicates of a single
standard solution were injected to check the system suitability.
The HPLC procedure
was optimized with a view to develop a suitable LC method for the
determination of Methylcobalamin, Epalrestat and Pregabalin in fixed dose
combined dosage form. The analytical method was developed to select preliminary reversed phase HPLC chromatographic conditions, including detection
wavelength, mobile phase, stationary phase, and sample preparation procedure. For this purpose, a
series of trials were performed
by varying the ratio of water and acetonitrile and optimizing the chromatographic conditions on Agilent C18, (150 x 4.6mm, particle size 5mm),
column. Mobile phase containing water: acetonitrile
taken in the ratio of 60:40v/v was selected.
Methylcobalamin, Epalrestat
and Pregabalin were found to show appreciable absorbance at 210nm when determined Spectro-photometrically and hence it was selected
as the detection wavelength. An optimized chromatogram showing the separation of Methylcobalamin,
Epalrestat and Pregabalin
was shown in figure 2.
Figure 2: Optimized chromatogram
A system suitability test was well defined based on results
found in representative chromatograms. The column efficiency was determined from the analyte peak, the tailing
factor was <2. According to ICH guidelines all the system suitability criteria
during validation of the study
were within the acceptance limit. The results
of system suitability are depicted in table 1
Table 1: System suitability data
|
Para-meters |
Methy- lcobalamin |
Epalrestat |
Pregabalin |
Acceptance Criteria |
|
Retention time |
2.224 |
2.716 |
3.284 |
>2 |
|
Theoretical Plates |
4262.5 |
4840 |
7204.5 |
>2000 |
|
Tailing factor |
1.15 |
1.38 |
1.32 |
<2 |
|
%RSD |
0.7 |
0.8 |
0.8 |
<2 |
Linearity:
The peak area obtained was plotted against
corresponding concentrations to obtain the calibration curve.
The results of linearity study gave linear relationship over the concentration range. The correlation coefficient was found to be 0.999. The linearity
data was represented in table
2.
Table 2: Linearity Date
|
Concentration (µg/ml) |
Methylcobalamin |
Epalrestat |
Pregabalin |
|
0 |
0 |
0 |
0 |
|
25 |
5070 |
591200 |
510664 |
|
50 |
10923 |
1180326 |
1110279 |
|
75 |
15683 |
1658384 |
1608412 |
|
100 |
20096 |
2277730 |
2162736 |
|
125 |
25677 |
2767411 |
2709007 |
|
150 |
30617 |
3308037 |
3203628 |
|
Statistical Analysis |
|||
|
Correlation coefficient |
0.999 |
0.999 |
0.999 |
|
Slope |
13360 |
14779 |
14416 |
|
Intercept |
8085 |
19510 |
17525 |
Accuracy of the method was confirmed by recovery study from marketed formulation at three
levels (50%, 100%, and 150%) of standard addition.
Percentage recovery for Methylcobalamin, Epalrestat and Pregabalin were
found to be in the range of 98-102%. The
results are depicted in table 3
Table 3: Accuracy
data
|
Recovery % |
Methylcobalamin |
Epalrestat |
Pregabalin |
|
50% |
100.3 |
99 |
98.76 |
|
100% |
100.92 |
99.32 |
99.86 |
|
150% |
98.86 |
100.2 |
100.8 |
Precision was evaluated
at the repeatability and intermediate precision levels. For
repeatability analysis, six independent portions of a sample solution
of Methylcobalamin, Epalrestat and Pregabalin were processed through the full analytical
method and results were evaluated
obtaining a % RSD value of 0.5, 0.9 and 0.3. Intermediate precision was
evaluated with a new series of six
portions of the same sample used in the repeatability assay, processed on a different
day, one week later and by a different analyst.
The corresponding % RSD was 0.7, 0.9 and 0.9.
The analytical method robustness was tested by evaluating
the influence of minor modifications in HPLC
conditions on system suitability parameters of the proposed method such as the composition
of the mobile phase, temperature and
flow rate. In all modifications, good separation of was achieved, and it
was observed that the percent of
recovery was within acceptable limits and
the %RSD was within limit of not more than 2.0%. The tailing factors and number of theoretical plates were found within acceptable limits as well. The results are summarized in table 4.
LOD is the lowest amount of the analyte that can be detected. The results are shown in table 5.
Table 4: Robustness data
|
Percentage RSD of the drug |
Flow rate |
Mobile phase |
Temperature |
|||
|
Flow rate plus |
Flow rate minus |
Mobile phase plus |
Mobile phase minus |
Temperature plus |
Temperature minus |
|
|
Methylcobalamin |
0.6 |
0.8 |
0.9 |
1.1 |
1.1 |
0.4 |
|
Epalrestat |
0.3 |
0.7 |
1.0 |
0.9 |
0.2 |
0.6 |
|
Pregabalin |
0.6 |
0.9 |
0.2 |
0.6 |
0.9 |
0.4 |
Table 5: LOD and
LOQ results
|
S No |
Drug name |
LOD |
LOQ |
|
1 |
Methylcobalamin |
0.2 |
0.5 |
|
2 |
Epalrestat |
0.9 |
1.5 |
|
3 |
Pregabalin |
1.2 |
0.9 |
The forced degradation study for the triple
combination drug revealed
that drug was degraded under the influence
of acid, alkali,
thermal, water, hydrogen
peroxide and photolytic conditions. Data was recorded in table
6
Table 6: Degradation results
|
S No |
Degradation condition |
%Drug degraded |
||
|
Methylcobalamin |
Epalrestat |
Pregabalin |
||
|
1 |
Acid |
7.30 |
8.04 |
6.64 |
|
2 |
Alkali |
4.45 |
7.50 |
7.62 |
|
3 |
Thermal |
3.58 |
6.94 |
5.03 |
|
4 |
Uv |
2.42 |
5.30 |
2.26 |
|
5 |
Water |
1.80 |
1.80 |
1.75 |
|
6 |
Peroxide |
0.82 |
0.50 |
0.46 |
The present research work was a simple, rapid,
accurate, precise and stability indicating RP-HPLC method coupled
with photo diode array detector
has been developed
for simultaneous estimation of Methylcobalamin, Epalrestat and Pregabalin. The developed method was completely
validated with respect to
linearity, accuracy, precision, robustness, sensitivity and specificity. The analytical method conditions and the mobile
phase solvents provided
good resolution. The main
features of the analytically developed method are short run time and retention
time. Hence, the simultaneous method can be successfully used for routine
analysis.
The authors are thankful to GITAM University for providing the necessary
facilities to carry out this work.
The authors declare
no conflict of interest.
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Received on 05.08.2020
Modified on 25.09.2020
Accepted on 30.10.2020
© RJPT All right reserved
Research J. Pharm.
and Tech. 2021; 14(10):5097-5100.
DOI: 10.52711/0974-360X.2021.00888