Development
and validation of RP-HPLC method for simultaneous estimation of Paracetamol and
Flupirtine Maleate
Sathish
Kumar Konidala*, Adinarayana
Penumala, Vinod Kumar Mugada, Govinda Rao Kamala
Aditya Institute of
Pharmaceutical Scienecs and Research, Surampalem, Kakinada,
East Godawary, Andhra Pradesh, India – 533 437
*Corresponding Author E-mail: sathishkonidala@gmail.com
ABSTRACT:
A simple, validated RP-HPLC method for the simultaneous estimation
of Paracetamol and Flupirtine maleate
in pharmaceutical dosage form was developed and validated. This method was
developed by selecting Inertsil, C18, (250 x 4.6mm,
5µ) column as stationary phase and Methanol: O-Phosporic
acid (85: 15 v/v) as mobile phase. Flow rate of mobile phase was maintained at
1 ml/min at ambient temperature throughout the experiment. Quantification was
achieved with ultraviolet (DAD) detection at 239 nm. The retention times of
Paracetamol and Flupirtine maleate
were found as 2.78 min and 3.47 min respectively. The detector response was
linear in the concentration range of 65-190 μg/ml
and 20-60 μg/ml for Paracetamol and Flupirtine maleate respectively,
and the regression coefficients found as 0.998 and 0.999 for Paracetamol and Flupirtine maleate respectively.
From recovery studies we concluded that the recovery of Paracetamol and Flupirtine maleate has no
interference with any excepients in the formulation.
This method has been validated according to ICH guidelines and shown to be
Specific, Sensitive, Precise, Accurate, Rugged and Robust. Hence, this method
can be applied for routine quality control of Paracetamol and Flupirtine maleate in dosage
forms as well as in bulk drug.
KEYWORDS: Paracetamol
and Flupirtine maleate;
RP-HPLC; Analytical method validation; Lupirtine- P
tablets.
INTRODUCTION:
Paracetamol
Paracetamol1 is a common analgesic and antipyretic
drug that is used for the relief of fever, headaches and other minor aches and
pains is a weak inhibitor of PG synthesis of COX-1 and COX-2 in broken cell systems, but,
therapeutic concentrations of Paracetamol inhibit PG synthesis in intact cells
in vitro. Their determination in pharmaceuticals is of paramount importance,
since an overdose of Paracetamol can cause fulminating hepatic necrosis and
other toxic effects. It has the structural formula and shown in Fig. 1.
Figure 1:
Chemical structure of Paracetamol
The
chemical name of Paracetamol is N-(4-hydroxyphenyl) acetamide.
The molecular formula of paracetamol is C8H9NO2
and it has the molecular weight of 151.1 g/mol. It is freely soluble in methyl
alcohol. It has the melting point of 169-171 oC,
pH of 5.5-6.5 and pKa of 9.38.
Flupirtine
Maleate
Flupirtine Maleate2 is a non-opioid centrally-acting analgesic agent, structurally
dissimilar from ther analgesics. It has the
structural formula and shown in Fig. 2.
Figure 2: Chemical
structure of Flupirtine Maleate
The
chemical name Flupirtine
Maleate is Ethyl
N-(2-amino-6-{[(4-fluorophenyl)methyl]amino}pyridin-3-yl)carbamate.
The molecular formula of Flupirtine Maleate is C15H17FN4O2
and it has the molecular mass of 304.31 g/mol. It is soluble in methanol. It
has the melting point of 115.5 o C, pH of 2.61 and pKa of 12.9.
From the literature survey, we found that Paracetamol and Flupirtine maleate were estimated
by different analytical methods like RP-HPLC, 3-8 LC-MS9
and UV spectrophotometry.10
Only one HPLC method for simultaneous estimation Paracetamol and Flupirtine maleate
is available, but it show high Rt Values (time
consuming) method.
So, there is a need to develop a validated RP-HPLC method
for simultaneous estimation of Paracetamol and Flupirtine maleate which can
complete the estimation in short time and with highly accurate recovery
results.
This study aimed to develop a simple, precise, accurate and
validated Reversed-Phase HPLC method for the simultaneous estimation of Paracetamol and Flupirtine maleate
in bulk and pharmaceutical dosage form as per ICH guidelines. The statistical
analysis proved that method is reproducible and selective for the simultaneous
analysis of Paracetamol and Flupirtine maleate in bulk and formulations.
MATERIALS AND METHODS:
Chemicals and Instruments:
Gift samples of Pharmaceutical grade Paracetamol and Flupirtine maleate
were supplied by Hetero Drugs Ltd., Hyderabad, India. The Methanol (HPLC
grade), Acetonirile (HPLC grade) and Ortho Phosphoric
acid were purchased from MERCK and triple distilled water was collected from
in- house production. The commercially available Lupirtine-
P tablets (one equivalent to 325mg of Paracetamol and 100mg of Flupirtine maleate)
manufactured by Lupin was purchased from market for
analysis.
The instruments that are used in proposed method are Waters
HPLC 2690 system connected to DAD detector and LC-GC AGN204PO balance was used
for all weighing.
Method Development
Chromatographic Conditions
Chromatographic separation was achieved by using Inertsil, C18, (250x4.6mm, 5µ) column as stationary phase
and composition of Methanol: O-Phosporic acid (85: 15
v/v) as mobile phase. Flow rate was maintained at 1 ml/min at ambient
temperature and the injection volume used was 20μl. The detection was
carried out at 239 nm. Mobile phase is used as diluent the samples were
filtered through Whatman filter paper (0.45μm) and degassed before
injected into the system. Typical chromatogram of blank, standard drug and Samples were
as shown in Fig. 3, 4 & 5.
Preparation of stock solution
10mg of Flupirtine maleate and 32.5mg of Paracetamol RS drugs were weighed
individually and taken into a 100mL clean dry volumetric flask and added about 70mL of diluent. It was sonicated to
dissolve completely and made volume up to the mark with the same diluents (100, 325 µg/mL). From
this, 4 ml of the solution was pipette into another 10ml volumetric flask and
diluted up to the mark with diluent (40, 130µg/mL).
Preparation of mobile phase
The mobile phase was prepared by mixing 850 ml of methanol,
150 ml of O-Phosphoric acid, filtered through Whatman filter paper
(0.45μm) and degassed before use.
Figure 3: Typical chromatogram of blank
Figure 4: Typical chromatogram of Standard drugs
Figure 5: Typical Chromatogram of Sample
Table 1: Analysis of Formulation
|
S.NO
|
Drug
|
Label claim(mg)
|
Amount found(mg)*
|
% of Assay
|
|
1
|
Paracetamol
|
325
|
324.49
|
99.84
|
|
2
|
Flupirtine maleate
|
100
|
100.93
|
100.93
|
|
|
|
|
|
|
* Mean of three readings
Preparation of working sample solution:
Twenty tablets containing Paracetamol and Flupirtine maleate combination of
marketed formulation was taken and powdered. The powder equivalent to 10mg of Flupirtine maleate and 32.5mg of
Paracetamol RS drugs were weighed individually and taken into a 100mL clean dry
volumetric flask and added about 70mL of diluent. It was sonicated
to dissolve completely and made volume up to the mark with the same diluents
(100, 325 µg/mL). From this, 4 ml of the solution was
pipette into another 10ml volumetric flask and diluted up to the mark with
diluent (40, 130µg/mL). A typical chromatogram of Lupirtine- P tablet formulation (sample) drug was shown in
Fig. 4. The assay results are shown in table 1.
METHOD VALIDATION:
The method was validated for its linearity range, accuracy,
precision, sensitivity and specificity. Method validation is carried out as per
ICH guidelines.11
System Suitability:
A standard solution was prepared by using Paracetamol and Flupirtine maleate working
standards as per test method and was injected five times into the HPLC system.
The system suitability parameters were evaluated from standard chromatograms by
calculating the % RSD from five replicate injections for Paracetamol and Flupirtine maleate retention
times and peak areas.
Specificity:
Blank and sample were prepared and are injected into chromatographic
system.
Linearity:
A Series of solutions are prepared using Paracetamol and Flupirtine maleate working
standards at concentration levels from 20ppm to 40ppm and 65-195µg/ml of target
concentration.
Precision:
i. Repeatability:
a. System precision: Standard solution prepared as per test method and
injected five times.
b. Method precision: Prepared six sample preparations individually using
single as per test method and injected each solution.
ii. Intermediate precision (analyst to analyst variability):
A study was conducted by two analysts as per test method.
Accuracy (Recovery):
A study of Accuracy was conducted. Drug Assay was performed in triplicate
as per test method with equivalent amount of Paracetamol and Flupirtine maleate into each
volumetric flask for each spike level to get the concentration of Paracetamol
and Flupirtine maleate equivalent
to 50%, 100%, and 150% of the labeled amount as per the test method. The
average % recovery of Paracetamol and Flupirtine maleate were calculated.
Robustness:
Effect of variation of flow rate:
A study was conducted to determine the effect of variation in flow rate.
Standard solution prepared as per the test method was injected into the HPLC
system using flow rates, 0.8ml/min and 1.2ml/min.
Limit of Detection and Quantitation (LOD
and LOQ):
From the linearity data calculate the limit of detection
and quantitation, using the following formula.
σ = Standard deviation
of the response
S = Slope of the calibration curve of the analyte
σ = Standard deviation
of the response.
S = slope of the calibration curve of the analyte.
RESULTS AND DISCUSSION:
System Suitability
Table 2: System Suitability Results
|
Parameter
|
Paracetamol
|
|
Rt
|
Area
|
USP
Tailing
|
Plate
Count
|
|
Mean*
|
2.78
|
1287211
|
0.88
|
3968.34
|
|
% RSD
|
0.16
|
0.22
|
0.08
|
0.35
|
|
Parameter
|
Flupirtine maleate
|
|
Rt
|
Area
|
USP
Tailing
|
Plate
Count
|
|
Mean*
|
3.47
|
786451.8
|
1.15
|
6079.15
|
|
% RSD
|
0.11
|
0.05
|
0.29
|
0.44
|
* Average of Five Readings
Acceptance
Criteria:
1)
The %
RSD for the retention times of principal peak from 5 replicate injections of
each Standard solution should be not more than 2.0 %
2)
2.
The % RSD for the peak area responses of principal peak from 5 replicate
injections of each standard Solution should be not more than 2.0%.
3) 3. The
number of theoretical plates (N) for Paracetamol and Flupirtine
maleate peaks is
NLT 3000.
4) 4. The Tailing factor (T) for Paracetamol
and Flupirtine maleate
peaks is NMT 2.0
Observation:
The %RSD for retention times and peak areas were found to be within the
limit.
Specificity:
Blank interference:
Fig.6: Interference of blank
Acceptance Criteria:
The peaks of blank should not interfere with peaks of Paracetamol and Flupirtine maleate.
Observation:
No peaks were interfered with Flupirtine Maleate and Paracetamol.
Linearity
Fig. 7: Calibration Curve of
Paracetamol
Fig. 8: Calibration Curve of Flupirtine maleate
Table 3: Linearity Results
|
S.No
|
Paracetamol
|
Flupirtine maleate
|
|
Conc.
(µg/ml)
|
Area
|
Conc.
(µg/ml)
|
Area
|
|
01
|
65
|
650897
|
20
|
385989
|
|
02
|
97.5
|
975648
|
30
|
598796
|
|
03
|
130
|
1287967
|
40
|
786757
|
|
04
|
162.5
|
1598098
|
50
|
976578
|
|
05
|
195
|
1897869
|
60
|
1167876
|
|
Slope
|
9831
|
Slope
|
19562
|
|
R2
|
0.9998
|
R2
|
0.9995
|
% of RSD for level 1 and
Level 6 should be not more than 2.0%.
Observation:
The linear fit of the system was
illustrated graphically. The results are presented in table and chromatograms
are shown in fig.
Precision
i. Reapeatability
Table 4: Repeatability Precision Results
|
Paracetamol
|
|
Conc. (µg/ml)
|
Peak Area*
|
SD
|
% RSD
|
|
130
|
1277112
|
10256.02
|
0.80
|
|
Flupirtine
maleate
|
|
Conc. (µg/ml)
|
Peak Area*
|
SD
|
% RSD
|
|
40
|
786683.8
|
149.52
|
0.019
|
* Average of Six Reading
Acceptance Criteria:
ü
The %
relative standard deviation of individual samples from the six units should be
not more than 2.0%.
ü
The assay of Paracetamol and Flupirtine maleate should be not
less than 98% and not more than 102.0%.
Observation:
Test results are showing that the test method is precise. Results are
given in tables and for system precision and method precision and chromatograms
are shown in fig. for system precision and for method precision.
ii. Intermediate Precision
Table 5: Intermediate Precision Results
|
Paracetamol
|
|
Conc. (µg/ml)
|
Peak Area
|
SD
|
% RSD
|
|
130
|
128582
|
4452.60
|
0.34
|
|
Flupirtine
maleate
|
|
Conc. (µg/ml)
|
Peak Area
|
SD
|
% RSD
|
|
40
|
786535.6
|
428.42
|
0.05
|
* Average
of Six Reading
Acceptance Criteria:
The individual assays of Paracetamol and Flupirtine
maleate should be not less than 98% and not more than
102% and %RSD of assay should be NMT 2.0% by both analysts.
Observation:
Individual %assays and %RSD of Assay are within limit and
passes the intermediate precision.
Accuracy
Acceptance Criteria:
The mean % recovery of Paracetamol and Flupirtine
maleate at each spike level should be not less than
98.0% and not more than 102.0%.
Observation:
The recovery results indicating that the test method
has an acceptable level of accuracy.
Table 6: Accuracy results for Paracetamol
and Flupiritine Maleate
|
Conc.
(µg/ml)
|
Paracetamol
|
Flupirtine maleate
|
|
Area
|
Amount
Added
(µg/ml)
|
%
Recovery*
|
%
RSD
|
Area
|
Amount
Added
(µg/ml)
|
%
Recovery*
|
%
RSD
|
|
50
|
650477.3
|
65
|
101.92
|
0.46
|
384853
|
20
|
99.08
|
0.87
|
|
100
|
1290803
|
130
|
101.12
|
1.03
|
786973
|
40
|
100.47
|
0.44
|
|
150
|
1898188
|
195
|
99.14
|
0.86
|
1174890
|
60
|
100.03
|
0.37
|
* Mean of three readings
Table 7: Robustness of Paracetamol and Flupirtine maleate
|
Parameter
Change
|
Paracetamol
|
Flupirtine maleate
|
|
Rt*
|
Peak
Area*
|
%
RSD
|
Rt
|
Peak
Area
|
%
RSD
|
|
Flow rate (±0.2 ml/min)
|
0.8ml
|
3.07
|
1275786
|
0.34
|
3.84
|
786798
|
0.47
|
|
1 ml
|
2.79
|
1287967
|
0.27
|
3.48
|
786757
|
1.04
|
|
1.2ml
|
2.53
|
1277112
|
0.86
|
3.15
|
790121
|
0.35
|
* Mean of six
readings, % RSD values for Peak Area
Robustness:
Acceptance Criteria:
The Tailing Factor of Paracetamol and Flupirtine
maleate standards should be NMT 2.0 for Variation in
Flow.
Observation:
The tailing factor for Paracetamol and Flupirtine
maleate were found to be within the limits
Limit of detection and limit of
quantification
Table 8: LOD and LOQ results for
Paracetamol and Flupirtine maleate
|
S.NO
|
Drug Name
|
LOD
|
LOQ
|
|
01
|
Paracetamol
|
8.23
|
24.93
|
|
02
|
Flupirtine Maleate
|
4.15
|
12.57
|
SUMMARY AND CONCLUSION
A new method was developed for simultaneous estimation of
Paracetamol and Flupirtine maleate
by RP-HPLC method. The sample preparation was simple and analysis time was
short. The analytical procedure was validated as per ICH guidelines and shown
to be accurate, precise and specific.
The analytical method was developed by studying different parameters. Maximum absorbance was found to be at 239nm for
Paracetamol and Flupirtine maleate
and the peak purity was excellent. Injection volume was selected
to be 20µl which gave a good peak area. The column used for study was Inertsil C18 chosen good peak shape. Ambient
temperature was found to be suitable for the nature of drug solution. The flow
rate was fixed at 1.0ml/min because of good peak area and satisfactory
retention time. Different
ratios of mobile phase were studied, and mobile phase with ratio of 85:15(methanol:o-phosporic acid) was fixed due to good
symmetrical peak. So this mobile phase was used for the proposed study.
Methanol was selected because of maximum extraction, sonication time was fixed to be
5min at which all the drug particles were completely soluble and showed good
recovery. Run time was selected to be 5min because analyse
gave peak around 2.78 and
3.47 also to reduce the total run time.
The present recovery
was found to be 98.86-100.73 was linear and precise over the same range. Both system
and method precision was found to be accurate and well within range. Detection limit was found to be 8.23 for PCM and 4.15 for Flupirtine maleate. Linearity study correlation coefficient 0.999 and curve fitting was found. The analytical method was
found linearity over the range of 20-60, 65-195ppm of the target concentration.
The analytical passed both robustness and ruggedness tests. On both cases, relative
standard deviation was well satisfactory.
This method represents a fast
analytical procedure for the simultaneous quantitation
of Paracetamol and Flupirtine maleate.
This method can be applicable to the routine analysis of large numbers of
sample with good precision and accuracy.
Hence this
method was found to be simple, accurate, economical and rapid and they can be
applied for routine analysis in laboratories and is suitable for the quality
control of the raw materials, formulations, dissolution studies and can be
employed for bioequivalence studies for the same formulation.
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