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International Standard Serial Number (ISSN): 2319-8141
International Journal of Universal Pharmacy and Bio Sciences 2(2): March-April 2013
INTERNATIONAL JOURNAL OF UNIVERSAL
PHARMACY AND BIO SCIENCES
Pharmaceutical Sciences
Research Article……!!!
Received: 06-03-2013; Accepted: 16-03-2013
DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR
SIMULTANEOUS ESTIMATION OF PARACETAMOL AND
PAMABROM IN BULK AND PHARMACEUTICAL DOSAGE FORM
Hardik Padaliya*, Hemant Patel
Babaria Institute of Pharmacy, Vadodara-Mumbai NH#8, Varnama, Vadodara-391240, Gujarat
(India).
KEYWORDS:
ABSTRACT
A simple, rapid and precise Reverse Phase High Performance
RP-HPLC,
Liquid Chromatographic method was developed for simultaneous
Paracetamol,
estimation of Paracetamol and Pamabrom in pharmaceutical
Pamabrom, Validation.
For Correspondence:
dosage form by reverse phase C18 column ODS HyperSil (250
Hardik Padaliya*
mm, 4.6 mm, and 5 μm). The sample was analyzed using
Address: Babaria
phosphate buffer (pH-4.8): Acetonitrile (85:15, v/v), as a mobile
Institute of Pharmacy,
phase at a flow rate of 0.8 ml/min. and detection at 277 nm. The
Vadodara-Mumbai
NH#8, Varnama,
retention time for paracetamol and pamabrom was found to be 5.7
Vadodara-391240,
min and 8.0 min, respectively. The linearity of developed method
Gujarat (India)
was achieved in the range of 5-50 μg/ml for paracetamol and 2-
Email:
hardik_padaliya@yahoo.
com
20μg/ml for Pamabrom. The method was validated in terms of
accuracy, precision, linearity, limit of detection, limit of
quantitation, robustness and ruggedness as per ICH guidelines.
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International Standard Serial Number (ISSN): 2319-8141
1. INTRODUCTION :
Paracetamol (N- 4-hydroxyphenyl acetamide) is an Analgesic-antipyretic drugs with poor
Anti-inflammatory action, belongs to Para-aminophenol derivative categories of NSAIDs.
The Main mechanism of action of paracetamol is considered to be the inhibition of
cyclooxygenase (COX), recent findings suggest that it is highly selective for COX-2.Because
of its selectivity for COX-2 it does not significantly inhibit the production of the pro-clotting
thromboxanes [1].
Pamabrom is a xanthine derivative drug and it might increase the renal blood flow by virtue
of their cardiac stimulant property and vasodilator action which promote filtration of fluid by
the glomeruli. They also produce dieresis by diminishing the tubular reabsorption of water.
The chief mechanism seems to be the interference in tubular reabsorption of Na+ and Cl- ,
perhaps by acting on the enzyme concerned with the transport of these ions. This eventually
leads to less absorption of water and favours its excretion [2].
Paracetamol and Pamabrom tablet is use for menstrual pain. Paracetamol is a official in
Indian Pharmacopoeia, British Pharmacopoeia, and United state Pharmacopoeia. Many
methods like spectroscopy, HPLC, and HPTLC method are reported for estimation
paracetamol. Pamabrom is a Official in United state Pharmacopoeia. Only HPLC method is
reported for estimation Pamabrom.
Paracetamol and Pamabrom combination is approved by CDSCO. But no method Reported
for estimation of Combination of Paracetamol and Pamabrom in pharmaceutical dosage form.
Fig. 1: Chemical structures of the Paracetamol
Fig. 2: Chemical structures of the Pamabrom
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International Standard Serial Number (ISSN): 2319-8141
MATERIALS AND METHODS:
Chemicals and solvents:
Potassium di hydrogen phosphate (AR grade) was used for preparing the buffer. HPLC grade
Acetonitrile, methanol was used. Pure sample of Pamabrom was a gift sample from suven
pharmaceutical (Hyderabad).
Chromatographic Conditions:
A High pressure liquid chromatography (Shimadzu L- 2400) with variable wavelength
programmable UV Visible detector and ODS HyperSil C-18 column [250mm, 4.6m, 5μm]
was used. A freshly prepared mixture of potassium di-hydrogen phosphate buffer (pH-4.8):
Acetonitrile (85:15 v/v) used as the mobile phase. Buffer solution was prepared by dissolving
6.8gms of potassium dihydrogen phosphate in 1000ml of water. Mobile phase was filtered
through a 0.45 μm membrane filter and sonicated before use. The flow rate of the mobile
phase was maintained at 0.8 ml/min [3, 6-21].
Preparation of standard stock solutions:
Reference standard of paracetamol 25 mg and pamabrom 10 mg was transferred to 10 ml
volumetric flask separately and dissolved in methanol. The flask was shaken for 30 min and
the volume was made up to the mark with mobile phase to obtain standard stock solution of
Paracetamol (2500 µg/ml) and Pamabrom (1000 µg/ml). Stock solution was filtered through
a 0.2 µm membrane filter. The working standard solution of paracetamol and Pamabrom was
prepared from suitable aliquots of stock solution.
Preparation of Sample Solution:
Take 10mg of PBM and 25 mg of PCM and transferred in to a 10 ml volumetric flask and
sonicated for 20 min. The solution was filtered through whatman filter paper and the volume
was adjusted up to the mark with mobile phase. This solution is expected to contain
1000µg/ml PBM and 2500µg/ml PCM. This solution (1 ml) was taken in to a 10 ml
volumetric flask and the volume was adjusted up to mark with mobile phase to get a
concentration of PBM (100µg/ml) and PCM (250 µg/ml). From this solution(1 ml) was taken
in to a 10 ml volumetric flask and the volume was adjusted up to mark with mobile phase to
get a final concentration of PBM (10µg/ml) and PCM (25 µg/ml).
Optimization of the HPLC method:
The pure drug solution of paracetamol and pamabrom were injected individually into HPLC
system and allow run in different mobile phases like methanol: phosphate buffer, methanol:
Acetonitrile: water and methanol: water were tried in order to find the optimum conditions
for the separation of paracetamol and pamabrom. It was found that mobile phase containing
Phosphate buffer(pH-4.8):Acetonitrile(85:15 v/v), at a flow rate of 0.8 ml/min with detection
at 277 nm gave satisfactory results with sharp well defined and resolved peaks with minimum
tailing as compared to other mobile phases. Under these conditions the retention times were
typically 5.7 min for paracetamol and 8.0 min for pamabrom (Fig. 3) [5].
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VALIDATION OF THE METHOD:
Validation of the optimized HPLC method was carried out with respect to the following
parameters [4].
Linearity and range:
From standard stock solution, aliquots of 0.2 ,0.4, 0.8 ,1.2 ,1.6 and 2.0 ml was transferred to
10 ml volumetric flask and the volume was made up to the mark with mobile phase to obtain
concentration of paracetamol 5-50 μg/ml and Pamabrom 2-20 μg/ml. The solution of 20 μl
was injected into column with the help of Hamilton syringe. All measurements were repeated
three times for each concentration. The calibration curves of the area under curve Vs
concentration were recorded for both drugs.
Precision:
The precision of the method was verified by repeatability, interday and intraday precision.
Repeatability studies were performed by analysis of three different concentrations of the drug
in six times on the same day. Intraday precision was determined by analyzing sample
solutions at different time intervals on the same day and on different day for interday
precision.
Accuracy:
Recovery studies were carried out by adding a known amount of standard solution of pure
drug (paracetamol and pamabrom) to a pre analysed sample solution. These studies were
carried out at 80%, 100% and 120% level.
Limit of detection and limit of quantitation:
The LOD and LOQ were separately determined based on the calibration curves. The
Standard Deviation of they- intercept and slope of the regression line were used.
The LOD and LOQ were calculated using the formulas,
LOD = 3.3 × D / S
LOQ = 10 × D / S
Where,
S = Slope of regression line
D = Standard deviation of y- intercept on the regression line.
Robustness of method:
To evaluate the robustness of the developed RP-HPLC method, minute variations in the
optimized method parameters were done. The parameters such as, effect of change in pH of
mobile phase, flow rate, effect of mobile phase ratio on the retention time, theoretical plates,
area under curve and percentage content of paracetamol and pamabrom were studied. The
solution containing, Paracetamol (25µg/ml) and Pamabrom (10µg/ml) was injected into
sample injector of RP- HPLC three times under the varied conditions.
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International Standard Serial Number (ISSN): 2319-8141
Ruggedness of method:
Standard solution containing mixture of paracetamol (25µg/ml) and pamabrom (10µg/ml)
was prepared from stock solution and analyzed by two different analyst using same
operational and environmental conditions. From the area, the amounts of both the drugs were
calculated.
System Suitability Parameters:
As per USP-24, system suitability tests were carried out on freshly prepared standard stock
solution of paracetamol and pamabrom of both drugs 20µl were injected under optimized
chromatographic condition and following parameters were studied to evaluate the suitability
of the system.
Analysis of a Synthetic Mixture:
The tablet powder equivalent to Paracetamol (250mg) and Pamabrom (100mg) was weighed,
transferred to a 100 ml volumetric flask and dissolved in methanol, shake for 30 min and the
volume was made up to the mark with mobile phase. The content was ultra sonicated for 20
min. The solution was filtered through a 0.2 µm membrane filter paper. This tablet solution
was further diluted with mobile phase to obtain mixed sample solutions in the Beer’s and
Lamberts range.
RESULTS AND DISCUSSION:
The results of validation studies on simultaneous estimation method developed for
paracetamol and pamabrom in the current study involving phosphate buffer(pH-4.8) and
Acetonitrile(85:15 v/v%), as the mobile phase for HPLC are given below.
Linearity:
The drug response was linear (r2 = 0.9994 for paracetamol and 0.9997 for pamabrom) over
the concentration range between 5-50 μg/ml for paracetamol and 2-20 μg/ml for pamabrom
(Table-1&2).
Precision:
The results of the repeatability, intra-day and inter-day precision experiments are shown in
Table -3 and Table -4. The developed method was found to be precise as the RSD values for
repeatability of intra-day and interday precision studies were < 2 %, respectively which is
under limit as per recommendations of ICH guidelines.
LOD and LOQ:
The LOD and LOQ were separately determined based on the calibration curves for
paracetamol and pamabrom. The LOD and LOQ were found to be 0.33µg/ml and 1.007
µg/ml for paracetamol and 0.092 µg/ml and 0.27µg/ml for Pamabrom respectively(Table-2).
Robustness and Ruggedness:
The standard deviation of the peak areas was calculated for each parameter and the % RSD
was found to be less than 2 %. Results shows low values of % RSD, as shown in Table -7 and
Table -8 signify the robustness and ruggedness of the method.
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International Standard Serial Number (ISSN): 2319-8141
Recovery studies:
As shown from the data in Table 6, good recoveries of the paracetamol and pamabrom in the
range from 99 to 101 % were obtained at various added concentrations.
Analysis of a Synthetic Mixture:
Experimental results of the amount of paracetamol and pamabrom in mixture, expressed as a
percentage of label claims were in good agreement, thereby suggesting that there is no
interference from any of the excipients which are normally present in tablets. In the replicate
analysis (n=6) of paracetamol and pamabrom by proposed method showed that the content of
paracetamol and pamabrom was 99.82% and 97.72% respectively. The retention times of
paracetamol and pamabrom was found to be 5.78 min. and 8.08min respectively and the
result of the analysis of tablet are given in Table 5.
System suitability studies:
The column efficiency, resolution and peak asymmetry were calculated for the standard
solutions and the results are expressed in Table 9. The values obtained demonstrated the
suitability of the system for analysis of this drug combination.
CONCLUSION:
The proposed RP-HPLC method for the simultaneous estimation of paracetamol and
pamabrom in combined tablet dosage forms is accurate, precise, linear, rugged, robust,
simple, rapid, and selective. It can be adopted efficiently and easily for routine quality control
(QC) analysis of raw materials, formulations and dissolution testing with accuracy and
repeatability of results.
ACKNOWLEDGEMENT:
The authors are thankful to Akshaya Laboratories Pvt. Ltd. And Suven Pharmaceutical,
Hyderabad, India and Babaria Institute of Pharmacy, Vadodara, Gujarat, India for providing
PBM and PCM respectively for research. The authors are highly thankful to Babaria Institute
of Pharmacy, Vadodara, and Gujarat, India for providing all the facilities to carry out the
work.
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solution using an unique liquid chromatography methodology.
Figure -1: Chromatogram of Paracetamol (tR 5.78min) and Pamabrom (tR 8.08 min).
40
8.083
UV
Retention Time
Pamabrom
20
20
5.783
mAU
Paracetamol
0
0
2
4
mAU
40
0
6
8
10
Minutes
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International Standard Serial Number (ISSN): 2319-8141
Table-1: Linearity Data of Paracetamol and Pamabrom by Propose method
Sr No.
Concentration of Drug
(ppm)
Peak Area ±SD
Paracetamol
Pamabrom
Paracetamol
Pamabrom
1
5
2
518038±323.4
530173±234.8
2
10
4
1143233±4532.7
1178773±2356.6
3
20
8
2205047±2345.9
2290788±3389.4
4
30
12
3511063±1239.7
3616993±4547.8
5
40
16
4591648±983.7
4796772±864.3
6
50
20
5823761±1287.3
6031277±1645.4
Table-2: Characteristics of HPLC method
Drug
Paracetamol
Pamabrom
137
Parameters Determined
Obtained Value
Linearity range (μg/ml)
5-50
Slope
117477
Intercept
69347
Regression Coefficient(r2)
0.9991
LOD(μg/ml)
0.33
LOQ(μg/ml)
1.007
Linearity range (μg/ml)
2-20
Slope
305275
Intercept
80377
Regression Coefficient(r2)
0.995
LOD(μg/ml)
0.092
LOQ(μg/ml)
0.27
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Table-3: Precision study (Repeatability)
Drug
Injection
Peak area
Concentration(µg/ml)
%R.S.D.
Paracetamol
Pamabrom
1
2857492
24.95
2
2841576
24.78
3
2847492
24.83
4
2892372
25.21
5
2872682
25.04
1
2881483
9.70
2
2895432
9.74
3
2910462
9.79
4
2903472
9.77
5
2921325
9.83
Table-4: Inter-day and Intra-day precision
Drug
Concentration
Inter-day precision
Paracetamol
Pamabrom
0.452
0.633
Intra-day precision
(µg/ml)
Mean* ± S.D
%R.S.D
Mean* ± S.D
%R.S.D
5
99.81±0.616
0.617
99.73±0.123
0.123
25
99.53±0.225
0.226
99.44±0.572
0.575
50
99.31±0.415
0.417
99.72±0.323
0.323
2
100.36±0.78
0.777
98.68±0.94
0.952
10
99.88±0.987
0.988
97.89±0.56
0.572
20
102.65±1.03
1.003
98.89±1.76
1.779
*Average of three determinations
Table -5: Analysis of mixture by proposed method
Drug
Amount
added(mg)
Observed
amount(mg)
%Amount
found* ±SD
% R.S.D.
Paracetamol
25
24.955
99.82±0.443
0.443
Pamabrom
10
9.777
97.73±2.27
2.32
*average of six determinations
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Table-6: Recovery study
Amount
Present(µg/ml)
Drug
Paracetamol
Pamabrom
Total Amount
amount recovered (µg/ml)
added(µg/ml) (µg/ml)
Amount
Recovery
(%)*± S.D.
15
12(80%)
27
26.89
99.27±0.76
15
15(100%)
30
29.92
99.47±0.97
15
18(120%)
33
33.03
100.2±0.49
6
4.8(0%)
10.8
10.65
97.5±1.02
6
6(100%)
12
11.88
98.0±0.98
6
7.2(120%)
13.2
13.06
97.66±1.12
Table-7: Robustness testing
Factor
Level
Retention
time
Theoretical
plate
Peak area
Flow
rate(ml/min)
PCM
PBM
PCM
PBM
PCM
0.6
-2
5.832
8.124
3453
4674
2865473 2960212
0.8
0
5.783
8.083
3764
4963
2866418 2961623
1.0
+2
5.583
7.892
3214
4423
2859638 2953432
1.3
-2
5.763
8.025
4523
5432
2845637 2943647
1.5
0
5.783
8.083
3764
4963
2866418 2961623
1.7
+2
5.789
8.082
3639
4887
2854656 2964630
4.6
-2
5.834
8.124
3147
4656
2844800 2936400
4.8
0
5.783
8.083
3764
4963
2866418 2961623
5.0
+2
5.981
8.239
3241
4638
2825471 2836400
139
PBM
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Table -8: Ruggedness studies
Drug
Label
Claim(mg)
Amount Found (%)
Analyst-1
Analyst-2
Paracetamol
250
99.45
99.29
Pamabrom
100
100.2
100.56
Table-9: System Suitability Studies
System Suitability
Parameters
Proposed Method
Paracetamol
Pamabrom
Retention Time (tR)
5.783
8.083
Capacity Factor (k)
56.83
79.83
Theoretical Plate Number (N)
3764
4963
1.88
1.62
0.00
5.50
Tailing Factor (T)
Resolution Factor (R)
140
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