International Journal of Chemical and
Natural Science
Vol. 3, No. 3 (2015): 258-263
Research Article
Open Access
ISSN: 2347-6672
Analytical Method Development and Validation of
Simultaneous Estimation of Ketorolac and
Phenylephrine by RP-HPLC Method in Bulk
Gujapaneni Swetha* and P. Jaya Chandra Reddy
Department of Pharmaceutical Analysis and Quality Assurance, Krishna theja Pharmacy College, renigunta road, Tirupathi, India.
* Corresponding author: Gujapaneni Swetha; email: [email protected]
Received: 01 March 2015
Accepted: 12 April 2015
Online: 01 May 2015
ABSTRACT
A simple, precise, accurate and rapid reverse phase high performance liquid chromatographic method had been
developed for simultaneous estimation of ketorolac and phenylephrine in bulk .Symmetry C18 kromasil column
having i.d of 150×4.6 mm and 5µm particle size (Make: Waters) was used. The method was carried out in isocratic
program using mobile phase, potassium dihydrogen ortho phosphate buffer: Acetonitrile (40:60). Flow rate was
adjusted to 0.9ml/min and effluents were monitored at 228nm. The retention time obtained for ketorolac and
phenylephrine was 3.623& 2.272min respectively. The calibration curves were linear in the concentration range of
25-150µg/ml and with correlation coefficients (r2) 0.999 and 0.999 for ketorolac and phenylephrine respectively.
The mean recoveries were found to be in the ranges of 100.41% -101.81%and 99.25%-100.88% for ketorolac and
phenylephrine respectively. The proposed method has been validated as per ICH guidelines and successfully applied
to the estimation of ketorolac and phenylephrine in their combined form.
Keywords: Phenylephrine, ketorolac simultaneous estimation, RP-HPLC.
1. INTRODUCTION
Ketorolac is a non-steroidal anti-inflammatory drug,
when administered systemically, has demonstrated
analgesic, anti-inflammatory and anti-pyretic activity.
Chemically
it
is
(±)-5-benzoyl-2,
3-dihydro1Hpyrrolizine- 1-carboxylic acid, 2-amino-2- (hydroxyl
methyl)-1,3-propanediol. Ketorolac acts by inhibiting
the bodily synthesis of prostaglandins. An ophthalmic
solution of ketorolac is available and is used to treat
eye pain and to relieve the itchiness and burning of
seasonal allergies.
Phenylephrine
hydrochloride
(PHE)
is
α1adrenoceptor agonist which stimulates postsynaptic
Alpha receptor causes vasoconstriction, systolic and
diastolic pressure. It is indicated for nasal Congestion,
minor
eye
irritations
and
open
angle
glaucoma.Phenylephrine hydrochloride (PHE), a
synthetic sympathomimetic agent, is used in the
treatment of sinusitis and bronchitis. Chemically it is
benzenemethanol,
3-hydroxy-α
[(methylamino)
methyl]-hydrochloride (R). Various analytical methods
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have been reported for the assay of PHE alone or in
combination with other sympathomimetic agents in
pharmaceutical formulations. They include UV
spectroscopy
high
performance
liquid
chromatography.[1-5] Several methods have been
studied for simultaneous determination of ketorolac
and phenylephrine, but there are limited reports on
method for combination. So the aim of our study is to
develop simple, fast, accurate and specific reversed
phase high performance liquid chromatographic
method for simultaneous determination of ketorolac
and phenylephrine in bulk drugs and Pharmaceutical
Dosage form.
Figure 1(a): Chemical structure of ketorolac.
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water bath for 5 minutes. Filter through 0.45 µ filter
under vacuum filtration.
Figure 1(b): Chemical structure of phenylephrine.
2. MATERIALS AND METHODS
2.1 Chemicals and solvents:
were obtained respectively from spectrum pharma
research solutions, Hyderabad, India. Acetonitrile,
Methanol and water used are of HPLC grade.
2.2 Instrumentation:
The chromatographic separations were performed
using HPLC-Waters alliance (Model-2996) consisting of
an inbuilt auto sampler, a column oven and 2996 PDA
detector. The data was acquired through Empower-2software. The column used was Symmetry C18
(150×4.6mm i.d, 5µm particle size) (make: Waters).
Meltronics sonicator was used for enhancing
dissolution of the compounds. Elico pH meter was used
for adjusting the pH of buffer solution. All weighing was
done on Sarotorious balance (model AE-160).
2.3 Chromatographic conditions:
Detector
:
229nm
Injection volume
:
20µl
Flow rate
:
0.9ml/min
Temperature
:
Ambient
Run time
:
7min
Mobile phase
:
Buffer and
Acetonitrile taken in the ratio 40:60A
Diluent
:
first dissolved in
Methanol and made up with water
2.4 Preparation of buffer solution (pH3.5):
Accurately weighed and transferred 2.72gm of
Potassium di-hydrogen Ortho phosphate in a 1000ml of
Volumetric flask add about 900ml of milli-Q water (4.5
pH), and was adjusted to 3.5 with Ortho phosphoric
acid.
2.5 Preparation of mobile phase:
Mix a mixture of above Buffer 250 ml (25%), 750ml of
Acetonitrile HPLC (75%) and degassed in ultrasonic
Concentration of
ketorolac in µg/ml
1
3ppm
2
6ppm
3
9ppm
4
12ppm
5
15ppm
6
18ppm
Correlation Coefficient
S.No
2.6 Preparation of standard stock solutions:
(120µg/ml Ketorolac, 400µg/ml Phenylephrine)
Accurately Weighed and transferred 6mg of Ketorolac
and 20mg of Phenylephrine working Standards into a
50ml clean dry volumetric flask, add 3/4th volume of
diluent, sonicated for 5 minutes and make up to the
final volume with diluents.
1ml from the above two stock solutions was taken into
a 10ml volumetric flask and made up to 10ml.
(12µg/ml Ketorolac, 40µg/ml Phenylephrine).
2.7 Preparation of sample stock solution: (120µg/ml
Ketorolac, 400µg/ml Phenylephrine)
5 tablets were weighed and calculate the average
weight of each tablet then the weight equivalent to 5
tablets was transferred into a 50ml volumetric flask,
30ml of diluent added and sonicated for 25 min, further
the volume made up with diluent and filtered.
From the filtered solution 0.5ml was pipette out into a
10 ml volumetric flask and made up to 10ml with
diluent. (12µg/ml Ketorolac, 40µg/ml Phenylephrine).
2.8 METHOD VALIDATION:
The developed method was validated as per the ICH
(International
Conference
on
Harmonization)
guidelines with respect to System suitability, Precision,
Specificity, Linearity, Accuracy, Limit of detection and
Limit of quantification.
2.8.1 Linearity:
Aliquots of 0.25, 0.5, 0.75, 0.1, 0.25 And 0.50ml were
taken from stock solution of concentration 120µg of
ketorolac and 400µg of phenylephrine, and then diluted
up to 10ml with diluents. Such that the final
concentrations were in the range 3-18µg for ketorolac
and 10-60µg for phenylephrine. Volume of 10µl of each
sample was injected in five times for each
concentration level and calibration curve was
constructed by plotting the peak area versus drug
concentration. The observations and calibration curve
were shown in Table 1 and Fig. 2, 3.
Table-1: Linearity data
Ketorolac Area
156083
308011
452295
623748
753757
923783
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Concentration of
phenylephrine in µg/ml
10ppm
20ppm
30ppm
40ppm
50ppm
60ppm
phenylephrine Area
223018
425092
643685
867725
1071512
1298345
0.999
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Figure 2: Phenylephrine calibration curve
Figure 3: Ketorolac calibration curve
2.8.2 Assay:
Accurately weighed powder equivalent to 12mg of
ketorolac and 40mg of phenylephrine was transferred
into a 100ml clean dry volumetric flask add Diluent and
sonicate to dissolve it completely and make volume up
to the mark with the same solvent.
Drug
ketorolac
phenylephrine
% linearity level
50
100
150
(Stock solution)
Further pipette 1ml of ketorolac and phenylephrine of
the above stock solution into a 10ml volumetric flask
and diluted up to the mark with diluents it gives 12µg
of ketorolac & 40µg of phenylephrine. The results were
shown in Table-2. The chromatograms were shown in
Fig-4.
Table-2: ketorolac and phenylephrine assay
Label claim
mg/tab
12
40
Amount found
mg/tab
59.5
99.7
Label
claim(% )
99.16
99.7
Table-3: Accuracy (ketorolac)
% recovery
100.41
100.27
101.81
101.83
99.91
100.26
99.25
100.79
100.88
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S.D*
% R.S.D
0.189754
0.191106
0.329008
0.329008
%mean recovery ± S.D
100.83± 0.86
% RSD
0.85
100.67± 1.02
1.01
100.31 ± 0.92
0.91
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Accuracy (phenylephrine)
% linearity level
50
% recovery
101.01
99.98
101.21
100
101.68
100.57
101.59
150
101.87
101.80
%mean recovery ± S.D
100.74± 0.66
% RSD
0.65
101.28± 0.62
0.61
101.37 ± 0.80
0.79
100.44
2.8.3 Accuracy:
Accuracy of the method was done by recovery study.
Sample solutions were prepared by spiking at about
50%, 100%, and 150% of specification limit to placebo
and analyzed by the proposed HPLC method. Results
are shown in Table-3(a) and (b).
2.8.4 Specificity:
The specificity of the method was performed by
injecting blank solution( without any sample) and then
a drug solution of 10µl injected into the column, under
optimized chromatographic conditions, to demonstrate
the separation of both ketorolac and phenylephrine
from any of the impurities, if present. As there was no
interference of impurities and also no change in the
retention time, the method was found to be Specific.
INJECTIONS
1
2
3
4
5
6
AVG
S.D
%R.S.D
Drug
phenylephrine
ketorolac
Injections
1
2
3
4
5
6
1
2
3
4
5
6
2.8.5 Limit of detection (LOD) and Limit of
quantification (LOQ):
The parameters LOD and LOQ were determined on the
basis of response and slope of the regression equation.
The linearity for ketorolac and phenylephrine was
performed from 3-18µg/ml and 10-60µg/ml
respectively.
2.8.6 System precision:
Precision is the measure of closeness of the data values
to each other for a number of measurements under the
same analytical conditions. Standard solution of
ketorolac (12µg/ml) and phenylephrine (40µg/ml)
were prepared as per test method and injected for 3
times. Results are shown in Table-4.
Table-4: System precision
AREAS(ketorolac)
653667
657914
652222
659062
659932
658899
656949
3200.4
0.5
AREAS(phenylephrine)
844171
846421
844985
841066
845330
848482
845076
2465.0
0.29
Table-5: Method precision
% Assay
100.22
99.91
99.61
100.33
100.33
99.58
99.51
99.88
99.96
100.05
99.97
99.93
2.8.7 Method precision:
Three samples were prepared and analyzed as per the
test method on same day and three different days and
calculated the % RSD for assay of five preparations.
Results were shown in Table- 5.
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Mean
S.D
% R.S.D
100.00
0.35
0.35
99.98
0.192
0.19
2.8.8 Robustness:
Robustness studies were carried out by variations in
flow rate, mobile phase compositions and temperature.
It was observed that the small changes in these
operational parameters did not lead to changes of
retention time of the peak interest. The degree of
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reproducibility of the results proven that the method is
robust.
2.8.9 System suitability test:
The system suitability was determined by making six
replicate injections from freshly prepared standard
Drug
phenylephrine
ketorolac
solutions. The observed RSD values were well within
usually accepted limits (≤2%). Theoretical plates,
tailing factor, resolution between ketorolac and
phenylephrine were determined. The results are all
within acceptable limits summarized in Table-6.
Table-6: Characteristics of HPLC method
Parameters defined
Linearity range (µg/ml)
Slope
Intercept
Regression coefficient(r2)
LOD ( µg/ml)
LOQ (µg/ml)
Tailing factor
Plate count
Linearity range (µg/ml)
Slope
Intercept
Regression coefficient(r2)
LOD (µg/ml)
LOQ (µg/ml)
Tailing factor
Plate count
Obtained value
10-60µg/ml
21552
483
0.999
0.03
0.09
1.09
3888
3-18 µg/ml
50981.5
836
0.999
0.01
0.02
1.09
13133
Figure 4: Standard chromatogram of phenylephrine and ketorolac.
3. RESULTS AND DISCUSSION
The nature of sample, its molecular weight and
solubility decides the proper selection of stationary
phase. The drugs ketorolac and phenylephrine were
preferably analyzed by reverse phase chromatography
and accordingly C18 column was selected. The elution of
the compounds from column was influenced by polar
mobile phase. The ratio of Potassium di-hydrogen
Orthophosphate to Acetonitrile was optimized to
(40:60) to give well resolved and good symmetrical
peaks with short run time. The retention time of
ketorolac and phenylephrine were found to be 3.623 &
2.272 min respectively. The calibration curve was
linear over the concentration range of 3-18µg/ml
(ketorolac) and 10-60µg/ml (phenylephrine).
The linearity of the method was statistically confirmed.
RSD values for accuracy and precision studies obtained
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were less than 2% which revealed that developed
method was accurate and precise. The system
suitability parameters were given in table-5. The
analytical recovery at five different concentrations of
ketorolac and phenylephrine was determined and the
recovery results were in the range of 100.41% 101.81% and 99.25%-100.88% µg/ml. Therefore
proposed validated method was successfully applied to
determine ketorolac and phenylephrine in tablet
dosage form.
4. CONCLUSION
The developed method is accurate, simple, rapid and
selective for the simultaneous estimation of ketorolac
and phenylephrine in pharmaceutical dosage form. The
excipients of the commercial sample analyzed did not
interfere in the analysis, which proved the specificity of
the method for these drugs. The sample preparation is
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simple, the analysis time is short and the elution is by
isocratic method. Hence the proposed method can be
conveniently adopted for the routine quality control
analysis in the combined formulation.
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