Department of pharmaceuticals Analysis Materials & Methods

MATERIALS AND METHODS

HPLC-Equipment

Table no. 5.1 HPLC equipment

S.no. Name Make / Model

1 Analytical balance,210gm Mettler, Bangalore

2 HPLC instrument A HPLC system

Series Shimadzu prominence
Software Spinchrom

3 Column Athene
C18(250mm,4.6mm,5µ)
4 Detector UV-Visible PDA detector
Shimadzu, Mumbai, India

5 Sonicator Sonica 2200MH

6 pH meter Metler Toledo

7 Vaccum filter Model XI 5522050 of

Millipore

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REAGENTS USED FOR THE STUDY

Table no. 5.2 Reagents used for the method development

REAGENT MAKE

Potassium di hydrogen ortho phosphate (HPLC grade-Merk),

Mol. Formula – KH2PO4 Mumbai.

Mol. Weight – 136.09

Ortho phosphoric acid (HPLC grade-Merk)

Mol. Formula – H3PO4 Mumbai.

Mol. Weight – 98

Ammonium acetate (HPLC grade-Merk)

Mol. Formula – CH3COONH4 Mumbai.

Mol. Weight –77.082

Methanol (HPLC grade-Merk)

Mol. Formula – CH3OH Mumbai

Mol. Weight –32.04

Acetonitrile (HPLC grade-Merk)

Mol. Formula – CH3CN Mumbai.

Mol. Weight –41.05

Water (Milli- Q grade)

Mol. Formula – H2O Mumbai.

Mol. Weight -18

METHOD DEVELOPMENT

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The objective of this experiment was to optimize the assay method for simultaneous
estimation of Ketorolac and Olopatadine based on the literature survey made and the methods
given in official pharmacopoeias. Trials done for optimization are as follows:

Trail and Error Report

Table no.5.3.1 Trial and error report of Ketorolac and Olopatadine

Trail Column Flow rate Temp Mobile phase Wavel pH Remark

(ml/min) ength
1 ZODIAC C18 1.0 Ambient Ammonium 212 6.0 It was not
C18(250x4.6mm, acetate: Acn satisfacto
5µ)Column Methanol ry
50:30:20
2 INERTSIL 1.2 30 ̊ C 0.01M 212 3.0 It was not
C18(250x4.6mm, KH2PO4: satisfacto
5µ)Column Methanol ry
30:70
3 KROMOCIL 1.5 Ambient KH2PO4: 223 4.0 It was not
C18(250x4.6mm, Ammonium satisfacto
5µ)Column acetate: Meoh ry
40:40:20
4 ZODIAC C18 1.0 45 ̊ C 1.0M 213 4.5 It was not
C18(250x4.6mm, KH2PO4: Acn satisfacto
5µ)Column 50:50 ry

5 KROMOCIL 1.2 45 ̊ C KH2PO4: 212 5.0 It was not

C18(250x4.6mm, Meoh satisfacto
5µ)Column ry
6 HYPERSIL 1.0 30 ̊ C Ammonium 212 5.0 It was not
BDS C18(250x acetate: Acn satisfacto
4.6mm,5µ)Colu 70:30 ry
mn

7 INERTSIL 1.5 Ambient KH2PO4: Acn 213 5.4 It was not

C18(250x4.6mm, Methanol satisfacto
5µ)Column 40:40:20 ry
8 ATHENE 1.0 Ambient KH2PO4: Acn 212 5.5 System
C18(250x4.6mm, 50:50 suitabilit
5µ)Column y factors
satisfied

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System Suitability
A Standard solution of Ketorolac & Olopatadine working standard was prepared as per
procedure and was injected six times into the HPLC system. The system suitability
parameters were evaluated from standard Chromatograms obtained by calculating the % RSD
of retention times, tailing factor, theoretical plates and peak areas from six replicate
injections.
1. The % RSD for the retention times of principal peak from 6 replicate injections of each
Standard solution should be not more than 2.0 %

2. The number of theoretical plates (N) for the Sum and Nap peaks should be NLT 2000.

3. The Tailing factor (T) for the Ketorolac and Olopatadine peaks shold be NMT 2.0.

Specificity

Preparation of blank solution:

Acetonitrile: Potassium di hydrogen phosphate Buffer P H 5.5(50:50) v/v was taken
as blank and the chromatogram was recorded.

Preparation of standard solution:

Standard stock solutions of Ketorolac & Olopatadine (mg/ml) were prepared by
dissolving 32mg of Ketorolac and 40mg of Olopatadine in 100ml of mobile phase. The above
solution is filtered by using 0.45-micron syringe filter and Sonicated for 5 min. Further dilution of
32µg/ml of Ketorolac and 40μg/ml Olopatadine was made by adding 1mlto 10 ml of mobile
phase.

The standard solution was injected and the chromatogram was recorded.

Tablet sample preparation:

16mg of Ketorolac and 20mg of Olopatadine combined tablets was taken into a
mortar and crushed to fine powder and uniformly mixed. Tablet stock solutions of Ketorolac
& Olopatadine (µg/ml) were prepared by dissolving equivalent weight 16mg of Ketorolac
20mg of Olopatadine in 50ml of mobile phase. The above solution is filtered by using 0.45-
micron syringe filter and Sonicated for 5 min. Further dilution of 32μg/ml of Ketorolac and
40μg/ml of Olopatadine was made by adding 1 ml of stock solution to 10ml of mobile phase.

The tablet sample solution was injected and the chromatogram was recorded.

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Accuracy
Accuracy is a measure of the closeness of test results obtained by a method to the
true value. Accuracy indicates the deviation between the mean value found and the true
value. It is determined by applying the method to samples to which known amounts of
analyte have been added. These should be analysed against standard and blank solutions to
ensure that no interference exists. The accuracy is then calculated from the test results as a
percentage of the analyte recovered by the assay.

Standard stock solution preparation:

Weigh and transfer 16mg Ketorolac and 20mg of Olopatadine into 50ml volumetric
flask, add 30ml of diluent and sonicate to dissolve and dilute to volume with diluent.

Standard preparation:
Transfer 10ml of standard stock solution into 100ml volumetric flask and dilute to
volume with diluent.

Preparation of Test Solutions:

1. Preparation of 80% Solution:
Finely grind pre weighed 40 tablets. Transfer grinded Sample quantitatively
equivalent to 16mg of Ketorolac and 20mg of Olopatadine in to 50ml volumetric flask add
30ml of diluent, sonicate to dissolve for 10 minutes and dilute to volume with diluent.
Further filter the solution through 0.45µ vaccum filter. Dilute 10ml of filtrate to 100ml
with mobile phase.

2. Preparation of 100% Solution:

Finely grind pre weighed 20 tablets. Transfer grinded Sample quantitatively
equivalent to 32mg of Ketorolac and 40mg of Olopatadine in to 50ml volumetric flask add
30ml of diluents, sonicate to dissolve for 10 minutes and dilute to volume with diluent.
Further filter the solution through 0.45µ vacuum filter. Dilute 10ml of filtrate to 100ml with
mobile phase.

3. Preparation of 120% Solution:

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Finely grind pre weighed 20 tablets. Transfer grinded Sample quantitatively

equivalent to 48mg of Ketorolac and 60mg of Olopatadine in to 50ml volumetric flask add
30ml of diluent, sonicate to dissolve for 10 minutes and dilute to volume with diluent.
Further filter the solution through 0.45µ vacuum filter. Dilute 10ml of filtrate to 100ml with
mobile phase.

Acceptance:
The mean % recovery at each level should not be less than 98% and should not be
more than 102%.

Conclusion:
The above results reveal that the method is accurate.

Precision

Standard stock solution preparation:

Weigh and transfer 16mg of Ketorolac working standard and 20mg of
Olopatadine working standard into 50ml volumetric flask, add 30ml of diluent and sonicate
to dissolve and dilute to volume with diluent.
Standard preparation:
Transfer 10ml of standard stock solution into 100ml volumetric flask and dilute to
volume with diluent.
Sample Preparation:
Finely grind pre weighed 40 tablets. Transfer grinded Sample quantitatively
equivalent to 16mg Ketorolac and 20mg of Olopatadine in to 50ml volumetric flask add
30ml of diluent, sonicate to dissolve for 10 minutes and dilute to volume with diluent.
Further filter the solution through 0.45µ vacuum filter. Dilute 10ml of filtrate to 100ml with
mobile phase.

Method precision:
Finely grind pre weighed 40 tablets. Transfer grinded Sample quantitatively
equivalent to 16mg Ketorolac and 20mg of Olopatadine in to 50ml volumetric flask add

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30ml of diluent, sonicate to dissolve for 10 minutes and dilute to volume with diluent.
Further filter the solution through 0.45µ vacuum filter. Dilute 10ml of filtrate to 100ml with
mobile phase. Peak areas were noted down .Average, Standard deviation, %RSD were
calculated
Linearity
Standard stock solution preparation:
Weigh and transfer 32mg of Ketorolac and 40mg of Olopatadine working
standard into 50ml volumetric flask, add 30ml of diluent and sonicate to dissolve and dilute
to volume with diluent.

Table no.5.6 Linearity dilutions

Linear solutions Stock solution taken Stock solution Diluted to volume
(%) in Ketorolac(mcg) taken in (ml) with diluent
Olopatadine(mcg)
60% 19.2 24 100
80% 25.6 32 100
100% 32 40 100
120% 38.4 48 100
140% 44.8 56 100

Procedure:

Inject each concentration in to the chromatographic system and measure the peak
area. Plot the graph of concentration on X-axis versus peak area on Y-axis. Calculate the
correlation coefficient.

Robustness

Standard stock solution preparation:

Weigh and transfer 32mg of Ketorolac working standard and 40mg of
Olopatadine working standard into 50ml volumetric flask, add 30ml of diluent and sonicate
to dissolve and dilute to volume with diluent.

Standard preparation:
Transfer 10ml of standard stock solution into 100ml volumetric flask and dilute to
volume with diluent.

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Wavelength was varied to 210nm and 214nm:

Weigh and transfer 32mg of Ketorolac working standard and 40mg of Olopatadine
working standard into 50ml volumetric flask, add 30ml of diluent and sonicate to dissolve
and dilute to volume with diluent. Transfer 10ml of standard stock solution into 100ml
volumetric flask and dilute to volume with diluent.

Flow rate was varied to 0.8ml/min and 1.2ml/min ( 0.2ml/min):

Weigh and transfer 32mg of Ketorolac working standard and 40mg of

Olopatadine working standard into 50ml volumetric flask, add 30ml of diluent and sonicate
to dissolve and dilute to volume with diluent. Transfer 10ml of standard stock solution into
100ml volumetric flask and dilute to volume with diluent.

Ruggedness
A wide range of the method was studied by carrying out the experiments by changing
the conditions such as
 Different operators in the same laboratory (using waters HPLC system & spectra
physics HPLC system).
 Different instruments in the same lab (using waters HPLC system & spectra physics
HPLC system.

Preparation of standard solution of Ketorolac & Olopatadine :

Weigh and transfer 32mg of Ketorolac working standard and 40mg of Olopatadine
working standard into 50ml volumetric flask, add 30ml of diluent and sonicate to dissolve
and dilute to volume with diluent. Transfer 10ml of standard stock solution into 100ml
volumetric flask and dilute to volume with diluent.

The solution was injected once into the present chromatographic system by Analyst
1.Again the solution was injected once into the same chromatographic system by analyst.

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2. The peak areas were noted down and %RSD were calculated. To evaluate the intermediate
precision (also known as Ruggedness) of the method, Precision was performed on different
day by using different make column of same dimensions.

Assay Of Ketorolac & Olopatadine

Standard stock solution preparation:

Weigh and transfer 32mg of Ketorolac working standard and 40mg of
Olopatadine working standard into 50ml volumetric flask, add 30ml of diluent and sonicate
to dissolve and dilute to volume with diluent.

Standard preparation:
Transfer 10ml of standard stock solution into 100ml volumetric flask and dilute to
volume with diluent.
Sample Preparation:
Finely grind pre weighed 40 tablets. Transfer grinded Sample quantitatively
equivalent to 32mg of Ketorolac working standard and 40mg of Olopatadine working
standard in to 50ml volumetric flask add 30ml of diluent, sonicate to dissolve for 10
minutes and dilute to volume with diluent. Further filter the solution through 0.45µ vacuum
filter. Dilute 10ml of filtrate to 100ml with mobile phase.
%Assay =

[ ( AT ÷ AS ) × (WS ÷ DS ) × ( DT ÷ WT ) × ( P ÷ 100 ) × ( Avg Wt ÷ Lablel Claim ) ]×100

Where: AT = Peak Area of obtained with test preparation.

AS = Peak Area of obtained with standard preparation.

WS = Weight of working standard taken in mg

WT = Weight of sample taken in mg

DS = Dilution of Standard solution

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DT = Dilution of sample solution

P = Percentage purity of working standard.

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OPTIMISED CHROMATOGRAPHIC CONDITIONS FOR SIMULTANEOUS

ESTIMATION OF KETOROLAC&OLOPATADINE BY RP HPLC:

Optimised chromatographic conditions

Table no.5.4 Optimised method conditions

Mode of separation Isocratic separation

Mobile phase pH 5.5 KH2PO4:ACN
Column Athene C18 (250mmX4.6mm,5µ)
Flow rate 1.0 ml/min
Detector wavelength 212 nm
Injection volume 20µl
Oven temperature Ambient
Run time 6 min

Figure no.5.1 Optimized Chromatogram of Ketorolac & Olopatadine

Tabie no.5.5 Optimized results of Ketorolac & Olopatadine

Name Rt TP Efficiency TF Resolution
Ketorolac 2.337 3189 31886 1.5 6.154
Olopatadine 3.783 6673 66728 1.476

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