Validated Spectrophotometric Method for the Estimation of Ketorolac Tromithamine in Bulk and Tablets Using Ninhydrin: A Modified Approach

Abstract

A new, accurate, precise ecofriendly and economical spectrophotometric method has been developed and validated for
the determination of Ketorolac Tromithamine in bulk drugs and tablet dosage forms. The proposed method is based on
the reaction of ninhydrin with primary amino group present in Ketorolac Tromithamine in the presence of Ammonium
Molybdate which forms Ruhemann’s purple colour by heating at 90 ± 1°C for 15 min and absorbs maximally at about
570 nm. Beer-Lambert’s law is obeyed in the concentration range of 50-250μg/ml and is described by the regression
equation Y = 0.002x – 0.186 with a regression coefficient (r2) = 0.998 (n = 6). The effects of variables such as
temperature, heating time, concentration of colour producing reagent and stability of colour were investigated to
optimize the procedure. For the proposed method, the value of molar absorptivity and Sandell’s sensitivity are 1.03434
x 103 L/mol/cm and 0.363995μg/cm2, respectively. The LOD and LOQ are found to be 15.369 μg/ml and 46.57μg/ml,
respectively. The statistically validated results indicate that the proposed method has good sensitivity, accuracy,
precision and stability. Along with its cost effect, it is eco friendly and used for routine quality control of Ketorolac
Tromethamine in bulk and tablet dosage forms.

Full text

Asian J. Research Chem. 7(1): January 2014
19
ISSN 0974-4169
www.ajrconline.org
RESEARCH ARTICLE
Validated Spectrophotometric Method for the Estimation of Ketorolac
Tromithamine in Bulk and Tablets Using Ninhydrin: A Modified Approach
Vandana P. Patil*, Subhash J. Devdhe, Sachidanand S. Angadi, Suwarna H. Kale ,
Shital D. Phalke, Santosh D. Shelke and Rushikesh H. Patil
Yash Institute of Pharmacy, Department of Pharmaceutical Analysis,Aurangabad-431134, India
*Corresponding Author E-mail: vandana2609@gmail.com
ABSTRACT:
A new, accurate, precise ecofriendly and economical spectrophotometric method has been developed and validated for
the determination of Ketorolac Tromithamine in bulk drugs and tablet dosage forms. The proposed method is based on
the reaction of ninhydrin with primary amino group present in Ketorolac Tromithamine in the presence of Ammonium
Molybdate which forms Ruhemann’s purple colour by heating at 90 ± 1°C for 15 min and absorbs maximally at about
570 nm. Beer-Lambert’s law is obeyed in the concentration range of 50-250µg/ml and is described by the regression
equation Y = 0.002x – 0.186 with a regression coefficient (r
2
) = 0.998 (n = 6). The effects of variables such as
temperature, heating time, concentration of colour producing reagent and stability of colour were investigated to
optimize the procedure. For the proposed method, the value of molar absorptivity and Sandell’s sensitivity are 1.03434
x 10
3
L/mol/cm and 0.363995µg/cm
2
, respectively. The LOD and LOQ are found to be 15.369 µg/ml and 46.57µg/ml,
respectively. The statistically validated results indicate that the proposed method has good sensitivity, accuracy,
precision and stability. Along with its cost effect, it is eco friendly and used for routine quality control of Ketorolac
Tromethamine in bulk and tablet dosage forms.
KEY WORDS:
Ketorolac Tromithamine; Ninhydrin; Ammonium Molybdate; UV-visible Spectrophotometry.
1.0 INTRODUCTION
Ketorolac Tromithamine (KT) is a NSAID in the family of
Heterocyclic Acetic Acid derivative. Chemically, it is 5-
Benzoyl-2,3-Dihydro-1H-Pyrolizine-1-Carboxylic Acid, 2
Amino-2-(hydroxymethyl)-1,3-Propaneddiol (Figure1)
1
. KT
acts by inhibiting the bodily synthesis of prostaglandins. It
is used in short term management of moderate to severe
pain
2
.
N
O
HO
NH2
OH
OH
OH
O
.
Figure 1: Structure of Ketorolac Tromithamine (KT).
European Pharmacopoeia describes assay of Ketorolac
Tromithamine by reversed phase high performance liquid
chromatography in bulk and pharmaceutical formulations
3
.
KT has been studied and determined by relatively other
methods such as Spectrophotometry
4-6
, HPLC
7-12
and
HPTLC
13-16
.
Received on 10.08.2013 Modified on 20.10.2013
Accepted on 06.11.2013 © AJRC All right reserved
Asian J. Research Chem 7(1): January 2014; Page 19-24
Many researchers have dealt with the development of
methods that quantify Ketorolac Trimithamine (KT) in pure
form and in tablets. Visible spectrophotometry is the
technique of choice even today because of its inherent
simplicity, sensitivity, selectivity, accuracy, precision and
cost-effectiveness. The scientific references found in the
CAS and SCI database, relating to green analytical
chemistry or environmentally-friendly analytical methods
have been growing significantly in recent years
17-18
. The
recent development of new analytical methods with good
characteristics such as selectivity and sensitivity are not
only sufficient but also modern analytical methods need to
be green. Hence, the purpose of the present study was to
improve these methods with respect to two experimental
objectives: (i) decrease the heating time, (ii) avoid the use
of organic solvent. Both factors are of great significance in
reducing time and cost of analysis. In the present
investigation, solution of distilled water has been employed
to solubilize a water soluble drug, KT forms a purple
colored product with ninhydrin in the presence of saturated
solution of Ammonium Molybdate
and further
spectrophotometric estimation was carried out maximum at
about 570 nm without employing any organic solvents.
These modifications resulted in increased linear range (50-
250µg/ml), molar absorptivity (ε = 1.034 x 10
3
L/mol/cm)

Asian J. Research Chem. 7(1): January 2014
20
and sensitivity (0.363901µg/cm
2
) compared too many
existing spectrophotometric methods.
2.0 EXPERIMENTAL
2.1 Apparatus
A Shimadzu 1800 double beam UV-VIS spectrophotometer
provided with 1 cm matched quartz cell was used for
absorbance measurements.
2.2 Materials and Reagents
Ketorolac Tromithamine was obtained as gift sample from
Wockhardt Pharmaceuticals Pvt. Ltd. (Aurangabad, India).
All other reagents used were of analytical grade.
2.3 Solubility Studies
Ketorolac Tromithamine is water soluble and the proposed
method was carried out in distilled water.
2.4 Preparation of Citrate buffer
10.5gm Citric acid was dissolved in 500ml of 1M NaOH.
2.5 Preparation of 0.2M Ninhydrin Solution
0.44gm mg of Ninhydrin was dissolved in 25 ml of Citrate
buffer.
2.6 Preparation of 0.2 M Ammonium Molybdate
Approximately 6.1gm was taken in a beaker containing 25
ml of citrate buffer and stirred with a magnetic stirrer for
twenty minutes. The solution was decanted and filtered
using quantitative filter paper.
2.7 Preparation of Standard solution of KT
100 mg pure Ketorolac Tromithamine was transferred to
1000 ml volumetric flask and diluted upto the mark with
distilled water to get a concentration of 100µg/ml solution.
2.8 Optimized Procedure
In a 10 ml volumetric flask, add 0.5 ml standard stock
solution (100µg/ml), 1.0 ml ninhydrin and 1.0 ml
ammonium molybdate solution and diluted upto the mark
with citrate buffer (10µg/ml). It was heated in boiling water
bath at 90 ± 1°C in 15 min and cooled to room temperature.
Scan the spectrum and absorbance was measured λmax at
570 nm versus the reagent blank (Figure 2).
Figure 2: Spectrum of Ketorolac Tromithamine (KT)
3.0 METHOD VALIDATION:
3.1 Stability
In order to demonstrate the stability of the Ninhydrin-
Ketorolac Tromithamine complex in presence of
Ammonium molybdate solution was analyzed over a period
of 12 hrs at room temperature. During analysis, the complex
was found to be stable over a period of 2 hr at room
temperature as shown in Figure 3.
Figure 3: Effect of time (min) on absorbance of KT- Ninhydrin
complex.
3.2. Linearity
From the Standard Stock solution (100µg/ml), different
aliquots (0.5, 1.0, 1.5, 2.0 and 2.5) were taken in a series of
10 ml volumetric flasks and 1.0 ml ninhydrin was added to it
followed by 1.0 ml Ammonium molybdate solution and
volume made up with Citrate buffer to get concentration 50-
250µg/ml. All flasks were heated for 15 min in boiling water
bath and cooled to room temperature and measure the
absorbance at 570 nm. Five replicates of analytes were
measured and record the absorbance. Plot a graph
concentration versus absorbance, a linear correlation was
found which obeys Beer Lambert’s Law in the concentration
range of 50-250 µg/ml (Figure 4). Regression analysis of
Beer’s law data using the method of least squares was made
to evaluate the slope (b), intercept (a) and the correlation
coefficient (r
2
).
Figure 4: Calibration curve of Ketorolac Tromithamine (KT).

Asian J. Research Chem. 7(1): January 2014
21
3.3 Limit of Detection (LOD) and Limit of
Quantification (LOQ)
The detection limit of an individual analytical procedure is
the lowest amount of analyte in a sample that can be
detected but not necessarily quantitated as an exact value.
The quantitation limit of an individual analytical procedure
is the lowest amount of analyte in a sample that can be
quantitatively determined with suitable precision and
accuracy. The quantitation limit is a parameter of
quantitative assays for low levels of compounds in sample
matrices and is used particularly for the determination of
impurities and/or degradation products. The valve of LOD
and LOQ are determined and given in Table 1.
Table 1: Optical characteristics data and validation parameters of
KT.
Parameter Analytical data
Linearity Range (µg/ml) 5-25
λ max (nm) 570
Molar extinction coefficient 1.034x10
3
Sandell’s sensitivity 0.363901
Slope 0.7 x 10
-
2
Intercept -205
Standard deviation about regression (Sy) ±0.241077
Standard deviation of Slope (Sb) ±0.001525
Standard deviation of intercept (Sa) ±0.252844
Correlation co-efficient (r
2
) 0.997
Limit of detection (LOD, µg/ml) 15.36995
Limit of quantification (LOQ, µg/ml) 46.5756
3.4. Precision
To determine precision, 7 days measurement (intra-days
and interday) were computed with relative standard
deviation (RSD %) for replicate samples (n = 5) using
concentration 50, 100and 150µg/ml both the intraday and
interday samples were calibrated with standard curve
concurrently prepared in the same day of analysis.
3.4.1. Intraday Precision
Intraday precision of test method is demonstrated by three
samples of the same batch (same concentration) at initial,
24 and 48 hrs (Table 2).
3.4.2. Interday Precision
Interday precision of test method is demonstrated by three
samples of the same batch (same concentration) on three
successive days (Table 2).
3.5. Accuracy
To determine the accuracy of the proposed method,
recovery study was carried out by adding different amount
(80%, 100%. 120%) of bulk sample of Ketorolac
Tromithamine within the linearity range and results
obtained are compiled in Table 3 and show good accuracy
for the method.
3.6 Assay
Assay of tablet dosage form was carried by same procedure
as mentioned in methodology to equivalent weight of
Ketorolac Tromithamine by proposed spectrophotometric
method. The percent purity was found out using regression
analysis (Table 4).
4.0 RESULTS AND DISCUSSION:
Preliminary studies were carried out to establish the
optimum conditions for assay of the Ketorolac
Tromithamine.
4.1 Effect of temperature
The effect of temperature on the complexation reaction at
80
0
, 90
0
and 97 ± 1°C were examined (Table 5). It was
observed that ninhydrin–AM complex in saturated
Ammonium Molybdate required 97 ± 1°C for obtaining
maximum and stable absorbance and remained constant for
about a further 2 hrs.
4.2 Effect of the reaction heating time
The effect of time on the complexation reactions at room
temperature was examined. It was observed that ninhydrin–
KT charge transfer complex in saturated sodium
bicarbonate required 15 min for obtaining maximum and
stable absorbance as shown in Figure 5.
Table 2: Evaluation of intra-day and inter-day accuracy and precision
KT taken
(µg/ml)
Intraday Accuracy and precision Interday Accuracy and precision
KT found(µg/ml) RE % RSD % KT found(µg/ml) RE % RSD %
100 89.29 89.29% 1.39 85.74 85.74 1.47
150 143.75 95.83% 0.87 139.62 93.08 0.89
200 191.24 95.60% 0.65 188.54 94.27 0.66
Table 3: Recovery Data
Level Amount of KT added (µg) Amount of KT found (µg) % Recovery % RSD
80 % 80 82.40 103 0.68
100 % 100 102.67 102.67 0.82
120 % 120 111.14 92.61 1.21
*An average value ± relative standard deviation of 5 observations.
Table 4: Assay Results of Tablet Dosage Form.
Formulation Actual amount of KT (µg) Amount found of KT(µg) % of KT Found
Tablet 10 10.13 101.3

Asian J. Research Chem. 7(1): January 2014
22
Table 5: Effect of temperature on absorbance.
Temperature (
0
C) Absorbance
80 ± 1 0.042
90 ± 1 0.904
97 ± 1 1.644
Figure 5: Effect of the reaction heating time on absorbance.
4.3 Effect of solvent
Distilled water was a solvent of choice for ninhydrin–KT
system because intense and stable colour was obtained only
after 10 min of reagent mixing.
4.4 Effect of reagents concentration
The optimum conditions for the method was established by
varying the concentration of reagent at a time and keeping
the fixed drug concentration and observing the effect
produced on the coloured species. To establish the optimum
experimental condition for ninhydrin–KT charge transfer
complex, the drug (100 µg /ml) was allowed to react with
varying volumes of 0.5%ninhydrin.The maximum
absorbance was obtained with 1.0 ml of the reagent (Figure
6); further addition caused no significant change in
absorbance. Therefore a volume of 1.0 ml was used as an
optimum value.
Figure 6: Effect of volume of 0.5% ninhydrin on the absorbance of
reaction product
O
O
O H
O H
2
N i n h y d r i n
O
O
O H
H O
H y d r i n d a n t in
O
O
R u h e m a n n ' s P u r p l e
A m m o n i u m M o l y b d a t e
+
+
1 5 M i n u t e s
9 0 ± 1 ° C
N
O
O
O
O
N
O
H O
N H
2
O H
O H
O H
O
.
N
O
H O
O H
O H
O H
O H
O
.
K e t o r o l a c T r o m i t h a m i n e
Scheme I: Formation of Ruhemann’s purple complex between KT and ninhydrin.

Asian J. Research Chem. 7(1): January 2014
23
Table 6: Comparison of Proposed and Published SpectrophotometricMethods
19-20
.
It was reported that in alkaline medium, ninhydrin is
reduced to an intermediate, hydrindantin and converted to
diketohydrindylidene diketohydrindamine which is
commonly referred as ‘Ruhemann’s purple’ The primary
amino group of Ketorolac Tromithamine reacted with
ninhydrin in presence of saturated Ammonium Molybdate
solution (alkaline medium) to give diketohydrindylidene
diketohydrindamine of Ketorolac Tromithamine reacts with
ninhydrin via oxidation deamination of the primary amino
group followed by the condensation of the reduced
ninhydrin to form the colored Ruhemann’s purple without
employing any organic solvent., which absorbs a maximum
at 570 nm as shown in Figure 2. The proposed reaction
between of Ketorolac Tromithamine and ninhydrin is
shown in the Scheme I.
To optimize the reaction conditions, different parameters
have been investigated such as temperature, heating time,
reagent concentration, and color stability. Reaction between
ninhydrin and of Ketorolac Tromithamine did not give any
colored product in the absence of Ammonium Molybdate,
not even after prolonged heating. It was observed that
complete colour development was attained at 90± 1°C. The
optimum reaction time was determined by heating the
reaction mixture on a water bath at 90± 1°C. It was noted
that complete colour development was attained in fifteen
minutes. The effect of ninhydrin concentration on the
colour development was investigated. 1 ml of 0.5%
ninhydrin reagent produced maximum colour intensity.
Interestingly, reaction was found to be specific in
Ammonium Molybdate
medium. The comparison between
the proposed and published method was shown in Table 6.
5.0 CONCLUSIONS:
By the comparison of proposed and published methods as
shown in Table 6, it can be concluded that
spectrophotometric method has found to be new for the
determination of Ketorolac tromethamine. In comparison
with the existing visible spectrophotometric methods for the
quantification of Ketorolac Tromethamine, the present
modified method can be considered green as it
demonstrates that visible spectrophotometry can be utilized
without the usage of organic solvent. Overall the proposed,
spectrophotometric method is new, accurate, precise,
economical and ecofriendly. Thus it can be suitable for
quality control of Ketorolac Tromethamine in bulk, fixed-
dose combination tablets.
6.0 ACKNOWLEDGEMENT:
The authors are gratefully acknowledging the receipt of
pure Ketorolac Tromethamine as gift sample from
Wockhardt Pharmaceuticals Pvt. Ltd.(Aurangabad, India).
Authors express their gratitude to Yash Institute of
Pharmacy, Aurangabad for providing the instrumental and
chemicals facility.
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Parameter Proposed Method Published Methods
I II III
Solvent Distilled Water Methanol Phosphate Buffer Methanol
Reagents
Citrate buffer pH
5.5
2,3-dichloro-5,6-dicyano-p-
benzoquinone (DDQ)
Methylene Blue 2,3-dichloro-5,6-dicyano-p-
benzoquinone (DDQ)
Ammonium
molybdate
2,4-dichloro-6-nitrophenol
(DCNP)
Saframine
Ninhydrin
Extraction No No Extracted with Chloroform Extraced with Methanol/
Acetone
Heating Time(Min.) 15 min No No No
Absorption maxima at
λmax
570 nm
392 nm with DDQ 640 nm with Methylene
Blue
460 nm with DDQ
425 nm with DCNP 515 nm with saframine
Linearity 50-250 µg/ml 10-80 µg/ml 5-50µg/ml 50-250µg/ml

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