Simultaneous determination of Ketorolac tromethamine and Fluorometholone
in Eye drops by spectrophotometry
Mukthinuthalapati Mathrusri Annapurna*, Vellanki S. V. Sevyatha,
Malineni Sushmitha
Department of Pharmaceutical
Analysis and Quality Assurance, GITAM Institute of Pharmacy, GITAM University,
Visakhapatnam-530045, India
*Corresponding
Author E-mail: mathrusri2000@yahoo.com
ABSTRACT:
Four simple spectrophotometric
methods have been developed for the simultaneous determination of Fluorometholone
and Ketorolac tromethamine in ophthalmic preparations. Simultaneous equation
method, absorbance ratio method (Q – Analysis), first derivative method and
multicomponent mode of analytical techniques were selected for the
determination of Fluorometholone and Ketorolac tromethamine in phosphate buffer
(pH 2.0) solution. Fluorometholone and Ketorolac tromethamine has followed
linearity over the concentration range 0.1-20 µg/mL and 1-20 µg/mL
respectively. The methods were validated and applied for the simultaneous
determination of Fluorometholone and Ketorolac tromethamine in the available
pharmaceutical formulations i.e. eye drops.
KEYWORDS: Fluorometholone,
Ketorolac tromethamine, Spectrophotometry, Simultaneous equation method,
Simultaneous derivative, Multicomponent analysis, Q-Analysis, Validation.
INTRODUCTION:
Ketorolac tromethamine1
(CAS No. 74103-06-3) chemically known as
(±)-5-benzoyl-2,3-dihydro-1H-pyrrolizine-1-carboxylic acid (C15H13NO3)with
molecular weight 255.27 g/mol. is freely soluble in methanol. An ophthalmic
solution of Ketorolac is available and is used to treat eye pain and to relieve
the itchiness and burning of seasonal allergies. The primary mechanism of
action responsible for Ketorolac's anti-inflammatory, antipyretic and analgesic
effects is the inhibition of prostaglandin synthesis by competitive blocking of
the enzyme cyclooxygenase (COX). Ketorolac tromethamine (KT) is a non-selective
COX inhibitor (Fig 1).
Fluorometholone (CAS No.
426-13-1) is chemically
6α-methyl-9α-fluoro-11β,17α-dihydroxypregna-1,4-diene-3,20-dione
(C22H29FO4) with molecular weight 376.462
g/mol (Fig 2). It is used in the treatment of steroid-responsive inflammatory
conditions of the palpebral and bulbar conjunctiva, cornea, and anterior
segment of the eye. Fluorometholone is available with brand names Flurisone,
FML Forte, FML. Very few spectrophotometric2 and liquid
chromatographic methods3 are available in the literature for the
simultaneous determination of FLM and KT. The authors have proposed four
spectrophotometric methods for the simultaneous determination of
Fluorometholone and Ketorolac tromethaminein ophthalmic preparations and the
methods are validated4.
[A]
[B]
Fig 1: Structures of
Fluorometholone (FLM) [A] Ketorolac tromethamine (KT) [B]
MATERIALS AND METHODS:
Chemicals and reagents:
Stock solutions of both
Fluorometholone and Ketorolac tromethamine were prepared in methanol and
dilutions were made with phosphate buffer (pH 2.0). The combination of
Fluorometholone and Ketorolac tromethamine is available with brand name
Eyetrust (Neiss labs Ltd, Mumbai) as eye drops containing Fluorometholone 0.1%
and Ketorolac tromethamine 0.5%.
Instrumentation:
UV-1800 double beam UV-VIS
spectrophotometer (Shimadzu) with a pair of 10mm path length matched quartz
cells is used for the study. All the solutions were scanned 200-400 nm with
medium scanning speed.
General procedure:
Four spectrophotometric
methods - simultaneous equation method (Method A), absorbance ratio method /
Q-Analysis (Method B), first derivative method (D1) (Method C) and
multicomponent mode (Method D)were developed for the simultaneous determination
of Fluorometholone and Ketorolac tromethamine.
Method A:
Simultaneous equation method:
Adequate dilute solutions of
Fluorometholone and Ketorolac tromethamine were prepared from their stock
solutions separately with phosphate buffer and scanned in UV region (200-400
nm) as their solutions were colorless. The absorption spectra so obtained shows
λmax at 241 nm for Fluorometholone and323 nm for Ketorolac
tromethamine. These two wavelengths were selected for the simultaneous equation
method. The absorptivity (ε) values were calculated from their individual
spectra and incorporated in simultaneous equation from which
individual concentrations of Fluorometholone and Ketorolac tromethamine were
determined.
Method B:
Absorbance ratio method
(Q-Analysis):
For this method, one of the
λmax of the drugs and an isosbestic point are required and the
absorbance at these two wavelengths were determined from their individual
spectra and the absorptivity (ε) values were calculated. These ε
values are incorporated in the equation and the individual concentrations of
Fluorometholone and Ketorolac tromethamine were determined. Fluorometholone has
shown λmax at 241 nm and Ketorolac tromethamine at 323 nm and
two isosbestic (iso-absorptive) points were observed at 214.63 and 279.34 nm in
the overlay spectra of Fluorometholone and Ketorolac tromethamine. For the
present Q-analysis method, the two wavelengths 323 nm (λmaxKT)
and 214.63 nm were selected and the individual concentrations of
Fluorometholone and Ketorolac tromethamine were determined from the equation.
Method C:
Simultaneous first derivative
method (D1):
The individual zero order
absorption spectra of Fluorometholone and Ketorolac tromethamine were converted
in to their first order derivative spectra with the help of inbuilt software.
The first order derivative spectra of FLM shows zero crossing points at 214.99,
241.68 and 305.67 nm and that of Ketorolac tromethamine at 228.52, 249.38,
271.66 and 322.85 nm. FLM can be quantified from the maxima observed at 228.52
nm (zero crossing point of KT) whereas KT can be quantified from the maxima
observed at 305.67 nm (zero crossing point of FLM).
Method D:
Multicomponent mode:
In this multicomponent mode
method FLM an KT were mixed in different ratios (>3) and the instrument scan
(200-400 nm) the solutions with the inbuilt software and finally the
concentration of the individual components i.e. FLM and KT were given directly
for the unknown combination of solutions (or formulation solutions). For the
present study five standard solutions containing Fluorometholone and Ketorolac
tromethamine were prepared in 1:5, 5:2, 10:20, 15:10and 30:20 ratio in
phosphate buffer (pH 2.0) and scanned in multicomponent mode.
Validation
Linearity:
0.1-20 µg/mL Fluorometholone
and 1-20 µg/mL Ketorolac tromethamine solutions were prepared from their
individual stock solutions separately and scanned against the reagent blank
i.e. phosphate buffer. The absorbance as well as the absorptivity values were
calculated at the selected wavelengths for both A and B methods as described in
the procedure. A graph was drawn by taking the concentration of the drug
solution on the x- axis and the corresponding absorbance values on the y-axis
at the selected wavelengths whereas for method C derivative absorbance was plotted
against the concentration of the drug solution. For method C the concentration
of the drug solutions is given directly.
Table. 1. Details of reported
analytical methods for Fluorometholone and Ketorolac tromethamine
|
Method |
Reagents/Mobile phase |
Linearity(µg/mL) |
λmax (nm) |
References |
|
Spectrophotometric |
Methanol |
10-70 (FLM) 10-70 (KT) |
226 296 |
2 |
|
HPLC |
Phosphate buffer: methanol |
2.5-7.5(FLM) 12.5-37.5(KT) |
260 |
3 |
|
Spectrophotometric |
Phosphate buffer (pH 2.0) |
0.1-20 (FLM) 1-20 (KT) |
241 323 |
Present work |
Precision and Accuracy
studies:
The intra-day and inter-day
precision studies were performed at three different levels (10, 15 and 20
µg/mL) and accuracy studies were carried out by standard addition method (80%,
100%, and 120%) and the % recovery was calculated.
Assay of Fluorometholone and
Ketorolac tromethamine:
The Eyetrust (Neiss labs Ltd,
Mumbai) eye drops containing Fluorometholone 0.1% and Ketorolac tromethamine
0.5% was procured from the local pharmacy store, extracted with methanol and
assayed after dilution with phosphate buffer for all the methods.
RESULTS AND DISCUSSION:
Four new spectrophotometric
methods, simultaneous equation method (Method A), Q-Analysis (Method B),
simultaneous derivative (Method C) and Multicomponent mode (Method D) were
proposed for the simultaneous determination of Fluorometholone and Ketorolac
tromethamine in eye drops using phosphate buffer (pH 2.0) and a detailed
reported methods were given in Table 1.
Validation:
Linearity:
Method A: Simultaneous
equation method:
The overlay zero order
absorption spectra of Fluorometholone and Ketorolac tromethamine and their
formulation was shown in Fig 2. The absorptivity values were calculated from
the absorbance and substituted in the simultaneous equation given below.
At 241
nm, A1
= 445.1 CFLM + 200.15 CKT
At 323
nm, A2
= 14.8 CFLM + 644.5 CKT
A1 and A2
represents the combined absorbance of the marketed formulation solution at 241
nm and 323 nm respectively; CFLM and CKT are the
concentrations of Fluorometholone and Ketorolac tromethamine (g/100ml)
respectively.
Absorptivity of FLM at 241nm =
445.1
Absorptivity of FLM at 323nm =
14.8
Absorptivity of KT at 241nm =
200.15
Absorptivity of KT at 323nm =
644.5
Method B: Absorbance ratio
method:
Two isosbestic points were
observed at 214.63, 279.34 nm in the overlay absorption spectrum and assay was
performed at 279.34 nm and 323 nm. The absorptivity values obtained at the
selected wavelengths were substituted in the given equation
Cx = Qm-Qy/Qx-Qy×A1/ax1
Cy = Qm-Qx/Qy-Qx×A2/ay1
Cx = Concentration of
Fluorometholone
Cy = Concentration of
Ketorolac tromethamine
A1= Absorbance at
isoabsorptive wavelength 279.34 nm.
A2 = Absorbance at
wavelength 323 nm.
ax1 = Mean
absorptivity of Fluorometholone at 279.34 nm. = 119.6
ay1 = Mean
absorptivity of Ketorolac tromethamine at 323 nm. = 125.5
Qm = Ratio of absorbance of
formulation solution at 279.34 and 323 nm.
Qx = Ratio of absorptivity of
Fluorometholone at 279.34 and 323 nm. = 0.1237
Qy = Ratio of absorptivity of
Ketorolac tromethamine at 279.34 and 323nm. = 5.1354
FLM and KT obeys
Beer-Lambert’s law 0.1-20 µg/mL and 1-20 µg/mL in all the methods (Figure 3A
and 3B) with linear regression equations y = 0.0458x + 0.0026 (R² = 0.9991) and
y = 0.0651x + 0.0055 (R² = 0.9995) for Fluorometholone and Ketorolac
tromethamine respectively.
Method C: Simultaneous first derivative method
(D1):
The overlay first order
derivative spectrum of Fluorometholone and Ketorolac tromethamine was shown in
Fig 4. Linearity was observed over the concentration range 0.1-20 µg/mL and
1-20 µg/mL for FLM and KT respectively. A graph was drawn by taking the drug concentration
(FLM or KT) on the x-axis and the corresponding derivative absorbance on the
y-axis and a straight line graph was obtained (Fig 5A and 5B).
Method D: Multicomponent mode:
In multicomponent mode method
FLM an KT were directly determined from the inbuilt loaded software. The
eyedrop formulation (1:5) has shown FLM: KT as 1.024: 5.0882 indicating that
FLM is 102.4 % and KT as 101.76%.
Fig 2: Overlay zero order
absorption Spectra of Fluorometholone (20 µg/mL), Ketorolac tromethamine (15
µg/mL) and FLM: KT formulation in phosphate buffer
(A) (B)
Fig 3: Calibration curves of
Fluorometholone (A) and Ketorolac tromethamine (B)
Fig4: Overlay first
derivative spectrum of Fluorometholone (____) and Ketorolac tromethamine (- - -
-) in phosphate buffer pH (2.0).
(A)
(B)
Fig 5. Calibration curves of
Fluorometholone (A) and Ketorolac tromethamine (B)
Precision and Accuracy
studies:
The % RSD in intra-day and
inter-day precision was found to be less than 2 for both FLM and KT indicating
that the method A, B and C are precise and the % RSD in accuracy studies was
also less than 2 showing that the methods are accurate (Table 2).
Table 2. Accuracy study of
Fluorometholone (FLM) and Ketorolac tromethamine (KT)
|
Drugs |
Spiked Conc (μg/ml) |
Formulation Conc. (μg/ml) |
Method A |
Method B |
Method C |
|||
|
%RSD |
%*Recovery |
%RSD |
%*Recovery |
%RSD |
%*Recovery |
|||
|
FLM |
0.8 (80%) |
1 |
1.12 |
99.56 |
0.67 |
99.4 |
0.97 |
98.24 |
|
1 (100%) |
1 |
1.09 |
99.09 |
0.82 |
98.37 |
1.07 |
99.37 |
|
|
1.2 120%) |
1 |
0.97 |
98.64 |
0.94 |
98.87 |
0.67 |
99.97 |
|
|
KT |
4(80%) |
5 |
1.25 |
98.57 |
1.04 |
99.20 |
1.34 |
99.57 |
|
5 (100%) |
5 |
0.19 |
99.87 |
1.25 |
99.59 |
1.57 |
98.91 |
|
|
6 (120%) |
5 |
1.05 |
99.20 |
0.97 |
98.10 |
1.09 |
99.02 |
|
*Mean of three replicates
Assay of Fluorometholone and
Ketorolac tromethamine:
Eyetrust eye drops available
in the local pharmacy store were procured and extracted with methanol, followed
by filtration and dilution with phosphate buffer and analysed by the above
methods. The % recovery obtained in the assay studies was shown in Table 3.
Table. 3. Assay of
Fluorometholone and Ketorolac tromethamine
|
Formulation brand |
Drug |
Labelclaim (%) |
*Amountfound (%) |
*% Recovery |
||||
|
Method |
Method |
|||||||
|
A |
B |
C |
A |
B |
C |
|||
|
Brand I |
KT Fluorometholone |
0.5 0.1 |
0.481 0.098 |
0.491 0.099 |
0.492 0.097 |
96.2 98.0 |
98.2 99.0 |
98.4 97.0 |
*Mean of three replicates
CONCLUSION:
The four spectrophotometric
methods are simple, precise and accurate for the routine simultaneous
determination of Fluorometholone and Ketorolac tromethamine in pharmaceutical
formulations successfully.
ACKNOWLEDGEMENT:
The authors are grateful to
M/s GITAM University, Visakhapatnam for providing the research facilities. The
authors have no conflict of interest.
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Budavari S. (ed). The Merck Index, An Encyclopedia of chemicals, drugs
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Shah JA and Maheshwari DG, development and validation of first order
derivative UV spectrophotometric method for simultaneous estimation of
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Indian Journal of Drugs, 2(2), 2014, 56- 64.
3.
Priti SC, Rajesh RP and Dushyant AS, Development and validation of
RP-HPLC method for simultaneous estimation of Ketorolac tromethamine and
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Received on
31.03.2017
Modified on 24.04.2017
Accepted on
30.04.2017 © RJPT All
right reserved
Research J. Pharm. and Tech.
2017; 10(4): 1179-1183.
DOI: 10.5958/0974-360X.2017.00225.6