Spectrophotometric determination and spectroscopic studies on Schiff base and charge transfer complex of ketorolac tromethamine
© Ismail and Narayana. 2015
Received: 23 September 2015
Accepted: 6 November 2015
Published: 11 November 2015
Ketorolac tromethamine is a versatile analgesic drug used extensively for the treatment of fever and moderate to severe pain. It is a potent non-steroidal anti-inflammatory drug that extends opioid-level analgesia to patients. Two new selective methods are proposed for spectrophotometric determination of ketorolac tromethamine (KT) in pure and pharmaceutical formulation.
The determination procedures are based on the condensation reaction of KT with 2,4-dinitrophenylhydrazine (DNPH) in strongly alkaline medium resulting in the formation of red-colored hydrazones which is quantitated at 424 nm for method A and method B involves the charge transfer reaction between KT as electron donor and 7,7′,8,8′-tetracyanoquinodimethane (TCNQ) as π-acceptor to form dark green-colored complex which is measured at 842 nm.
Linear correlation is obtained in the range 0.50–18.50 μg mL−1 and 2.00–50.00 μg mL−1 with detection limits of 0.1579 and 0.3721 μg mL−1 for method A and method B, respectively. The optimum analytical conditions are analyzed, and the proposed assay is validated as per the ICH guidelines.
The developed methods are effectively applied to the determination of KT in tablets, injections, and ophthalmic formulations with good percentage recoveries. The final reaction products are characterized by spectral analysis (FT-IR and 1H NMR). In addition, the surface morphology of the reaction products are also studied using scanning electron microscopy.
KeywordsSchiff base 2,4-Dinitrophenylhydrazine Charge transfer TCNQ Spectral characterization
KT is an off-white crystalline powder freely soluble in methanol, and it exists in the anionic form, in particular, at physiological pH. It is a racemate of [−]S- and [+]R-enantiomeric forms, with the S-form exhibiting the therapeutic activity. To enhance the water solubility and render the drug fairly appropriate for parenteral administration (Buckley and Brogden 1990), it is formulated as tromethamine salt and administered intramuscularly, intravenously, as a topical ophthalmic solution and also orally as a continuation therapy to injection (Sinha et al. 2009). The analgesic efficacy of KT appears to be superior to morphine or meperidine because it lacks the depressant effects of opioids. Because of its enhanced importance over morphine, it is widely preferred as an analgesic drug over opiate comparators in controlling postsurgical pain [DeAndrade et al. 1994].
The great clinical significance of KT in medicinal field has led to an extensive literature on its determination in pharmaceutical formulation. A survey of literature revealed that the KT has been analyzed in pharmaceutical preparations either alone or in combination with other drugs by HPLC (Kumaraswamy et al. 2012; Razzaq et al. 2012; Tsvetkova et al. 2012; Dave et al. 2013; Dubey et al. 2012), HPTLC (Devarajan et al. 2000; Vandana et al. 2013), voltammetry (Squella et al. 1997), and micellar electrokinetic chromatography (Orlandini et al. 2004). Though these analytical procedures are sensitive, it entails a disadvantage of not being simple and requiring sophisticated experimental setup, increased cost of analysis and high consumption of solvents by the mobile phase for the chromatographic techniques. Therefore, it is essential to develop simple, rapid, and reproducible methods that can be adapted for routine analysis of KT in quality control laboratories. In recent times, spectrophotometry has been successfully employed as a quantitative technique for the determination of drugs in bulk and formulations.
Derivative spectrophotometric methods (Jitendra et al. 2009; Yatri et al. 2013; Ramzia et al. 2013) and quite a few visible spectrophotometric methods (Rajasekaran 1995; Pratapareddy and Chakravarthi 2012; Kamath et al. 1994; Arshiya et al. 2012; Dattaray and Uday 1997; Sawsan et al. 2013) are reported in literature for quantitative determination of KT in bulk and dosage forms. The purpose of this study is to develop simple, accurate, and economical methods for the determination of KT using 2,4-dinitrophenylhydrazine (DNPH) and 7,7′,8,8′-tetracyanoquinodimethane (TCNQ) as reagents. Optimization of various experimental conditions, stoichiometry, and mechanism of the proposed reactions are studied. The suggested methods have high sensitivity and are validated as per ICH guidelines and rely on the use of non-expensive technique and affordable easily available chemicals. The application of the proposed methods is explored by employing these methods for the microdetermination of KT in its pharmaceutical dosage forms. Attempts have also been made to synthesize and characterize the reaction products using spectroscopic techniques.
Instrumentation and physical measurements
Spectrophotometric measurements were performed on SHIMADZU UV-2550 double beam spectrophotometer equipped with 1-cm matched quartz cells. The infrared spectra within the range of 4000–400 cm−1 for the free reactants and the reaction products were recorded using KBr disc on SHIMADZU FT-IR-Prestige-21 spectrometer. 1H NMR spectra (400 MHz) were recorded on AV400 spectrometer using DMSO-d6 as a solvent and TMS as the internal reference standard. Scanning electron microscopy (SEM) images were taken in Zeiss Sigma instrument equipped with GEMINI column, with an accelerating voltage of 2.00 kV.
Materials and reagents
Pure sample of ketorolac tromethamine (KT) was provided by CAD Pharma Inc., Bangalore, India. The following pharmaceutical formulations were procured from commercial source: Ketorol™ tablets (Dr. Reddy’s Laboratories Ltd., India) labeled to contain 10 mg of ketorolac tromethamine per tablet, Ketorol™ vials (Dr. Reddy’s Laboratories Ltd., India) labeled to contain 30 mg of ketorolac tromethamine in 1 mL of sterile solution, and Ketlur® sterile ophthalmic solution (Sun Pharmaceutical Industries Ltd., India) labeled to contain 5 mg of ketorolac tromethamine per milliliter of aqueous solution.
Analytical reagent grade 2,4-dinitrophenylhydrazine and potassium hydroxide were purchased from BDH chemicals. The reagent 7,7′,8,8′-tetracyanoquinodimethane was obtained from Sigma-Aldrich. Spectroscopic grade methanol and acetonitrile solvents were supplied by Spectrochem.
Preparation of standard stock and reagent solutions
The stock standard solutions of KT (100 μg mL−1) were prepared by dissolving precisely weighed 10 mg of pure drug in 100 mL of methanol and acetonitrile for method A and method B, respectively. The working concentrations were prepared by approximate dilution of standard drug solution.
The reagent solution of DNPH was prepared at a concentration of 5 × 10−3 M in methanol, and TCNQ was prepared at a concentration of 5 × 10−4 M in acetonitrile solvent. The stock solutions were freshly prepared and protected from light.
General analytical procedures
Determination of KT based on the measurement of Schiff base (method A)
Aliquots containing 0.50–18.50 μg mL−1 of standard KT (100 μg mL−1) were transferred quantitatively to 10 mL calibrated standard flasks. To that, 1 mL of DNPH solution was added followed by addition of 1 mL of 2 N methanolic potassium hydroxide and the volume was brought to 5 mL with methanol. The solutions were incubated in water bath at 60 °C for 15 min. The solutions were cooled to room temperature and diluted up to the mark using methanol, and absorbances were measured at 424 nm against the reagent blank.
Determination of KT based on the measurement of charge transfer (method B)
Aliquots containing 2.00–50.00 μg mL−1 of standard KT (100 μg mL−1) were transferred quantitatively to 10 mL calibrated standard flasks. To each flask, 2 mL of TCNQ was added and the reaction mixture was heated at 50 °C for 10 min. The solutions were then cooled and completed to the volume with acetonitrile solvent, and absorbances were measured at 842 nm against the reagent blank.
Analysis of pharmaceutical formulation
The contents of 20 tablets were pulverized to fine powder, and an amount equivalent to 10 mg was accurately weighed and dissolved in 50 mL methanol and acetonitrile for method A and B, respectively. The solutions were swirled for 10 min and filtered through Whatmann No. 40 filter paper into two separate 100-mL standard flasks which was then diluted to the mark with respective solvents. A convenient aliquot from the resulting solutions was then subjected to analysis.
The contents of two injection vials, each having an amount equivalent to 30 mg of active ingredient was filtered through Whatmann No. 40 filter paper and transferred into two separate 100-mL calibrated flask. It was then dissolved with methanol and acetonitrile for method A and B, respectively. The solutions were mixed well and completed to the volume with respective solvents. A convenient aliquot from resulting solutions was then subjected to analysis.
An accurately measured volume equivalent to 5 mg was dissolved in 25 mL methanol and acetonitrile for method A and B, respectively. The solutions were then mixed well and filtered through Whatmann No. 40 filter paper. The filtrates were collected in two separate 50-mL calibrated flasks and diluted to the mark with respective solvents. A convenient aliquot from resulting solutions was then subjected to analysis.
Synthesis of solid reaction products
The synthesis of KT-DNPH product was done by refluxing a mixture of KT (1 mmol, 0.3764 g) and DNPH (1 mmol, 0.1981 g) in 50 mL of methanol in basic medium for 4 h. The reaction mixture was allowed to cool and filtered to obtain red-colored solid precipitate, which was then washed with cold water and recrystallized from acetonitrile solvent. The solid charge transfer complex KT-TCNQ was synthesized by mixing an equimolar concentration of KT (0.50 mmol, 0.1882 g) and TCNQ (0.50 mmol, 0.1020 g) in 40 mL of acetonitrile, and the mixture was stirred well at room temperature for half an hour. The product was then filtered, washed with acetonitrile, and dried.
Results and discussion
Optimization of reaction parameters
In order to achieve maximum sensitivity and selectivity, the proposed spectrophotometric methods are optimized by carefully studying the different reaction variables affecting the reaction. The optimum experimental conditions are established by studying the influence of one parameter on the absorbance values of colored species and in turn keeping the other parameter constant.
Effect of solvent
Effect of heating time and temperature
Effect of reagent volume
For method A, the effect of different volumes of particular concentrations of DNPH and potassium hydroxide on fixed concentration of KT is investigated. It is found that reproducible absorbance value is obtained with 1 mL of 0.005 M DNPH and 1 mL of 2 N potassium hydroxide. Excess addition of base is avoided because stable results are not obtained and also the color of blank solution remains dark for long time. For method B, various volumes of TCNQ solutions are added to fixed drug concentration and the results show that 2 mL of 0.0005 M is adequate for reproducible and maximum color absorbance.
Stoichiometry of reaction
Validation of methods
The developed methods are validated with respect to linearity, accuracy and precision, specificity, limit of detection, and quantification according to the guidelines set by International Conference on Harmonization (ICH Q2 R1 2005).
Linearity and sensitivity
Spectral parameters and statistical data of the regression equation
λ max (nm)
Beer’s law limits (μg mL−1)
Molar absorptivity (L mol−1 cm−1)
0.7829 × 105
0.9033 × 104
Sandell sensitivity (μg cm−2)
0.4807 × 10−2
4.1666 × 10−2
Limit of detection (μg mL−1)a
Limit of quantification (μg mL−1)a
Y = a + bX
Y = a + bX
Correlation coefficient (r)
Limit of detection and quantification
The detection limit (LOD) and quantification limit (LOQ) is determined by evaluating the minimum level at which the active ingredient can be detected and quantified, respectively, with reliable accuracy and precision. The following expression was used: LOD = 3.3 σ/s and LOQ = 10 σ/s where σ is the standard deviation of the values obtained by replicate determination of blank and s is the slope of the calibration curve. The calculated values of LOD and LOQ are summarized in Table 1.
Accuracy and precision
Accuracy and precision data for the determination of KT by the proposed methods
KETO taken (μg mL−1)
Intraday (n = 5)
Interday (n = 5)
KETO founda ± SD (μg mL−1)
KETO founda ± SD (μg mL−1)
2.47 ± 0.02
2.51 ± 0.03
6.46 ± 0.02
6.51 ± 0.06
10.51 ± 0.05
10.47 ± 0.06
10.04 ± 0.08
10.01 ± 0.13
26.02 ± 0.12
25.88 ± 0.11
34.02 ± 0.12
33.82 ± 0.08
Determination of KT in dosage forms by the proposed methods
Labeled amount (mg)
Amount founda ± SD
10.04 ± 0.09
9.99 ± 0.08
t testb = 0.95
t testb = 0.50
% Recc = 100.40
% Recc = 99.90
30.01 ± 0.01
30.01 ± 0.03
t testb = 0.78
t testb = 0.99
% Recc = 100.03
% Recc = 100.03
Ketlur sterile eye drops
5.01 ± 0.05
5.00 ± 0.06
t testb = 1.74
t testb = 0.17
% Recc = 100.20
% Recc = 100.00
Application to the analysis of formulation
The developed methodology is applied to the determination of KT in three different pharmaceutical formulations. As can be seen from Table 3, the results of the assay are in good accordance with the label claim. The accuracy and reliability of the proposed assay are confirmed by applying the statistical tests such as Student’s t test. It is found that experimental t-value is smaller than the theoretical tabulated value at 95 % confidence interval and five degrees of freedom. Appreciable percentage recovery values in the range of 100.03–100.40 % and 99.90–100.03 % are obtained for method A and method B, respectively, which strongly suggests non-interference of pharmaceutical adjuvant in the proposed assay.
Characterization of reaction products
1H NMR spectra
The proposed analytical methods for the determination of KT in pure form and pharmaceutical formulation proved to be simple, accurate, and precise compared to the previously reported methods. The assay is rapid and requires simple sample preparation and economical since it depends on the commercially available laboratory instruments and low consumption of readily available inexpensive reagents. Furthermore, the excipients present in the formulations gave minimal interference with the proposed assay. These advantages permit the successful evaluation of these methods in pharmaceutical quality control, and so, it can be routinely applied to the pharmaceutical sample without prior treatment in the determination of KT in bulk and dosage forms. The formation of solid reaction products of KT is confirmed by FT-IR and 1H NMR, and features of surface morphology are investigated by scanning electron microscopy.
The authors gratefully acknowledge CAD Pharma Inc., Bangalore, for giving the drug sample. The authors would also like to thank the University Grants Commission for the financial assistance through BSR one time grant for the purchase of chemicals and Mangalore University for providing the required research facilities to carry out this work.
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