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

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.

 

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 pain².

 

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 formulations³. KT has been studied and determined by relatively other methods such as Spectrophotometry^4-6, HPLC^7-12 and HPTLC ^13-16.

 

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 10³ L/mol/cm) and sensitivity (0.363901μg/cm²) 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 (r²).

 

Figure 4: Calibration curve of Ketorolac Tromithamine (KT).

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.

 

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 80⁰, 90⁰ 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

 

 

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

 

Table 5:  Effect of temperature on absorbance.

 

 

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

 

 

Scheme I: Formation of Ruhemann’s purple complex between KT and ninhydrin.

Table 6: Comparison of Proposed and Published SpectrophotometricMethods^19-20.

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

 

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.

 

7.0. REFERENCES:

1.        USP Vol. 1.United States Pharmacopoeial Convention; 2003:1049.

2.        Norvasc, Online Drug Description and Clinical Pharmacology of ketorolac, The Internet Drug Index, Rx List Inc., 2008. Website:http://www.rxlist.com/cgi/generic/keto2.htm.

3.        Tripathi K.D., “Essential of Medical Pharmacology”,5^th Edn., Jaypee Brothers Medical Publisher; New Delhi;

4.        KumaraSwamy G., Kumar JMR., Sheshagirirao J.V.L.N.; Simultaneous Estimation of Febuxostat and Ketorolac in Pharmaceutical Formulations by Spectroscopic Method; International Journal of ChemTech; 2012; 4:847-850.

5.        Fegade J.D., Mehta H.P., Choudhari R.Y., Patil V.R.; Simultaneous Spectrophotometric Estimation of Ofloxacin and Ketorolac Tromethamine in Opthalmic Dosage Form; International Journal of ChemTech; 2009;1;189-194.

6.        Dewani A.P., Bakal R.L., Dr.Shiradkar M.R., Dr.Chandewar A.V.;Absorpion Ratio Method  for the Estimation of Moxifloxacin HCL and Ketorolac Tromethamine in their Combined Dosage Form by UV-Visible Spectroscopy; International Journal of Pharmaceutical Research and Development; 2011; 3(7);21-26.

7.        Sunil G., Jambulingam M., Thangadurai S. A., Kamalkannan D., Sundaraganapathy R., Jothimanivannam C., Development and Validation of Ketorolac Tromithamine in Eye Drop Formulation by RP-HPLC Method; Arabian Journal of Chemistry; 2013.

8.        Irene Tsina, Yuen Ling Tam, Aeelin Boyd, Cynthia Rocha, Ian Massey, Thomas Tarnowski; An Indirect and Direct HPLC Method for the determination of the Enantiomers of Ketorolac in Plasma; Journal of Pharmaceutical and Biomedical Analysis; 1996; 15(3); 403-417.

9.        Yang Guo Ping, HE Bin,LIU Shi Kun,et al; Determination of Content of Ketorolac Tromethamine in Injection by HPLC; Chinese Journal of Hospital Pharmacy; 2012.

10.     Tsvetkova Boyka G., Pencheva, Ivanka P., Peikov, Plamen T.; HPLC Determination of Ketorolac Tromethamine in Tablet Dosage Forms; Der Pharmacia Sinica; 2012; 3(4); 400.

11.     Kumaraswami Gandla, JMR Kumar, DVRN B Bikshapati, R Spandana; A Validated RP-HPLC Method for the Simultaneous Estimation of Febuxostat and Ketorolac Tromethamine in Pharmaceutical Pharmulations; Journal of Drug delivery and Therapeutics; 2013; 2(3); 173-176.

12.     Syed Naeem Razzaq, Muhammad Ashfaq, Islam ullah Khan, and Irfana Marium; Stability Indicating HPLC Method for Simultaneous Determination of Ofloxacin and Ketorolac Tromethamine in Pharmaceutical Formulations; Royal Society of Chemistry; 2012; 4(7); 2121-2126.

13.     Padma V. Devrajan, Subhash P. Gore, Sunil V. Chavan; HPTLC Determination of Ketorolac Tromithamine; Journal of Pharmaceutical and Biomedical Analysis; 2000; 22(4); 679-683.

14.     Rao P.L.K.M., Venugopal V., Teja S.G., Radhika D.V.N.S., Kavita K., Lavanya S., Manasa T., Pavani A; Validation and Analytical Evaluation of Ketorolac Tromethamine by HPTLC Using Reflectance Scanning Densitometry; International Journal of Research in Pharmacy and Chemistry; 2011; 1(2); 130-132.

15.     Lopez B.E., Castaneda H.G., Gonzalez-De La Parra M., Namur S.; Development and Validation of High Performance Thin Layer Cromatographic Method with densitometry for Quantitative Analysis of Ketorolac Tromethamine in Human Plasma; Journal of AOAC; 2008; 91(5); 1191-1195.  

16.     Gandhi S.P., Devani M.G., Borole T.C., Damle M.C.; Development and Validation of Stability Indicating HPTLC Method for Determination of Ofloxacin and Ketorolac Tromethamine in Combination; Journal of Advanced Scientific Research; 2011; 2(3); 77-82.

17.     Patil V.P., Gaikwad A.D., Kulkarni V.S., Kavade R.V., Kale S.H.; Green Analytical Chemistry; An overview; Inventi Rapid; Pharma Ana and Qua Assur; 2012; 2;294.

18.     Armenta S., Garrigues S., and Della Guardia; Green Analytical Chemistry; Tr.Ana.Chem.27:M.2008; 497-511.

19.     Kamath B.V., Shivram K., Vangani S.; Spectrophotometric Determination of Ketorolac Tromithamine by Charge Trancefer and Ion-Pair Complexation; Analytical letters; 1994 ;27(1):103-112.

20.     Pratapreddy A.J. and Chakravarthy I.E., New Spectrophotometric Determination of Ketorolac Tromithamine Bulk and Pharmaceutical Dosage Form; International Journal of Pharmaceutical Science and Research; 2012; 3(12); 4848-4850

 

 

 

Received on 10.08.2013         Modified on 20.10.2013

Accepted on 06.11.2013         © AJRC All right reserved