1. INTRODUCTION:

Ketorolac tromethamine (KT), chemically it is, 2-Amino-2-(hydroxymethyl)propane-1,3-diol-(1-RS)-5-benzoyl-2, 3-dihydro-1 H-pyrrolizine-1-carboxylate is a selective COX-I inhibitor, highly potent class of nonsteroidal anti-inflammatory drugs (NSAIDs) are used for short term treatment of post operative pain and local inflammation associated with musculoskeletal, joint disorders and some operative procedures.^1-5 When administered as eye drops it demonstrated analgesic, anti-histaminic, anti-inflammatory and anti-pyretic activity. The mechanism of action is to inhibit prostaglandin biosynthesis and given systemically does not cause pupil constriction.^6-8 In India, it is marketed under the trade name of Acular LS and Ketorol gel. Several studies for the estimation of the Ketorolac Tromethamine drug using various techniques have been utilized like Simultaneous Reverse phase high pressure liquid chromatographic (RP-HPLC) method used for estimation of ketorolac tromethamine in ophthalmic dosage forms⁹, Simultaneous spectrophotometric estimation of Ofloxacin and Ketorolac Tromethamine in tablet dosage forms¹⁰; Simultaneous estimation of combination diclofenac sodium, famotidine and ketorolac tromethamine has also been reported.¹¹

Spectrophotometric method used for determination of ketorolac tromethamine bulk and pharmaceutical dosage form¹². Two-dimensional liquid chromatography and ion trap mass spectrometry used for the simultaneous estimation of ketorolac enantiomers and paracetamol in human plasma, which help in pharmacokinetic study¹³. Proniosomes are nonionic surfactant based vesicles that have ability to deliver the hydrophobic and hydrophilic drugs¹⁴. Simple and sensitive method used for the analysis of ketorolac in human plasma using high-performance liquid chromatography¹⁵. The preparation and evaluation of KT Gel containing genipin for periodontal diseases¹⁶. The present study planned to estimation of ketorolac tromethamine in proniosomal gel and marketed gel formulation using RP-HPLC method.

2. EXPERIMENTAL PROCEDURES:

2.1 Instruments

The RP-HPLC system consisted of Shimadzu LC-20A (LC Solution Software) system equipped with model LC-20AT pump, SPD-20A prominence UV-visible detector (set at 306 nm) and a Rheodyne injection valve with a 20-μL loop. Sartorius Electronic Analytical balance, Crest sonicator was used.

2.2 Chemicals and Reagents:

The HPLC grade of methanol and o-phosphoric acid in were obtained from Merck, India. Potassium dihydrogen phosphate AR grade was obtained from HmBG Chemicals, Malaysia. A gift sample of Ketorolac Tromethamine was obtained from Piramal Health Care Pvt., Ltd. (Pithampur, India). Ketorol gel was purchased from local vendor, formulated at Dr. Reddy’s Pharmaceutical Ltd. India. HPLC grade deionized water was used throughout the experiment.

2.3 Chromatographic Conditions:

The experimental conditions were optimized on a Shimizu C₁₈ column (250X4.6 mmX5 μm) using the mobile phase methanol and water with 0.1% o-phosphoric acid in the ratio of 60:40 v/v. It was filtered through 0.45 μm nylon membrane filter and degassed. It was used as diluents for the preparation of sample and standard.

Chromatographic separation was achieved at 30 °C, the detection was carried at 306 nm at a flow rate of 1.5 mL min⁻¹ and run time was kept at 20 min. Prior to the injection of drug solution column was equilibrated for 60 min with the mobile phase flowing through the system. The flow rate of mobile phase was set at the rate of 1ml/min while the injection volume was 20µL. Blank containing the mobile was injected to check the solvent interference.

2.4 Wavelength Detection:

Accurately weighed KT equivalent to 100 mg in 100 mL volumetric flask, 100 mL of mixture of methanol and water in the ratio of 60:40 V/V used as diluents in sample preparation, pH 3.0 was adjusted with 0.1% o-phosphoric acid was added, sonicated for 5 min and filtered through 0.45 μm nylon membrane filter. Pipette out 1 mL of the above solution and dilute to 10 mL with diluents in 10 mL volumetric flask and scanned between 200 and 400 nm by UV spectroscopy.

Figure 1. UV absorbance spectra.

2.4 Standard Preparation:

KT (10 mg) was weighed and transferred into a 10 mL volumetric flask and make up the volume with diluent. From this, 0.05, 0.10, 0.15, 0.20 and 0.25 mL were taken and diluted with diluent up to 10 mL for preparation of 5, 10, 15, 20 and 25 μg/mL respectively. A representative chromatogram of the standard was shown in Figure 2.

Figure 2: Standard chromatogram of KT

2.5 Samples Preparation:

2.5.1 Ketorol Gel of Marketed Preparation

Ketorol gel sample equivalent to 10 mg of KT was weighed and transferred to 10 mL volumetric flask and make up the volume with diluents. To the above solution was added 70 mL of the diluent, it was filtered through 0.45 μm nylon membrane filter and degassed. A representative chromatogram of the marketed ketorol gel sample was shown in the Figure 3.

Figure 3: Chromatogram of marketed preparation

2.5.2 Formulated Proniosomal Gel of KT:

Proniosomal gel sample equivalent to 10 mg of KT was weighed and transferred to 10 mL volumetric flask and make up the volume with diluents. To the above solution was added 70 mL of the mobile phase, sonicated for about 15 min and filtered through 0.45 lm nylon membrane filter. A representative chromatogram of the formulated ketorol gel sample was shown in the Figure4.

Figure 4: Chromatogram of Proniosomal gel of KT

Table 1: Analysis of marketed formulation ketorol gel and Proniosomal gel of KT.

Formulation	Ingredients	Labelled amount (mg)	Amount found (mg)	% Amount found
Ketorol gel	Ketorolac Tromethamine	0.4	0.42	101
KT Proniosomal gel	Ketorolac Tromethamine	0.4	0.41	101

3. System Suitability Parameters:

The 20 μl of standard solution was injected in five duplicate before and after the analysis and the chromatogram were recorded. System suitability parameter like%RSD, column efficiency, plate count and tailing factor were calculated. The column efficiency less than 2000, USP Tailing for the same peak was not more than 2.0 and % RSD of five injection of the standard solution is not more than 2.0% the chromatogram was shown in Table 2.

Table 2: System Suitability Parameters Standard Preparation

Injection	RT	Peak Area	USP Plate Count	USP Tailing Factor
1.	6.974	6101397	1020	1.37
2.	6.884	6100185	1102	1.22
3.	6.962	6200132	1223	1.16
4.	6.889	6103114	1073	1.24
5.	6.971	6201052	-	1.28
Mean	6.936	6141176	-	1.25
SD	0.045	54250.1	-	-
% RSD	0.655	0.883	-	-

4. Analytical Method Validation^17-20

4.1. Specificity

Placebo solution was prepared as per the test solution using equivalent weight of the placebo in a portion. Placebo solution was injected into the HPLC system following the test conditions, the chromatogram was recorded and measured the responses of the peaks were noted for any interference of the excipient at the retention time of Ketorolac tromethamine (Figure 5).

Figure 5: Placebo (Specificity) chromatogram.

4.2. Precision:

Precision was measured in terms of repeatability of application and measurement. Repeatability of standard application (system precision) was carried out using six replicates of the sample injection (100 μg mL⁻¹). Repeatability of sample measurement (method precision) was carried out in six different sample preparations from the same homogenous blend of the marketed sample (100 μg mL⁻¹). The percentage RSD for repeatability of standard preparation was 1.17% whereas the % RSD for repeatability of the sample preparation was 0.02%. This shows that the precision of the method is satisfactory as percentage RSD is not more than 2% the chromatogram as shown in Figure 6 and 7 and Table 3.

Figure 6: Precision chromatogram.

Figure 7: Intermediate precision chromatogram.

Table 3: Precision study of the method

S. no	Sample area	% Assay	Amount present (mg)
1	2606385	100.3	0.47
2	2698064	103.8	0.48
3	2530365	97.82	0.43
4	2591486	98.87	0.46
5	2597270	99.49	0.45
6	2587679	99.34	0.46
MEAN		99.94	0.45
SD		54253.1247
% RSD		1.17

4.3. Linearity:

The linearity of Ketorolac tromethamine was determined by preparing and injecting a solution with a concentration of about 50-150 μg mL⁻¹. The calibration curve indicates the response is linear over the concentration range studied for Ketorolac Tromethamine with a correlation coefficient (r) of 0.999.

4.4. Stability of Sample Solution:

The sample solution was prepared as per the test method, and analyzed initially at different time intervals by keeping the solution at room temperature. The percentage response between the initial and different time intervals shows that the sample solutions were stable for at least 24 h at room temperature (Table 4).

Table 4: Stability study:

S. no	Time interval (h)	Peak response
1	0	2606836
2	2	2698061
3	6	2530365
4	10	2559486
5	16	2577201
6	24	2656773
Mean		2596031.33
SD		51277.56
% RSD		0.019

4.5. Robustness:

Robustness of the method was determined by analyzing the standard solution at normal operating conditions by changing some operating analytical conditions such as flow rate, column, oven temperature, detection wavelength and the mobile phase. The conditions with variation and their results were shown in Table 5. The tailing factor is around unity indicative of peak symmetry and theoretical plate counts were also above 2000. Hence robustness of the extent of variations applied to analytical conditions was shown in Figure 9.

Table 5: Robustness studies

System suitability parameters (variations)		% RSD of peak area response (n=3)	Mean tailing factor (n=3)	Mean retention time in min (n=3)
Change in (%)	+5	1.20	0.67	6.01
Organic phase	−5	1.23	0.86	6.05
Change in pH	+3	0.019	1.28	6.06
Change in pH	−3	0.017	1.30	6.08
Change in flow	+1.5	0.004	1.22	6.75
Change in flow	−1.5	0.003	1.25	6.85
Change in temp.	+25	0.003	1.19	5.18

Figure 9: Robustness chromatogram.

4.6. Accuracy:

The percentage recovery experiments were performed by adding a known quantity of pure standard drug into the pre-analyzed sample. The solution equivalent to 100 mg of Ketorolac Tromethamine was accurately weighed into a 100 mL volumetric flask. The sample was then spiked with standard at level 50%, 100% and 150% of test concentration. The resulting spiked sample solutions were assayed in triplicate and the results were compared and expressed as percentage. The mean percentage recovery of Ketorolac Tromethamine was found to be in the range between 98.54 and 102.5 which are within the acceptance limits as shown in Table 6 and Figure 10.

Figure 10: Accuracy chromatogram

4.7. Limit of detection (LOD) and limit of quantification (LOQ):

The limit of detection (LOD) is the lowest amount of analyte in a sample that can be detected, but not necessarily quantified, under the stated experimental conditions. LOD and LOQ were calculated by using standard deviation and slope values obtained from the calibration curve by using the formula LOD=3.3 (SD/S) and LOQ=10 (SD/S). The LOD and LOQ values for Ketorolac Tromethamine were found to be 0.116 μg mL⁻¹ and 0.32 μg mL⁻¹ respectively as shown in Figure 11 and 12 and Table 7.

Figure 11: LOD

Figure 12: LOQ

Table 7: LOD and LOQ

S. no	Concentration (μg mL⁻¹)	Peak area
1	50	1341056
2	70	1814323
3	100	2582490
4	120	3087955
5	150	3877114

5. RESULTS AND DISCUSSION:

A different combination of mobile phases and chromatographic conditions were tried and a mobile phase containing methanol and water with 0.1% O-phosphoric acid (60:40 V/V), Grace C18 (250 cm x 4.6 mmx5 μ) column, 1.0 mLmin-1 flow rate, 20 μL injection volume, 314 nm wavelength and 16 min run time was found to be suitable for all formulations. RP-HPLC method used for estimation and comparison of marketed preparation, and the area for standard preparation was found to be 6101397 and retention time was 6.974 min. The mean area for standard preparation was found to be 6141176 and % RSD for standard preparation was not more than 2. The area for proniosomal preparation was found to be 6275490 and retention time was 6.888 min. The mean area for the proniosomal preparation (n=5) was found to be 6240921 and % RSD for proniosomal preparation was not more than 2 and The area for marketed preparation was found to be 6299492 and retention time was 6.888min. The mean area for the marketed preparation (n=5) was found to be 6202598 and % RSD for marketed preparation was not more than 2. Specificity of the method was checked by injecting the placebo solution, no peaks were found at the retention time of Ketorolac Tromethamine. The stability of the sample solution was evaluated by preparing a sample solution as per the proposed method and analyzed initially and at 1 h intervals up to 24 h by keeping the sample solution at room temperature. The results of the stability studies showed that the solution of the drug was found to be stable for 24 h at room temperature. System precision and method precision results showed the%RSD of 1.16 and 0.02, respectively. A good linearity relationship indicated by correlation coefficient (r) value 0.999 was observed between the concentrations of 50–150 μg mL−1 of Ketorolac Tromethamine. Intermediate Precision was done by changing the analyst, column, with the same chromatographic conditions and the obtained results were within the limits. The Robustness method was evaluated by deliberately varying the chromatographic conditions of the method such as mobile phase methanol content, flow rate, column temperature and wavelength. The parameters like tailing factor and retention time showed adherence to the limits. The accuracy of the method was determined and the percentage recovery was calculated. The data indicate an average of 102.5% recovery of the standard sample.

6. CONCLUSION:

A RP-HPLC method utilized for estimation ketorolac tromethamine proniosomal gel and marketed gel formulation. The method for quantitative estimation of KT results indicated that the method was specific, accurate, linear, precise, rugged and robust for marketed and sample formulation, which can be possible by the reverse phase chromatographic method utilizing C₁₈ column and methanol and water with 0.1% O-Phosphoric acid in the ratio of 60:40 v/v used as a mobile phase. All the area of standard preparation, sample preparation of proniosomal gel and marketed preparation were found in seven digits and approx. same and repeated results were found same. Therefore, we can conclude that ketorolac tromethamine proniosomal gel and marketed preparation are same in drug concentration profile. The concentration of marketed gel and formulated proniosomal gel preparation were calculated by the calibration curve. The assay of the sample of formulated preparation and marketed gel were performed as per pharmacopoeial specification and % purities were observed in the acceptable limits. The method developed for Ketorolac Tromethamine was found to be a simple process and the procedure does not involve any experimental conditions. The validation results indicated that the method was specific, accurate, linear, precise, rugged and robust. The runtime was relatively 20 min which enabled a rapid quantification of many samples in routine and quality control analysis of ophthalmic formulation.

7. ACKNOWLEDGEMENTS:

The authors are highly thankful to the Principal, Indore Institute of Pharmacy, Indore for proving necessary facility and to Piramal Health Care Pvt. Ltd., Pithampur, India for providing a gift sample of Ketorolac tromethamine to carry out this work.

8. REFERENCES:

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Received on 12.02.2018 Accepted on 20.03.2018

Asian J. Pharm. Ana. 2018; 8(3): 157-163.

DOI: 10.5958/2231-5675.2018.00029.7

Spike level (%)	Amount added in (μg mL⁻¹)	Peak area	% Recovery	%Mean recovery
50	0.12	1347374	103.90	102.5
50		1308694	100.83
50		1336027	102.93
100	0.25	2635598	101.54	99.44
100		2575802	99.32
100		2530358	97.48
150	0.36	3532611	96.23	98.54
150		3858796	99.10
150		3906218	100.31

4. Analytical Method Validation17-20