Research & Reviews: Journal of Pharmaceutical Analysis
Menue-ISSN: 2320-0812
e-ISSN: 2320-0812
Vrushali Tambe*, Manaswi Patil, Ujjwala Kandekar, and Shrivastava SK
PES’s, Modern College of Pharmacy (For Ladies), Dehu-Alandi Road, Borhadewadi, Moshi, Pune- 412105, Maharashtra, India.
Received Date: 18/11/2013; Accepted Date: 12/12/2013; Published Date: 16/12/2013
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Ketorolac Tromethamine (KET) and Ofloxacin (OFL) from bulk and eye drop formulation were precisely and accurately determined using developed high performance thin layer chromatographic (HPTLC) method. Analysis was done on silica gel 60F254 as stationary phase with aluminum backed TLC plates. Dichloromethane: Methanol: Ammonia 25% (6:3:1) was used as a mobile phase. The two drugs were satisfactorily resolved with Rf 0.79± 0.03 and 0.41± 0.02 for KET and OFL respectively. The method performance was validated as per ICH guidelines. Linearity was found in the range of 25-75ng/band (r2 = 0.999) for KET and 15- 45ng/band (r2 = 0.9998) for OFL. The assay results were found to be 99.14±0.413 %w/w and 98.11±0.232 % w/w for KET and OFL respectively. The % Recovery for KET was in the range of 99.74-99.95% w/w and that of OFL was 99.78-99.87% w/w. This validated method can be used for simultaneous estimation of KET and OFL from bulk and ophthalmic formulation.
Ketorolac Tromethamine, Ofloxacin, HPTLC, Validation, ICH Guidelines
Ketorolac Tromethamine (Figure 1) is 5-benzoyl-2, 3-dihydro-1H-pyrrolizine-1-carboxylicacid, 2- (hydroxymethyl)-1, 3 propanediol1. It is a potent analgesic and with moderate anti-inflammatory activity.
Figure 1: Ketorolac Tromethamine
Ofloxacin (Figure 2) chemically is 9-fluro-2, 3-dihydro-3-methyl-10-(4-methyl-1-piperizinyl)-7-oxo-7H-pyrido [1, 2, 3-de]- 1, 4- benzoxaine- 6 -carboxylicacid [1]. Ofloxacin is a synthetic chemotherapeutic antibiotic of the fluoroquinolone drug class considered to be a second-generation fluoroquinolone.
Figure 2: Ofloxacin
KET has been estimated by UV Spectroscopy [2], HPTLC [3] and HPLC [4,5,6,7,8]. OFL was estimated alone or in combination with other drugs using techniques like UV Spectrophotometry [9,10,11,12,13,14,15], HPLC [16-27] and electrochemical analysis [28]. KET (5 mg/ml) and OFL (3 mg/ml) combination is available in the market in the form of ophthalmic solution (KETLUR). This paper provides simple, economic and validated method for simultaneous estimation of KET and OFL in eye drop formulation.
Chemicals and Reagents
Pure drugs of Ketorolac Tromethamine (Lupin Pharma Ltd, Pune) and Ofloxacin (Litaka Pharma Ltd., Pune) were obtained as a gift samples. All the other chemicals were of analytical grade and were purchased from Merck Chemicals.
Instrumentation and Chromatographic Conditions
Aluminum-backed TLC plates (20 cm × 10 cm), coated with 250 μm layer of silica gel 60 F254 (E. Merck, Darmstadt, Germany) were used. Plates were prewashed by methanol and activated at 110°C for 5 min. Samples were applied as 8 mm bands using 25 μl Linomat V applicator (Camag, Muttenz Switzerland) equipped with a Hamilton syringe (Bonaduz, Switzerland). A constant application rate of 1 μl/s was used and the space between two bands was 10 mm.
The optimized chamber saturation time for mobile phase was 10 min at room temperature (25°C±2) at 60±5% RH. Ascending development of the plates was performed with Dichloromethane: Methanol: Ammonia 25% (6:3:1) as mobile phase, in a Camag 20×10 cm twin trough glass chamber with chromatogram run of 8 cm. HPTLC plates after development were dried in current of air with the help of air dryer in a wooden chamber with adequate ventilation. A unidirectional air flow was maintained in the laboratory. The densitometric measurement was performed using a slit dimensions of 6.00×0.30 mm, the scanning speed of 20mm s-1 and monochromator bandwidth of 20 nm. Each track was scanned thrice using Camag HPTLC scanner III in the reflectance absorbance mode at 307 nm.
Preparation of standard solutions
An accurately weighed KET and OFL (50 mg of each) were dissolved separately in 20 ml methanol in 50 ml volumetric flask. The solutions were further diluted to the volume with methanol to the concentration of 1 mg ml-1 for both the drugs. Standard stock solutions were further diluted with methanol to concentration of 100 μg/ml for each drug.
Optimization of Mobile Phase
Various binary mobile phases as shown in Table 1 were used in initial few trials wherein OFL showed tailing and KET was found to have Retention factor close to 1. The ternary mobile phase were also tried to minimize the tailing factor of OFL.
Table 1: Optimization of Mobile phase for Simultaneous estimation of KET and OFL by HPTLC.
Analysis of Eye drop formulation
For analysis, 1 ml of the marketed formulation containing KET (5 mg) and OFL (3mg) was diluted to 10 ml with methanol to produce a concentration of 0.5 mg/ml for KET and 0.3 mg/ml for OFL. The solution was sonicated for 10 min. and was filtered through whatmann filter paper No. 41. Stock solution was diluted to get final concentration of 50ng/band for KET and 30ng/band for OFL. The plate was developed in an optimized mobile phase. The analysis was repeated in triplicate. The possibility of excipient interference with the analysis was examined.
Method Validation
Validation of the TLC method was carried out as per ICH guidelines [29] with the following parameters:
Linearity and range
Calibration curves were prepared using a standard solution containing 50 μg/ ml and 30 μg/ ml KET and OFL respectively in methanol. Six concentrations were spotted on the TLC plate to obtain the final concentration in the range of 25-75 ng/band (KET) and 15-45 ng/band (OFL). Each solution was applied thrice on the plate and average peak area of each concentration was measured at 307 nm. It was plotted against the corresponding concentrations to obtain the calibration curves. Residuals for both the drugs were plotted against the concentration of analyte to confirm linearity of method.
Precision
System Precision was performed by six repeated applications of standard solutions containing 50 ng/band for KET and 30 ng/ band for OFL. The precision of the method was established in terms of repeatability, Intraday and Interday studies. Repeatability studies were performed by applying standard solutions containing 50 ng/band for KET and 30 ng/ band for OFL respectively (represents 100% of the test concentration) six times, to a TLC plate followed by development. Each solution was separately prepared.
The intra-day precision was assessed by analyzing standard drug solutions at 100% of the test concentration (50 ng/band for KET and 30ng/ band for OFL) three times on the same day. Each solution was applied six times. The Interday precision of the method was checked by repeating a study on three different days and % RSD of peak area was calculated.
Accuracy
Accuracy of the method was carried out by applying the method to the sample (KET and OFL combination in ophthalmic solution) to which a known amount of KET and OFL standard solution corresponding to 80, 100, and 120% of label claim has been added in the initial phase of sample preparation, mixed, and analyzed by running chromatogram in an optimized mobile phase. % Recovery at each level was calculated.
Limit of detection (LOD) and limit of quantitation (LOQ)
LOD is the concentration of the analyte that yield signal-to-noise ratio of 3 and of 10 at LOQ. The LOD and LOQ were determined by measuring the magnitude of analytical background by spotting a blank and calculating the signal-to-noise ratio for KET and OFL by spotting a series of solutions until the S/N ratio of 3 was obtained for the LOD and 10 for the LOQ.
Specificity
The specificity of the method was ascertained by analysis of drug standards and samples. The identities of the bands for KET and OFL were confirmed by determining peak purity and also by comparing the Retention factor of the bands with those of standards.
Robustness
Small deliberate changes in the experimental parameters were done. These include time from spotting to chromatography and from chromatography to scanning were varied by + 5 mins, Ultra-sonication time of the mixture was varied by + 3 min. Plates from different lot number were used.
The developed HPTLC method for simultaneous estimation of KET and OFL in eye drops was found to be simple and convenient for the routine analysis of two drugs.
Optimization of other chromatographic conditions
The proposed method uses silica gel 60 F254 as a stationary phase, sample application in the form of 8 mm band at the rate of 1 μl/s, 10 mm as a space between two bands, chamber saturation time of 10 min at room temperature, 8 cm chromatogram run, the scanning speed of 20mm s-1, detection wavelength at 307nm and the average development time of 20 min.
Preparation of standard solution
Both drugs exhibit same absorptivity at 307 nm as indicated in Figure 3 was selected as analytical wavelength.
Figure 3: Overlay spectrum of KET and OFL
Optimization of mobile phase
The development of chromatogram in binary mobile phase like Chloroform: Ethyl acetate (3:8), Chloroform: Methanol (9:1) was unsuccessful due to tailing of OFL and Retention factor close to 1 for KET as shown in Table 1. Use of Dichloromethane: Methanol: Ammonia 25% (6:3:1) has resulted in a dense, compact and well resolved spots. Chromatogram is shown in Figure 4.
Figure 4: Chromatogram of KET and OFL at 307nm with Rf value of 0.79± 0.03 and 0.41± 0.02 respectively. At 307 nm
Analysis of Marketed formulation
The assay results as shown in Table 2 were found to be 99.14±0.813 9 %w/w and 98.11±0.232 % w/w for KET and OFL respectively.
Table 2: It shows Assay results for KET and OFL in ophthalmic formulation
Linearity and Range
Linearity of the method was studied by spotting six concentrations of the drugs in the range of 25-75 ng/band and 15-45 ng/band for KET and OFL respectively. Calibration curves as found in Figures 5 and 6 were found to be linear. Residuals do not show either of the positive or negative values and are randomly distributed around the regression function as shown in Figure 7. This proves the linearity of the method. The correlation coefficient of these drugs was found to be close to 1.00 indicates linearity of the method. Linearity data is presented in Table 3.
Figure 5: Calibration curve of KET
Figure 6: Calibration curve of OFL
Figure 7: Residuals of KET and OFL obtained from regression data at 307 nm by plotting distance of response from regression line.
Table 3: Linearity data of KET and OFL
Precision
Results of System Precision, Repeatability, intra-day studies and inter-day precision studies are shown in Table 4-6. % RSD of peak area after six subsequent injections of the same standard solution for both the drugs was not more than 2% indicating system precision. Percentage RSD of the peak area after application of six independent solutions of the same concentration was less than 2%. This proves method precision.
Table 4: It shows statistical analysis for System Precision.
Table 5: It shows method precision
Table 6: It shows data for Intraday and Interday Precision of the proposed method.
Accuracy
Percent recovery of the proposed method when applied for marketed formulation after spiking with standard was found to be 99.74-99.95 % for both drugs (Table 7).
Table 7: It shows statistical recovery of the proposed method.
Sensitivity
Sensitivity of the method as shown in Table 8 was proved by low limit of detections 0.077/0.2165 ng/band (KET/OFL) and limit of quantitation 0.2357/0.656 ng/band (KET/OFL).
Table 8: It shows statistical analysis for LOD and LOQ of the proposed methods
Specificity and robustness
Sensitivity of the method as shown in Table 8 was proved by low limit of detections 0.077/0.2165 ng/band (KET/OFL) and limit of quantitation 0.2357/0.656 ng/band (KET/OFL).
Specificity and robustness
The low values of % RSD in peak area after doing deliberate minor changes in the method parameters indicates robustness of the method (Table 9). Values of r as shown in Table 1 are greater than 0.99 indicates peak purity of both analytes. From peak purity and absence of additional peaks other than drugs proves specificity of the method.
Table 9: Robustness study of KET and OFL
This method utilizes simple mobile phase. As compared to other reported chromatographic methods proposed method is economic. Hence it can be used for routine analysis of two drugs in combined dosage forms.
The developed TLC technique for simultaneous determination of KET and OFL was validated for precision, specificity, robustness, accuracy, Limit of quantitation and Limit of detection. It was found to comply with all validation parameters. Statistical analysis proves that the method is suitable for the analysis of KET and OFL from the bulk drug and ophthalmic formulations without any interference from the excipients.
The authors are thankful to Modern College of Pharmacy for Ladies, Moshi and Anchrom Lab, Mumbai for providing facilities for the research work. The authors also acknowledge Lupin Pharma Ltd., Litaka Pharma Ltd. for providing gift sample of drugs for analysis.