International Journal of Chemical and Natural Science
Transcription
International Journal of Chemical and Natural Science
International Journal of Chemical and Natural Science Vol. 3, No. 3 (2015): 258-263 Research Article Open Access ISSN: 2347-6672 Analytical Method Development and Validation of Simultaneous Estimation of Ketorolac and Phenylephrine by RP-HPLC Method in Bulk Gujapaneni Swetha* and P. Jaya Chandra Reddy Department of Pharmaceutical Analysis and Quality Assurance, Krishna theja Pharmacy College, renigunta road, Tirupathi, India. * Corresponding author: Gujapaneni Swetha; email: [email protected] Received: 01 March 2015 Accepted: 12 April 2015 Online: 01 May 2015 ABSTRACT A simple, precise, accurate and rapid reverse phase high performance liquid chromatographic method had been developed for simultaneous estimation of ketorolac and phenylephrine in bulk .Symmetry C18 kromasil column having i.d of 150×4.6 mm and 5µm particle size (Make: Waters) was used. The method was carried out in isocratic program using mobile phase, potassium dihydrogen ortho phosphate buffer: Acetonitrile (40:60). Flow rate was adjusted to 0.9ml/min and effluents were monitored at 228nm. The retention time obtained for ketorolac and phenylephrine was 3.623& 2.272min respectively. The calibration curves were linear in the concentration range of 25-150µg/ml and with correlation coefficients (r2) 0.999 and 0.999 for ketorolac and phenylephrine respectively. The mean recoveries were found to be in the ranges of 100.41% -101.81%and 99.25%-100.88% for ketorolac and phenylephrine respectively. The proposed method has been validated as per ICH guidelines and successfully applied to the estimation of ketorolac and phenylephrine in their combined form. Keywords: Phenylephrine, ketorolac simultaneous estimation, RP-HPLC. 1. INTRODUCTION Ketorolac is a non-steroidal anti-inflammatory drug, when administered systemically, has demonstrated analgesic, anti-inflammatory and anti-pyretic activity. Chemically it is (±)-5-benzoyl-2, 3-dihydro1Hpyrrolizine- 1-carboxylic acid, 2-amino-2- (hydroxyl methyl)-1,3-propanediol. Ketorolac acts by inhibiting the bodily synthesis of prostaglandins. An ophthalmic solution of ketorolac is available and is used to treat eye pain and to relieve the itchiness and burning of seasonal allergies. Phenylephrine hydrochloride (PHE) is α1adrenoceptor agonist which stimulates postsynaptic Alpha receptor causes vasoconstriction, systolic and diastolic pressure. It is indicated for nasal Congestion, minor eye irritations and open angle glaucoma.Phenylephrine hydrochloride (PHE), a synthetic sympathomimetic agent, is used in the treatment of sinusitis and bronchitis. Chemically it is benzenemethanol, 3-hydroxy-α [(methylamino) methyl]-hydrochloride (R). Various analytical methods http://ijcns.aizeonpublishers.net/content/2015/3/ijcns258-263.pdf have been reported for the assay of PHE alone or in combination with other sympathomimetic agents in pharmaceutical formulations. They include UV spectroscopy high performance liquid chromatography.[1-5] Several methods have been studied for simultaneous determination of ketorolac and phenylephrine, but there are limited reports on method for combination. So the aim of our study is to develop simple, fast, accurate and specific reversed phase high performance liquid chromatographic method for simultaneous determination of ketorolac and phenylephrine in bulk drugs and Pharmaceutical Dosage form. Figure 1(a): Chemical structure of ketorolac. 258 G. Swetha et al. / Int J Chem Natur Sci. 2015, 3(3): 258-263 water bath for 5 minutes. Filter through 0.45 µ filter under vacuum filtration. Figure 1(b): Chemical structure of phenylephrine. 2. MATERIALS AND METHODS 2.1 Chemicals and solvents: were obtained respectively from spectrum pharma research solutions, Hyderabad, India. Acetonitrile, Methanol and water used are of HPLC grade. 2.2 Instrumentation: The chromatographic separations were performed using HPLC-Waters alliance (Model-2996) consisting of an inbuilt auto sampler, a column oven and 2996 PDA detector. The data was acquired through Empower-2software. The column used was Symmetry C18 (150×4.6mm i.d, 5µm particle size) (make: Waters). Meltronics sonicator was used for enhancing dissolution of the compounds. Elico pH meter was used for adjusting the pH of buffer solution. All weighing was done on Sarotorious balance (model AE-160). 2.3 Chromatographic conditions: Detector : 229nm Injection volume : 20µl Flow rate : 0.9ml/min Temperature : Ambient Run time : 7min Mobile phase : Buffer and Acetonitrile taken in the ratio 40:60A Diluent : first dissolved in Methanol and made up with water 2.4 Preparation of buffer solution (pH3.5): Accurately weighed and transferred 2.72gm of Potassium di-hydrogen Ortho phosphate in a 1000ml of Volumetric flask add about 900ml of milli-Q water (4.5 pH), and was adjusted to 3.5 with Ortho phosphoric acid. 2.5 Preparation of mobile phase: Mix a mixture of above Buffer 250 ml (25%), 750ml of Acetonitrile HPLC (75%) and degassed in ultrasonic Concentration of ketorolac in µg/ml 1 3ppm 2 6ppm 3 9ppm 4 12ppm 5 15ppm 6 18ppm Correlation Coefficient S.No 2.6 Preparation of standard stock solutions: (120µg/ml Ketorolac, 400µg/ml Phenylephrine) Accurately Weighed and transferred 6mg of Ketorolac and 20mg of Phenylephrine working Standards into a 50ml clean dry volumetric flask, add 3/4th volume of diluent, sonicated for 5 minutes and make up to the final volume with diluents. 1ml from the above two stock solutions was taken into a 10ml volumetric flask and made up to 10ml. (12µg/ml Ketorolac, 40µg/ml Phenylephrine). 2.7 Preparation of sample stock solution: (120µg/ml Ketorolac, 400µg/ml Phenylephrine) 5 tablets were weighed and calculate the average weight of each tablet then the weight equivalent to 5 tablets was transferred into a 50ml volumetric flask, 30ml of diluent added and sonicated for 25 min, further the volume made up with diluent and filtered. From the filtered solution 0.5ml was pipette out into a 10 ml volumetric flask and made up to 10ml with diluent. (12µg/ml Ketorolac, 40µg/ml Phenylephrine). 2.8 METHOD VALIDATION: The developed method was validated as per the ICH (International Conference on Harmonization) guidelines with respect to System suitability, Precision, Specificity, Linearity, Accuracy, Limit of detection and Limit of quantification. 2.8.1 Linearity: Aliquots of 0.25, 0.5, 0.75, 0.1, 0.25 And 0.50ml were taken from stock solution of concentration 120µg of ketorolac and 400µg of phenylephrine, and then diluted up to 10ml with diluents. Such that the final concentrations were in the range 3-18µg for ketorolac and 10-60µg for phenylephrine. Volume of 10µl of each sample was injected in five times for each concentration level and calibration curve was constructed by plotting the peak area versus drug concentration. The observations and calibration curve were shown in Table 1 and Fig. 2, 3. Table-1: Linearity data Ketorolac Area 156083 308011 452295 623748 753757 923783 http://ijcns.aizeonpublishers.net/content/2015/3/ijcns258-263.pdf Concentration of phenylephrine in µg/ml 10ppm 20ppm 30ppm 40ppm 50ppm 60ppm phenylephrine Area 223018 425092 643685 867725 1071512 1298345 0.999 259 G. Swetha et al. / Int J Chem Natur Sci. 2015, 3(3): 258-263 Figure 2: Phenylephrine calibration curve Figure 3: Ketorolac calibration curve 2.8.2 Assay: Accurately weighed powder equivalent to 12mg of ketorolac and 40mg of phenylephrine was transferred into a 100ml clean dry volumetric flask add Diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. Drug ketorolac phenylephrine % linearity level 50 100 150 (Stock solution) Further pipette 1ml of ketorolac and phenylephrine of the above stock solution into a 10ml volumetric flask and diluted up to the mark with diluents it gives 12µg of ketorolac & 40µg of phenylephrine. The results were shown in Table-2. The chromatograms were shown in Fig-4. Table-2: ketorolac and phenylephrine assay Label claim mg/tab 12 40 Amount found mg/tab 59.5 99.7 Label claim(% ) 99.16 99.7 Table-3: Accuracy (ketorolac) % recovery 100.41 100.27 101.81 101.83 99.91 100.26 99.25 100.79 100.88 http://ijcns.aizeonpublishers.net/content/2015/3/ijcns258-263.pdf S.D* % R.S.D 0.189754 0.191106 0.329008 0.329008 %mean recovery ± S.D 100.83± 0.86 % RSD 0.85 100.67± 1.02 1.01 100.31 ± 0.92 0.91 260 G. Swetha et al. / Int J Chem Natur Sci. 2015, 3(3): 258-263 Accuracy (phenylephrine) % linearity level 50 % recovery 101.01 99.98 101.21 100 101.68 100.57 101.59 150 101.87 101.80 %mean recovery ± S.D 100.74± 0.66 % RSD 0.65 101.28± 0.62 0.61 101.37 ± 0.80 0.79 100.44 2.8.3 Accuracy: Accuracy of the method was done by recovery study. Sample solutions were prepared by spiking at about 50%, 100%, and 150% of specification limit to placebo and analyzed by the proposed HPLC method. Results are shown in Table-3(a) and (b). 2.8.4 Specificity: The specificity of the method was performed by injecting blank solution( without any sample) and then a drug solution of 10µl injected into the column, under optimized chromatographic conditions, to demonstrate the separation of both ketorolac and phenylephrine from any of the impurities, if present. As there was no interference of impurities and also no change in the retention time, the method was found to be Specific. INJECTIONS 1 2 3 4 5 6 AVG S.D %R.S.D Drug phenylephrine ketorolac Injections 1 2 3 4 5 6 1 2 3 4 5 6 2.8.5 Limit of detection (LOD) and Limit of quantification (LOQ): The parameters LOD and LOQ were determined on the basis of response and slope of the regression equation. The linearity for ketorolac and phenylephrine was performed from 3-18µg/ml and 10-60µg/ml respectively. 2.8.6 System precision: Precision is the measure of closeness of the data values to each other for a number of measurements under the same analytical conditions. Standard solution of ketorolac (12µg/ml) and phenylephrine (40µg/ml) were prepared as per test method and injected for 3 times. Results are shown in Table-4. Table-4: System precision AREAS(ketorolac) 653667 657914 652222 659062 659932 658899 656949 3200.4 0.5 AREAS(phenylephrine) 844171 846421 844985 841066 845330 848482 845076 2465.0 0.29 Table-5: Method precision % Assay 100.22 99.91 99.61 100.33 100.33 99.58 99.51 99.88 99.96 100.05 99.97 99.93 2.8.7 Method precision: Three samples were prepared and analyzed as per the test method on same day and three different days and calculated the % RSD for assay of five preparations. Results were shown in Table- 5. http://ijcns.aizeonpublishers.net/content/2015/3/ijcns258-263.pdf Mean S.D % R.S.D 100.00 0.35 0.35 99.98 0.192 0.19 2.8.8 Robustness: Robustness studies were carried out by variations in flow rate, mobile phase compositions and temperature. It was observed that the small changes in these operational parameters did not lead to changes of retention time of the peak interest. The degree of 261 G. Swetha et al. / Int J Chem Natur Sci. 2015, 3(3): 258-263 reproducibility of the results proven that the method is robust. 2.8.9 System suitability test: The system suitability was determined by making six replicate injections from freshly prepared standard Drug phenylephrine ketorolac solutions. The observed RSD values were well within usually accepted limits (≤2%). Theoretical plates, tailing factor, resolution between ketorolac and phenylephrine were determined. The results are all within acceptable limits summarized in Table-6. Table-6: Characteristics of HPLC method Parameters defined Linearity range (µg/ml) Slope Intercept Regression coefficient(r2) LOD ( µg/ml) LOQ (µg/ml) Tailing factor Plate count Linearity range (µg/ml) Slope Intercept Regression coefficient(r2) LOD (µg/ml) LOQ (µg/ml) Tailing factor Plate count Obtained value 10-60µg/ml 21552 483 0.999 0.03 0.09 1.09 3888 3-18 µg/ml 50981.5 836 0.999 0.01 0.02 1.09 13133 Figure 4: Standard chromatogram of phenylephrine and ketorolac. 3. RESULTS AND DISCUSSION The nature of sample, its molecular weight and solubility decides the proper selection of stationary phase. The drugs ketorolac and phenylephrine were preferably analyzed by reverse phase chromatography and accordingly C18 column was selected. The elution of the compounds from column was influenced by polar mobile phase. The ratio of Potassium di-hydrogen Orthophosphate to Acetonitrile was optimized to (40:60) to give well resolved and good symmetrical peaks with short run time. The retention time of ketorolac and phenylephrine were found to be 3.623 & 2.272 min respectively. The calibration curve was linear over the concentration range of 3-18µg/ml (ketorolac) and 10-60µg/ml (phenylephrine). The linearity of the method was statistically confirmed. RSD values for accuracy and precision studies obtained http://ijcns.aizeonpublishers.net/content/2015/3/ijcns258-263.pdf were less than 2% which revealed that developed method was accurate and precise. The system suitability parameters were given in table-5. The analytical recovery at five different concentrations of ketorolac and phenylephrine was determined and the recovery results were in the range of 100.41% 101.81% and 99.25%-100.88% µg/ml. Therefore proposed validated method was successfully applied to determine ketorolac and phenylephrine in tablet dosage form. 4. CONCLUSION The developed method is accurate, simple, rapid and selective for the simultaneous estimation of ketorolac and phenylephrine in pharmaceutical dosage form. The excipients of the commercial sample analyzed did not interfere in the analysis, which proved the specificity of the method for these drugs. The sample preparation is 262 G. Swetha et al. / Int J Chem Natur Sci. 2015, 3(3): 258-263 simple, the analysis time is short and the elution is by isocratic method. Hence the proposed method can be conveniently adopted for the routine quality control analysis in the combined formulation. 5. REFERENCES 1. 2. 3. 4. Khushbu B, Krupa C and maheshwari. (2014). Stability indicating hplc method for simultaneous estimation of ciprofloxacin and phenylephrine in pharmaceutical dosage form. Pharmacophore. 5(2): 262-272. B. Raja and Lakshmana Rao. (2013). 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(1986) “Instrumental methods of analysis”, 5th edition, Van Nostrand Publisher, New Delhi, 175-250. Skoog, Holler, Nieman, (2005) “Principles of Instrumental Analysis”, 5th edition, Saunders Coll Publisher, 345-356. Munson. (2001) Modern Methods of Pharmaceutical Analysis, Medical book distributors, Mumbai, 17-54. © 2015; AIZEON Publishers; All Rights Reserved This is an Open Access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ***** http://ijcns.aizeonpublishers.net/content/2015/3/ijcns258-263.pdf 263