untitled European Journal of Chemistry 4 (1) (2013) 58‐60 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2013 EURJCHEM DOI:10.5155/eurjchem.4.1.58‐60.678 European Journal of Chemistry Journal homepage: www.eurjchem.com Validation of analysis method for determining ketoprofen concentration in pharmaceutical dosage form using high performance liquid chromatography Tadjuddin Naid a, Wahyu Rizandi Syukur a, Amran Ilyas a, Seniwati Dali b and Baharuddin Hamzah c, * a Faculty of Pharmacy, Hasanuddin University, Makassar, 90245, Indonesia b Chemistry Department, Faculty of Mathematics and Natural Sciences, Hasanuddin University, Makassar, 90245, Indonesia c Chemistry Education Study Program, Faculty of Teacher Training and Education, Tadulako University, Palu, 94118, Indonesia *Corresponding author at: Chemistry Education Study Program, Faculty of Teacher Training and Education, Tadulako University, Palu, 94118, Indonesia. Tel.: +62.853.27646270; fax: +62.451.429763. E‐mail address: hamzahhb@yahoo.com (B. Hamzah). ARTICLE INFORMATION ABSTRACT Received: 14 September 2012 Received in revised form: 28 October 2012 Accepted: 02 November 2012 Online: 31 March 2013 KEYWORDS The study was conducted with the purpose to develop and validate a high performance liquid chromatography method with UV detector to determine ketoprofen content in tablet preparation using ethanol‐phosphate buffer (pH = 6.0, 80:20, v:v). The method was validated toward parameters of accuracy, precision, linearity, selectivity, LOD and LOQ. The results obtained fulfill the validation requirement of the ketoprofen tablet in the form of LOD = 0.5302 ppm and LOQ = 1.7676 ppm. Conformity Optimization Ketoprofen Phosphate buffer Validation method High performance liquid chromatography 1. Introduction Ketoprofen is an non‐steroidal anti‐inflammation drug, used in this study by considering various aspects [1,2], namely solubility, weak acid compound and easily dissolve, in ethanol, chloroform and ether [3]. Ketoprofen dissolution can also be increased by adding 0.2 M phosphate buffer with pH = 5‐7 up to 3‐105 times compared to its solubility in water [4]. High performance liquid chromatograph (HPLC) is often used to validate method of analysis by first optimizing various parameters [5‐10]. The optimization is important for the effectiveness and efficiency of instrument. The validation parameters reported in this paper are accuracy, precision, selectivity, detection limit, linearity, and robustness of validated for the estimation of ketoprofen standard as well as tablet dosage form [11,12]. 2. Experimental HPLC instruments consist of phase isokratil type Shimadzu brand version 6.1 joined with SCL‐10 AVP control system, detector UV‐VIS SPD‐10 AMP, pump LC 10 ADVp, oven column CTO‐10 ACVP. Shim‐pack column VP‐ODS 250 x 4.6 mm. Filtration unit for HPLC (Shibata), ultrasonic bath (Shibata type SU‐2 TH), and spectrophotometer UV‐Vis (Shimadzu type 1601). Ketoprofen tablet 50 mg from the factory sample, ketoprofen standard of comparison from PT Pharos with LSA: 14901595 and gross weight: 0.051 kg. Difference: 0.001 kg, Net weight: 0.050 kg, Phosphate buffer pH = 5.6 and 7.0 with degree of pro analysis reaction, ethanol 99.99% gradient grade for liquid chromatography. 2.1. Preparation of Ketoprofen standard solution About 10 mg ketoprofen standard was weighed, then dissolved with ethanol in flask of 10 mL up to the sign limit, and homogenized to obtain concentration of ketoprofen standard solution 1000 ppm. 2.2. Optimization of mobile phase volume Ketoprofen standard solution 1000 ppm, pipetted 0.02mL was put into the flask of 10 mL, diluted with ethanol up to the sign limit, then injected as much as 25 μL into the HPLC with mix mobile phase of ethanol phosphate buffer pH = 5 (95:5, v:v) using speed flow 1 mL/min with 10 minutes retention time. The experiment was replicated 3 times and also the mix mobile phase of ethanol phosphate buffer (90:10, v:v; 85:15, v:v and 80:20, v:v), then the mix mobile phase was chosen to provide the best separation based on HETP and number of plats theory (N). 2.3. Test of system conformity Ketoprofen standard solution 1000 ppm was pipetted 0.2 mL and put into the flask of 10 mL and diluted with ethanol up to the sign limit, homogenized to obtain ketoprofen standard concentration each 20 ppm with three replications made. Then each replication was injected 25 mL into HPLC with optimum mix mobile phase, optimum pH phosphate buffer, the detector was arranged at the selected wave length and used speed flow 1 mL/min with 10 minutes retention time to obtain chromatogram for each concentration to be used in determining the repetition of standard solution change stated in relative standard deviation. Naid et al. / European Journal of Chemistry 4 (1) (2013) 58‐60 59 Table 1. Accuracy of test result method of standard additions of ketoprofen in the sample (concentration 80%) in ethanol‐dafarfosfat mobile phase pH = 6 (80:20, v:v) (n=5). No Tablets (mg) Standard (mg)(CA) Sample (mg)(CA) Total Ketoprofen (mg) Rt (minutes) Area (mAU*S) Content (CF) Recovery (%) 1 129 13 28.165 41.165 6.517 16176097 41.237 100.553 2 129 12 28.165 40.165 6.342 15709253 40.005 98.666 3 128 12 27.947 39.947 6.358 15716001 40.023 100.633 4 130 14 28.384 42.384 6.392 16653791 42.497 100.807 5 130 14 28.384 42.384 6.250 16569073 42.273 99.207 Average 99.973 Standard deviation 0.752 % Average standard deviation 0.867 Table 2. Repeability of test result assay of ketoprofen (a concentration of 20 ppm) in ethanol‐dafarfosfat pH = 6 (80:20, v:v) (n=5). No Area (mAU*S) Peak Height (mAU) 1 5354423 374391 2 5374777 375422 3 5360187 372357 4 5302888 374121 5 5150702 364068 Average 5322948 307021.8 Standard deviation 96702.570 4608.250 % Average standard deviation 1.739 1.238 Table 3. Repeability of test result assay of ketoprofen (a concentration of 100 ppm) in ethanol‐dafarfosfat pH = 6 (80:20, v:v) (n=5). No Area (mAU*S) Peak Height (mAU) 1 23835932 1610843 2 23703430 1630495 3 23252063 1635901 4 24447081 1650709 5 23418695 1692778 Average 23907026.8 1644145 Standard deviation 477493.809 30706.53 % Average standard deviation 1.944 1.867 Figure 1. Calibration curve by Ketoprofen by HPLC. 2.4. Linearity In order to determine linearity between ketoprofen concentration and size of area, the solution series were prepared: 20, 40, 60, 80 and 100 ppm from ketoprofen standard solution 1000 ppm. The result can provide equation of regression line y = 236904.255 x + 545295.9. The calculation of ketoprofen content used the equation line. The correlation value indicates that there is a good correlation between ketoprofen concentration and the size of area (Figure 1). 2.5. Accuracy The method applied was the addition of sample standard and then analyzed. The difference of result was compared with added standard content as percentage of re‐planning. The accuracy method is said to be good if the range of regain 98‐ 102% (Table 1). The recovery according to data was arranged with concentration 80% each was replicated 5 times and the average obtained is 99.973‐50.752 % with range 98.686‐ 100.807 % and value of averagedeviation standard 0.867. 2.6. Precision The test was done by examining low concentration as much as 20 ppm and high concentration 100 ppm each was replicated 5 times in method range. By using the result of standard dissolution in different concentration, the size of area can be known and peak height provided by HPLC shows that the method used to determine ketoprofen content is precise since the Average Deviation Standard of reexamination < 20% as can be seen in Table 2 and 3. 60 Naid et al. / European Journal of Chemistry 4 (1) (2013) 58‐60 Table 4. Test result of content uniformity of Ketoprofen inthe factory sample 50 mg (n=10). No Tablets (mg) Sample (mg) Rt (minutes) Area (mAU*S) Content of Ketoprofen each tablet Content of Ketoprofen (%) 1 229 50 6.383 18254098 46.719 93.438 2 231 50 6.433 19217996 49.262 98.524 3 228 50 6.533 20148247 51.715 103.431 4 239 50 6.533 19148247 49.078 98.156 5 224 50 6.417 19956240 51.209 102.419 6 231 50 6.650 20342563 52.229 104.458 7 228 50 6.667 18245556 46.696 93.393 8 230 50 6.683 20229079 51.929 103.859 9 229 50 6.342 18380439 47.052 94.105 10 230 50 6.308 18221926 46.634 93.268 Average 49.252 98.5051 Standard deviation 2.371 4.742 % Average standard deviation 4.814 4.814 Table 5. Test result of content uniformity of Ketoprofen in the factory sample 50 mg (n=6). No Tablets (mg) Sample (mg) Rt (minutes) Area (mAU*S) Content of Ketoprofen each tablet Content of Ketoprofen (%) 1 229 50 6.700 20196206 51.842 103.684 2 231 50 6.383 19870053 50.982 101.964 3 230 50 6.675 20047037 51.449 102.898 4 230 50 6.342 20093247 51.571 103.142 5 231 50 6.325 19535645 50.100 100.200 6 232 50 6.392 20593911 52.892 105.784 Average 51.427 102.945 Standard deviation 0.925 1.851 % Average standard deviation 1.798 1.798 3. Results and discussion The application of content determination method in 10 tablets each contains 50 mg ketoprofen indicates the uniformity of average content 49.252 mg/tablet and ketoprofen content 98.51%(Table 4). Based on the uniformity test of content, the content uniformity is good enough since the determination of content of each tablet, the ketoprofen content ranges from 93.29 to 104.46% with %average deviation standard = 4.814 if it is calculated with the data of peak area size. The value fulfills the general requirement of content uniformity of quality matter that is at the range 90‐100% with % average deviation standard < 6%. The result of ketoprofen content determination in tablet with six replications can be seen in Table 5. Table 5 shows that the average ketoprofen content in sample tablet preparation with 50 mg/tablet based on chromatogram area is 57.472 ± 0.925 mg/tablet (%R.S.D. = 1.798). This result fulfills the requirement that is 90‐110% of the table of good precision since it meets the acceptance criteria < 2%. The test of accuracy includes the regaining of concentration 80% (98.666‐100.807%), concentration 100% (99.073‐ 101.795%), concentration 120% (99.325‐100.379%) all meet the criteria. The test of precision provides the repetitive value less than 2% that is % R.S.D. for each concentration tested 1.739% and 1.949%. The test of linearity provides equation line that is y = 236904.255x + 545295.9 with the value of detection limit (LOD = 0.5302 ppm and limit of quantity (LOQ= 1.7676 ppm).The procedure of determining ketoprofen content is found in tablet preparation using HPLC Shimadzu brand version 6.1 with ethanol mobile phase: phosphate pH=6 with comparison 80:20 (v:v), speed flow 1 mL/min, using Shim‐Pack column VP‐ODS 250 x 4.6 mm at room temperature 27 oC and automatic column pressure. 4. Conclusion Validation levels of ketoprofen in tablet dosage form using newly developed method is reliable and can be used routinely. Acknowledgements Thanks addressed to the president of the Hasanuddin University for funding this research paper. We also thank to the head of Pharmaceutical Chemistry Laboratory of Hasanuddin University for sample preparation. References [1]. Aznan, H. J.; Juli, S. Use of Anti‐Inflammatory Non‐Steroidal Rational at Tackling Rheumatic Pain. 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