SIMPLE AND VALIDATED RP-HPLC METHOD FOR THE ESTIMATION OF CARBOPLATIN IN BULK AND FORMULATION DOSAGE FORM Subhashini.Edla* B.Syama sundhar Abstract Dept of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar. GUNTUR A simple, rapid, specific, accurate and precise reverse phase high performance liquid chromatographic method was developed for the estimation of Carboplatin in formulation dosage form. A Kromosil C18 5mm column having 250 x 4.6mm id in Isocratic mode with mobile phase containing Acetonitrile: Water (85:15v/v) at ph: 3.5 was used. The flow rate was 1ml/min and effluents were monitored at 225nm. The retention time of Carboplatin was 4.5min. The concentration curves were linear in the concentration range of 30 to 180µg/ml. The developed method was validated for specificity, precision, linearity, accuracy, ruggedness, robustness and solution stability. Recovery of Carboplatin in formulations was found to be in the range of 98.09% to 101.63%. Proposed method was successfully applied for the quantitative determination of Carboplatin in formulations. Drug introduction Carboplatin or cis-diammine(1,1-cyclobutanedicarboxylato)platinum(ii) is in a class of medications known as platinum-containing compounds and interact with DNA to interfere with DNA repair. Carboplatin was discovered at Michigan State University, [1,2] at the Institute of Cancer Research in London. Carboplatin is used alone or in combination with other medications to treat cancer of the ovaries that has spread to other parts of the body, not improved, or that has worsened after treatment with other medications or radiation therapy. Carboplatin is also sometimes used to treat lung, bladder, breast, and endometrial cancer; head and neck cancer; cancer of the cervix and testicles, certain types of brain tumors; neuroblastoma, Wilms' tumor. It works by stopping or slowing the growth of cancer cells. Figure: A. Structure of Carboplatin Two theories exist to explain the molecular mechanism of action of carboplatin with DNA-Aquation, or the like-cisplatin hypothesis, Activation, or the unlike-cisplatin hypothesis. The former is more accepted owing to the similarity of the leaving groups with its predecessor cisplatin, while the latter hypothesis envisages a biological activation mechanism to release the active Pt 2+ species. A recent study in mutant mice suggests that in the subset of women with breast cancer due to BRCA1 and BRCA2 genes (these cause a variety of familial breast cancer) carboplatin may be as much as 20 times more effective than the usual breast cancer treatments. [3] Carboplatin has also been used to treat testicular cancer 254
patients with stage 1 seminoma. Recent research indicates that this treatment is more effective and has fewer side effects than adjuvant radiotherapy. [4-5] It is as effective as radiotherapy at preventing development of seminoma in the remaining testicle. [6] Relative to cisplatin, the greatest benefit of carboplatin is its reduced side effects, particularly the elimination of nephrotoxic effects. Nausea and vomiting are less severe and more easily controlled. The main drawback of carboplatin is its myelosuppressive effect. This causes the blood cell and platelet output of bone marrow in the body to decrease quite dramatically, The side effects are common (occurring in greater than 30%) for patients taking Carboplatin include Low blood counts (including red blood cells, white blood cells and platelets), Nausea and vomiting, Taste changes, Hair loss, weakness, Blood test abnormalities. Very few analytical methods have been described for analysis of carboplatin [7-9]. This paper describes validated RPHPLC method for estimation of carboplatin in bulk and pharmaceutical formulations. The proposed method was optimized and validated as per the ICH guidelines. EXPERIMENTAL 2.1. Reagents and chemicals Working standard of carboplatin was obtained from well reputed research laboratories. HPLC grade water, Methanol, Acetonitrile was purchased from E. Merck (Mumbai, India). The pharmaceutical dosage forms used in the study was injection vial labeled containing 150mg of carboplatin purchased from the retail shop. 2.2. Apparatus A Series HPLC system PEAK LC 7000 isocratic HPLC with PEAK 7000 delivery system. Rheodyne manual sample injector with switch (77251), analytical column chromosil C18, 250 4.6mm, Electronic balance-denver (SI234), manual Rheodyne injector with a 20 μl loop was used for the injection of sample. PEAK LC software was used. UV 2301 Spectrophotometer was used to determine the wavelength of maximum absorbance 2.3. Chromatographic conditions Analysis was carried out using PEAK HPLC system, Consisting of LC 7000 pump. A chromosil C18 5mm column having 250 x 4.6mm id in isocratic mode with mobile phase containing Acetonitrile: Water 85:15 (V/V); ph: 5.1) was used at a flow rate 1ml/min. A Rheodyne injector with 20 ml loop was used for injecting the sample. Detection was carried out using UV/VIS detector at 225nm. 2.4. Preparation of mobile phase 150ml of water was mixed with 850ml of Acetonitrile in the ratio of 15:80 (%v/v). Then sonicated for 15min and filtered through a 0.45µm Millipore filter. 255
2.5. Standard solutions Weighed accurately about 100mg of carboplatin and was dissolved in 100 ml of mobile phase in to the 100ml light resistant volumetric flask. The final solutions containing 1mg/ml of carboplatin. Further dilutions are prepared from above solution. 2.6. Sample preparations A commercially available injection Cytocarb (150mg) of Cipla India Ltd, was chosen for this purpose. The contents of five vials of Cytocarb each containing 150 mg of Carboplatin were pooled up and reconstituted with the mobile phase in a 100 ml volumetric flask. An aliquot from this solution (1mg/ml) was taken in a separate 10 ml volumetric flask and was diluted with mobile phase to get final concentration 90μg/ml. The sample was injected into the column for five times and the mean peak area ratio of the drug to the internal standard was calculated from the chromatogram. The drug content was quantified using the regression equation obtained for the pure sample. 3. RESULT AND DISCUSSION 3.1 Method development and optimization The Chromatographic conditions were optimized for the Carboplatin content uniformity and assay method within a short analysis time (10 min) using simple mobile phase and an acceptable peak tailing (<1.5). In order to achieve these goals, the Chromatographic mobile phase and column were chosen first. The dissociation constant (pka) of Carboplatin is 4.51. The 10 µg/ml Carboplatin was scanned between 200 400 nm in a UV spectrophotometer. The maximum absorbance (λmax) was obtained at 225 nm as shown in Fig. 2 and hence the wavelength 285nm was selected in further development. Columns of Zodiac C18 and Hypersil BDS were tried, no better peak shape and non reproducible retention time was observed. The good peak shape (Tailing about 1.3) and retention time (about 4.5min) were observed with Kromosil C18 column having with a flow rate of 1.0ml/min. The system suitability tests were carried out on freshly prepared standard solution of Carboplatin to check various parameters. System suitability results are as follows: Retention time 4.51min, Asymmetric factor 1.3 Theoretical plates 12219 Calibration range 30-180µg/ml. 3.2. Method validation The proposed method has been validated for the assay of Carboplatin in formulation using following parameters: 3.2.1. Linearity: Linearity was studied by preparing standard solution at different concentration levels. The linearity range was found to be 30-180mg/ml. The regression equation was found to be y = 7351x + 25224 with coefficient of correlation R2 = 0.9993 (Fig.D.) 3.2.2. Precision Precision was studied to find out intra and inter day variations in the proposed method of Carboplatin with 90µg/ml on the same day and three different days respectively. The percentage RSD was calculated for intra and inters day precision and found to be less than 2%. The results of precision are presented in the table 2. 256
3.2.3. Recovery Study The accuracy of the method was determined by calculating recovery of Carboplatin 50%, 100% and 180% was added to a pre-quantified sample solution. The recovery studies were carried out three times over the specified concentration range and the percentage recovery of Carboplatin was found to be in the range of 98.09% to 101.63% and the results are presented in the table 3. 3.2.4. Robustness Robustness of the method was studied by changing the wavelength from 225 to 220, 230 and the mobile phase composition of organic phase changed by ± 5% and ph± 2. The results showed that the retention time and peak area of Carboplatin is remains almost unchanged and no significant degradation was observed. 3.2.5. Assay The standard and sample solutions were injected three times separately; chromatograms and the peak areas were recorded. Representative chromatograms of sample have been given in Fig B, C. The amount of drug present per injection vial was determined. Thus obtained results were presented in the table 4. 4. CONCLUSION Proposed study describes new reverse phase high performance liquid chromatographic method for the estimation of Carboplatin in formulations the method was validated and found to be simple, sensitive, accurate and precise. Percentage of recovery studies shows that, the method is free from interference of the other active ingredients and additives used in the formulation. Therefore proposed method can be used for routine analysis for the estimation of Carboplatin in bulk and formulation dosage form. Fig. B: Standard chromatogram of Carboplatin 257
Fig.C: Formulation chromatogram of Carboplatin Table.1: Linearity results of Carboplatin S.No Concentration (µg/ml) Area 1 30 270251 2 60 468625 3 90 680863 4 120 925817 5 150 1128318 6 180 1333851 Slope Intercept CC 7351.046 25223.68 0.9993 Peak Area 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 Carbiplatin Linearity y = 7351x + 25224 R² = 0.9993 0 50 100 150 200 Concentration Fig.D: Calibration curve of Carboplatin 258
Table.2: precision results of Carboplatin Sample (90µg/ml) Intraday precision Interday precision 1 698795 726883 2 695715 716051 3 702675 719299 4 697097 704640 5 693966 719155 6 696486 704613 RSD 0.43 1.24 Table.3: Recovery results of Carboplatin % Recove ry Target Conc., (µg/ml) Spiked conc, (µg/ml) Final Conc, (µg/ml) Conc., Obtained % of Recovery 50% 60 30 90 59.67 99.44 60 30 90 59.078 98.46 60 30 90 59.41 99.02 100% 60 60 120 119.26 99.38 60 60 120 117.71 98.09 60 60 120 121.00 100.83 150% 60 90 150 150.50 100.34 60 90 150 152.44 101.63 60 90 150 150.87 100.58 R.S.D 0.495 1.377 0.674 Table.4: formulation results of Carboplatin Formulation Dosage Concentration Amount found % Assay Cytocarb 150mg 90µg/ml 88.77µg/ml 98.63 References 1. Wheate NJ, Walker S, Craig GE, Oun R (September 2010). "The status of platinum anticancer drugs in the clinic and in clinical trials". Dalton Transactions 39 (35): 8113 27. 2. ONS Clinical Practice Committee. Cancer Chemotherapy Guidelines and Recommendations for Practice. Pittsburgh, PA: Oncology Nursing Society; 1999:32-41. 3. Henderson, Mark (May 1, 2006). "Lung cancer drug may fight breast tumour in women". Times Online. 4. "Testicular cancer drug effective". BBC News. 22 July 2005. 5. Rose, David (October 6, 2008). "Chemotherapy drug, carboplatin, is safer cure for testicular cancer ". The Times. 259
6. Nelson, Roxanne (June 10, 2008). "Carboplatin as Effective as Radiation in Preventing Relapse in Testicular Cancer". Medscape Medical News. 7. Villarino N, Determination of carboplatin in canine plasma by high-performance liquid chromatography. Biomed Chromatogr. 2010 Aug;24(8):908-13/ 8. Mittal A, HPLC method for the determination of carboplatin and paclitaxel with cremophorel in an amphiphilic polymer matrix. J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Aug 15;855(2):211-9. Epub 2007 May 16. 9. Ping Guo, Determination of carboplatin in plasma and tumor by high-performance liquid chromatography mass spectrometry, Journal of Chromatography B, Volume 783, Issue 1, 5 January 2003, Pages 43 52. 260