ANALYTICAL METHOD DEVELOPMENT FOR IMPURITIES PRESENT IN HYDROXY CHLOROQUINE SULPHATE FROM ANTIMALARIAL TABLET FORMULATION

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1 WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES SP. Karuppiah et al. SJIF Impact Factor Volume 3, Issue 7, Research Article ISSN ANALYTICAL METHOD DEVELOPMENT FOR IMPURITIES PRESENT IN HYDROXY CHLOROQUINE SULPHATE FROM ANTIMALARIAL TABLET FORMULATION SP. Karuppiah*, K. Anver Basha Department of chemistry, Sathyabama University, Chennai ,Tamilnadu,India. Article Received on 05 May 2014, Revised on 25 May 2014, Accepted on 25 June 2014 *Correspondence for Author SP. Karuppiah* Research Scholar, Sathyabama University, Rajiv Gandhi Road, JPR nagar,chennai ABSTRACT The study reveals that stability indicating analytical method development for the estimation of Impurities present in Hydroxy chloroquine Sulphate (HCS) from anti malarial tablet formulation.the developed method is specific,linear,accurate,robust and can be used for regular analysis. The known impurities are identified and estimated quantitatively as i) Hydroxy Chloroquine O-Sulphate (HCOS) ii)4,7- Dichloroquinoline (4,7 DCQ) iii)desethyl Hydroxy Chloroquine (DEHC) iv) Desethyl Chloroquine (DEC) by RP-HPLC method.the Relative Response Factor(RRF) and Relative Retention Time (RRT) has established for the quantification of impurities without using known impurities every time. Gradient elution has set up in proper proportion with buffer: methanol (90:10) and acetonitrile (100) in two different channels using Inertsil C8-3 (150 X 4.6), 5µm column in a detection wavelength of 380 nm. The range is covered from 50 to 150 of working concentration of 2ppm which is the range of Hydroxy chloroquine sulphate tablets 200 mg. The recovery study of each impurity is carried out and the result obtained is well within acceptance criterion of 90 to 110. Sulphate, 4,7-Dichloroquinoline, Desethyl Hydroxy Chloroquine, Desethyl Chloroquine. Keywords: Hydroxy chloroquine sulphate, impurity profiling, Hydroxy Chloroquine O- INTRODUCTION The chemical name of Hydroxychloroquine Sulphate is (RS)-2-N-[4-(7-chloro-4- quinolylamino) Pentyl ] - N-ethylaminoethanol sulphate.(c 18 H 26 ClN 3 O, H2SO4) having molecular weight which is white or almost white crystalline powder.(figure-1) Vol 3, Issue 7,

2 Figure1 : Chemical structure of Hydroxy Chloroquine sulphate Figure2 : Chemical structure of Hydroxy Chloroquine-O- sulphate Figure3 : Chemical structure of 4,7-Dichloroquinoline Figure4 : Chemical structure of Desethyl hydroxyl chloroquine Figure5 : Chemical structure of Desethyl chloroquine The chemical name of Hydroxy Chloroquine O-Sulphate is ( 2-[[4-[(7-Chloro-4- quinolinyl)amino]pentyl]ethylamino]ethanol 1-(Hydrogen Sulfate); 2-[[4-[(7-Chloro-4- quinolyl)amino]pentyl]ethylamino]ethanol Sulfate (C18H26CLN3O4S) having molecular weight (Figure-2) The chemical name of 4,7-dichloroquinoline is ( 4-[(7-chloroquinolin-4-yl)amino]-2- [(diethylamino)methyl]phenol}, (C 9 H 5 Cl 2 N) having molecular weight (Figure-3) The chemical name of desethyl hydoxychloroquine is [4-[(7-Chloro-4-quinolyl) amino] pentyl]amino]ethanol (C16H22ClN3O) having molecular weight (Figure-4) Vol 3, Issue 7,

3 The chemical name of desethylchloroquine is N4-(7-Chloro-4-quinolinyl)-N1-ethyl-1,4- pentanediamine (C16H22ClN3) having molecular weight (Figure-5) The above four metabolites of hydroxy chloroquine sulphate is separated each other by a single HPLC method to determine quantitatively the amount present in tablet formulation. Specificity by selectivity is performed to prove the method is specific and stability indicating. MATERIALS AND METHODS The materials used for this study is formulated tablets, active pharmaceutical ingredient, placebo used for making tablets, working /reference standards, Impurity standards, chemicals, solvents and laboratory glassware. RESULTS AND DISCUSSION Table No.1: Drug Product Specification Product Name Batch Number Manufacturer Plaquebaf(Hydroxychloroquine FD/HCQS/054 In-house Sulphate tablets 200 mg) Plaquenil(Hydroxychloroquine Sulphate tablets 200 mg) IW0015 Sanofi, Fawdon manufacturing centre. Table No.2: Chemicals & Reagents Specification Chemicals Name Reagent Grade Manufacturer Mono basic sodium phosphate Analytical reagent Ranchem anhydrous Di basic potassium phosphate Analytical reagent Ranchem Methanol HPLC Qualigen Acetonitrile HPLC Qualigen Triethylamine HPLC Ranchem Water HPLC Ranchem Table No.3: Drug Substance & Impurities Specification Name of the standards Category Manufacturer Hydroxychloroquine Sulphate API IPCA Laboratories Hydroxychloroquine-O-Sulphate Impurity Analytica chemie Inc., 4,7-Dichloroquinoline Impurity Analytica chemie Inc., Desethyl Hydroxy-chloroquine Impurity Analytica chemie Inc., Desethyl Chloroquine Impurity Analytica chemie Inc., Vol 3, Issue 7,

4 Table No.4: Drug Products Formula Name of the Ingredients Quantity per tablet in mg Grade DRY MIX Hydroxychloroquine Sulphate Pharmacopeial Lactose monohydrate Pharmacopeial Maize Starch Pharmacopeial BINDER Polyvinylpyrrolidone (K-30) Pharmacopeial Methocel E-5 (Hypromellose 5Cps) Pharmacopeial Purified Water Pharmacopeial LUBRICATION Maize Starch Pharmacopeial Magnesium stearate Pharmacopeial Coating Opadry White 04F58804 (Hypromellose 15 Cps, Macrogol 6000, Titanium dioxide) Pharmacopeial Total Coated tablet weight Analytical Methods The method established for the separation of all the impurities present in Hydroxy chloroquine sulphate tablets 200 mg is by using the analytical column Inertsil C8-3,150 X 4.6mm, 5µm at 30 C column oven condition and 320 nm UV absorbance with 1 ml flow rate of mobile phase A & mobile phase B in gradient elution. i) Gradient Programme TIME(MINS) MOBILE PHASE-A IN MOBILE PHASE-B IN ii) Mobile phase Preparation Transfer about 4 g of disodium hydrogen phosphate anhydrous, 2 g of potassium dihydrogen phosphate and 0.5ml triethylamine in to beaker containing 1000ml water. Filter the solution through 0.45µ membrane filter. Prepare Mobile phase A by mixing filtered and degassed Vol 3, Issue 7,

5 mixture of buffer and methanol in the ratio of 90:10(V/V).Use 100 acetonitrile as Mobile phase B.The diluent is prepared by mixing water and methanol in the ratio of 50:50(V/V). iii) Analytical Procedure The diluted standard solution is prepared by dissolving known quantity of Hydroxy chloroquine sulphate in diluent to get 2 ppm concentration.the resolution solution is prepared by dissolving 10 ppm each of hydroxy chloroquine sulphate and hydroxyl chloroquine-o- sulphate.to check the interference due to placebo 2ppm solution of placebo is prepared. Finally 20 tablets fine powder is used to prepare 2ppm concentration of sample solution. Inject 20 microlitre each of Blank, Placebo, Resolution solution, Standard preparation and sample preparation in to the chromatograph and record the response for all the peaks. The system suitability is evaluated as RSD of peak area responses of Hydroxychloroquine sulphate from five replicate injections of standard solution should not be more than 5.0, RSD of peak RT responses of Hydroxychloroquine sulphate from five replicate injections of standard solution should not be more than 1.0,the tailing factor for standard peak is NMT 2.0,The theoretical plates for standard peak is NLT 2000 and The resolution between Hydroxychloroquine and O-sulphate is NLT 3.0. Figure5 : Blank Chromatogram Vol 3, Issue 7,

6 Figure6 : 4,7-Dichloroquinoline Chromatogram Figure7 : Desethyl chloroquine Chromatogram Figure8 : Hydroxychloroquine-O- sulphate Chromatogram Vol 3, Issue 7,

7 Figure9 : Resolution solution Chromatogram Figure10 : sample spiked Chromatogram Figure11 : sample Unspiked Chromatogram Vol 3, Issue 7,

8 Figure12 : Hydroxy chloroquine standard Chromatogram Figure13 : Peak Identification Solution Chromatogram Calculate the content of each known impurity,unknown impurity and total impurities by taking the expression that divide the area of each known impurity present is test preparation by mean area of diluted standard preparation and is multiplied by respective concentration. Table No.5: The RRT & RRF Impurities Name of the Impurity RRT RRF Hydroxychloroquine sulphate 1.00 Desethyl Hydroxy-chloroquine Desethyl chloroquine Hydroxychloroquine-O-sulphate , 7-Dichloroquinoline Vol 3, Issue 7,

9 1) Known impurity Calculation (Desethyl Hydroxy-chloroquine) AT1 Wtstd 1 50 p X X X X X X Wtavg X RRF1 AS Wtspl 100 LC 2) Known impurity Calculation (Hydroxychloroquine-O-sulphate) AT2 Wtstd 1 50 p X X X X X X Wtavg X RRF2 AS Wtspl 100 LC 3) Known impurity Calculation (4, 7-Dichloroquinoline) AT3 Wtstd 1 50 p X X X X X X Wtavg X RRF3 AS Wtspl 100 LC 4) Known impurity Calculation (Desethyl chloroquine) AT4 Wtstd 1 50 p X X X X X X Wtavg X RRF4 AS Wtspl 100 LC 5) Highest Unknown impurity Calculation AT5 Wtstd 1 50 p X X X X X X Wtavg AS Wtspl 100 LC 6) Total Unknown impurity Calculation AT6 Wtstd 1 50 p X X X X X X Wtavg AS Wtspl 100 LC AT1 = peak area due to Desethyl Hydroxy-chloroquine obtained with RS test preparation AT2 = peak area due to Hydroxychloroquine-O-sulphate obtained with RS test preparation AT3 = peak area due to 4, 7-Dichloroquinoline obtained with RS test preparation AT4 = peak area due to Desethyl chloroquine obtained with RS test preparation Vol 3, Issue 7,

10 AT5 = peak area due to Highest Unknown impurity peak obtained with RS test preparation AT5 = peak area due to Highest Unknown impurity peaks obtained with RS test preparation AS = Mean peak area due to Hydroxychloroquine Sulphate obtained with diluted standard Preparation Wtstd =Weight of Hydroxychloroquine Sulphate working standard in mg Wtspl =Weight of sample taken in mg Wtavg = Average weight of capsule in mg. L.C. =Label claim in mg per tablet P =Potency of Hydroxychloroquine Sulphate working standard in percentage on as is basis RRF1 =0.99 RRF2 =1.09 RRF3 =0.84 RRF4 =0.56 Total impurities = sum of all known impurities and unknown impurities The impurities present in hydroxy chloroquine tablets have separated quantitatively to determine content estimation in percentage by HPLC. The method precision value obtained for six sets of sample preparation of the same batch is well within the acceptance criteria of 90 to 110 of label claim. The resolution between closely eluting impurity peaks are increased by optimizing the mobile phase gradient programme. Table No.6: Method precision value Name of the Impurity SET1 SET2 SET3 SET4 SET5 SET6 Limits Desethyl Hydroxychloroquine Desethyl chloroquine Hydroxychloroquine-Osulphate , 7-Dichloroquinoline Total unknown Impurities Total Impurities Vol 3, Issue 7,

11 Specificity of the product has performed by selectivity to prove the method is specific. The peak purity value of each impurity peak as well as main peak is passing and the system suitability parameters are well within the acceptance criteria. The method is linear in the range of 0.50 mcg to 3.0 mcg. The RRT and RRF has determined for this impurity separation to eliminate the use of impurity standard for each analysis by applying the factors in calculation part. The developed method is specific, linear, accurate and robust over a set of validation parameters as per ICH guidelines. The method is suitable for the determination of related substances estimation of Hydroxy chloroquine sulphate tablets. ACKNOWLEDGEMENTS Dr.K.Anver Basha, research guide, Sathyabama university for his expert guidance and assistance. Department of chemistry, Sathyabama University, Chennai ,Tamilnadu,India,for providing all the facilitation to complete this project. IPCA Laboratories LTD., Chemical Research & Development Centre,142 AB, Kandivli Industrial Estate, Kandivli (West),Mumbai , Maharashtra, for providing Active pharmaceutical ingredients used for this development. REFERENCES 1. Snyder. L.R, Kirkland. J.J, Glajch. J.L, Practical HPLC method development, Wiley inter science publication, New York, Edition 2,1997:Vol.1-9, The United states Pharmacopoeia, Unites States Pharmacopoeia Convention, Inc, Rockville, thirty-six Revision and national formulary thirty-first edition, Dr.chowdry KPR, Devalo Rao G, Himabindu G, Validation of analytical methods, The Eastern Pharmacist,1999:39-41: Sethi, Quantitative Analysis of drugs in Pharmaceutical formulations, CBS publishers and distributors, NewDelhi, Edition 3,1986: : CRC handbook of chemistry and Physics, Edition 88, Clarke s isolation and identification of drugs, Pharmaceutical press, 2nd Edition, 1986: International journal of pharmaceutics,volume 43,Issue3,May1988,Pages Journal of Chromatography B:Biomedical science and applications, Vol581, Issue1, 2, October 1998,Pages Vol 3, Issue 7,

12 9. Analytical and semi-preparative High performance liquid chromatographic separation and assay of Hydroxy chloroquine enantiomers, Dion R.Brocks, Franco M.Pasutto, Fakhreddin Jamali. 10. Journal of analytical toxicology,volume 18,Issue5,Pages Journal of Chromatography A, Vol.391,1987,Pages Vol 3, Issue 7,