A Validated Stability-Indicating UPLC Method for the Determination of Impurities in Maraviroc

Journal of Chromatographic Science 2014;52:609– 616 doi:10.1093/chromsci/bmt085 Advance Access publication July 3, 2013 Article A Validated Stability-Indicating UPLC Method for the Determination of Impurities in Maraviroc Mohanareddy Chilukuri1,2*, Katreddi Hussainreddy2, Papadasu Narayanareddy1 and Madireddi Venkataramana1 1 Hetero Labs, Ltd., Hetero House, Sanathnagar, Hyderabad-500078, AP, India, and 2Department of Chemistry, Sri Krishna Devaraya University, Anantapur-515003, India *Author to whom correspondence should be addressed. Email: Received 16 October 2012; revised 8 April 2013 Maraviroc is an antiretroviral drug in the CCR5 receptor antagonist class, which is used in the treatment of HIV. Maraviroc has six impurities. A novel, stability-indicating reversed-phase ultraperformance liquid chromatography (RP-UPLC) method has been developed for the quantitative determination of maraviroc in active pharmaceutical ingredients, along with its six impurities. The method is applicable to the quantification of related compounds and the assay of maraviroc. Efficient chromatographic separation was achieved on a BEH Shield RP-18 column, 100 3 2.1 mm, 1.7 mm, in isocratic elution within 12 min. The mobile phase was 0.01 M ammonium acetate in water and acetonitrile in the ratio of 63:37 (v/v). The flow rate was 0.4 mL/min, column oven temperature was maintained at 4088 C and detection was conducted at 210 nm. Stress degradation conditions were established for maraviroc by subjecting it to acid, base, oxidation, water, humidity, thermal and photolysis stress. The stress samples were assayed against a qualified reference standard and the mass balance was close to 98.0%. The developed UPLC method was validated according to the current International Conference on Harmonization guidelines for specificity, detection limit, quantitation limit, linearity, accuracy, precision, intermediate precision and robustness. The resolution between maraviroc and its six impurities was greater than 3.0. A regression analysis showed that the correlation coefficient value was greater than 0.999 for maraviroc and its six impurities. Introduction During the past decade, HIV infection, although an incurable disease, has become largely manageable. This is attributable to the advent of highly active antiretroviral therapy (HAART), in which patients are treated with a cocktail of drugs designed to reduce their viral loads to extremely low levels (1). Maraviroc, 4,4-difluoro-N-[(1S)-3-[(3-exo)-3-[3-methyl-5-(1-methylethyl)-4H-1,2,4-triazol-4-yl]-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl] -cylclohexane carboxamide, is an antiretroviral drug in the CCR5 receptor antagonist class, which is used in the treatment of HIV infection (2, 3). A new category of separation technique, ultra-performance liquid chromatography (UPLC), is one of the most promising developments in the area of fast chromatographic separations, with its unique characteristics of satisfactory chromatographic resolution (Table I), high speed and sensitive analysis (4 –7). This study identified six process-related impurities: the current (1S)-3-[(3-exo)-3-[3-methyl-5-(1-methylethyl)-4H-1,2, 4-triazol-4-yl]-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropanamine, hydrochloride (Imp-1), tert-butyl(1S)-3-[3-(3-isopropyl-5-methyl -4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropylcarbamate (Imp-2), 4,4-difluoro-N-[(1S)-3-[(3-endo)-3[3-methyl-5-(1-methylethyl)-4H-1,2,4-triazol-4-yl]-8-azabicyclo [3.2.1] oct-8-yl]-1-phenylpropyl]-cylclohexanecarboxamide (Imp-3), 1-((R)-3-(3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-8-azabicyclo [3.2.1]octan-8-yl)-1-phenylpropyl)-3-((1S)-3-(3-(3-Isop ropyl-5methyl-4H-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-1phenylpropyl) urea (Imp-4), 1-chloro-4,4-difluoro-N-[(1S)-3[(3-exo)-3-[3-methyl-5-(1-methylethyl)-4H-1,2,4-triazol-4-yl]-8azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl]-cyclohexane carboxamide (Imp-5) and N-((S)-3-((1R,3R,5S)-3-(3,5-diisopropyl-4H1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl)-1-phenylpropyl)4,4-difluorocyclohexane carboxamide (Imp-6). The chemical structures of maraviroc and its impurities are shown in Figure 1. A liquid chromatographic method has been described in the literature for the determination of maraviroc by reversed-phase high-performance liquid chromatography (RP-HPLC) (8). However, there are no methods in the literature for the quantification of maraviroc and its related compounds. Further, no official or draft monograph on maraviroc has been published in any of the pharmacopoeia for compendial applications. The dearth of pharmacopoeial methods and established stress stability conditions necessitated the development of a stabilityindicating UPLC method to separate all six impurities and degradants from maraviroc. The objective of the work was to develop a cheap, efficient RP-UPLC method to and demonstrate its stability-indicating capabilities by forced degradation. Forced degradation of maraviroc was conducted under acid hydrolysis, base hydrolysis, and oxidative, thermal, humid and photolytic stress conditions, because this is a part of developmental strategy under the recommendations of the International Conference on Harmonization (ICH). These studies provide valuable information on the stability, shelf life and storage of the drug and its degradation pathways. This paper also deals with the validation of the developed method, as per ICH guidelines. Experimental Materials and reagents Samples of maraviroc, maraviroc reference standard and its six impurities were received from Hetero Research Foundation (Hyderabad, India), along with their purity contents. All impurities and maraviroc reference standard were more than 95% pure; individual purity levels were as follows: maraviroc (99.7%), Imp-1 (95.8%), Imp-2 (99.3%), Imp-3 (97.4%), Imp-4 (96.2%), # The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: Compound Resolution Tailing factor RT RRT* Imp-1 Imp-4 Maraviroc Imp-2 Imp-6 Imp-3 Imp-5 — 5.758 9.256 10.052 3.878 8.637 14.378 1.103 1.117 1.388 1.007 1.001 1.013 1.011 0.887 1.637 3.194 4.640 5.191 6.416 8.912 0.28 0.51 1.00 1.45 1.63 2.01 2.80 Equipment A UPLC (Waters Aquity H Class, Milford, MA) equipped with a photodiode array detector (PDA) with an autosampler was used. The output signal was monitored and processed by using Empower software on a Pentium computer (Digital Equipment Co.). Photostability studies were conducted in a photostability chamber (Atlas Suntest CPS þ , Altenhasslau, Germany). Thermal stability studies were conducted in a dry hot air oven (Cintex precision hot air oven, Hyderabad, India). Imp-5 (95.6%) and Imp-6 (95.4%). In addition, HPLC grade acetonitrile and acetic acid were purchased from Merck (Darmstadt, Germany). Ammonium acetate was purchased from Rankem (Mumbai, India). Highly pure water was prepared with a Millipore Milli-Q Plus water purification (...truncated)