Development and Validation of Stability Indicating UPLC–PDA/MS for the Determination of Imiquimod and its Eight Related Substances: Application to Topical Cream

Journal of Chromatographic Science, 2019, Vol. 57, No. 3, 249–257 doi: 10.1093/chromsci/bmy108 Advance Access Publication Date: 19 December 2018 Article Article Venu Balireddi1,2,*, Siva Rao Tirukkovalluri2, Krishna Murthy Tatikonda1, Bhanu Teja Surikutchi1, and Parmita Mitra1 1 Dr. Reddy’s Laboratories Limited, Bachupally, Survey No. 42, 45 & 46, Qutubullapur Mandal Hyderabad 500072, India and 2Department of Inorganic & Analytical Chemistry, Chinna Waltair Main Road, South Campus, Andhra University, Vishakapatnam 530003, India * Author to whom correspondence should be addressed. Dr. Reddy’s Laboratories Limited, IPDO, Bachupally, Hyderabad 500072, India. Email: Received 9 August 2017; Revised 17 October 2018; Editorial Decision 19 November 2018 Abstract A stability indicating analytical method for imiquimod and its related impurities was developed by ultra-pressure liquid chromatography (UPLC) using design of experiments. This method could quantify imiquimod and all its eight known related impurities in a single run. The optimum separation was achieved on reverse phase Acquity UPLC column (2.1 mm × 100 mm, 1.7 μm) using 0.1% trifluoroacetic acid and acetonitrile as mobile phase. Preventing the use of ion pair reagents assured the compatibility of this method to liquid chromatography in tandem with mass spectrometry. All components were separated within 9 minutes, maintaining good resolution. The stability indicating nature of the developed method was assessed by analyzing the samples of imiquimod which were exposed to various environments such as acid, alkali, peroxide, light and heat. This method was found to be sensitive, precise and accurate. The method achieved the lower detection limit of 0.04 μg/mL and the quantification limit of 0.08 μg/mL for all analytes. Introduction Imiquimod is an imidazoquinolone derivative, which resembles a nucleoside. It shows potent induction of cell mediated immune response through endogenous antiviral pro-inflammatory mediators. It is a topically administered immune response modifier used for the treatment of external genital and perianal warts caused by human papillomavirus (HPV) (1, 2). It is available in the US market as Aldara® cream (5%, w/w) and Zyclara® cream (3.75 and 2.5%, w/ w). Zyclara® and Aldara® are approved for external genital warts (3, 4) and actinic keratosis (Aldara cream 5% 1997; Zyclara cream 1997). Aldara® is also approved for superficial basal cell carcinoma. Being a successful drug candidate, imiquimod receives prominence in terms of clinical usage as well as research interests pertaining to analytical determinations. USP enlists imiquimod and its five impurities (Imiquimod USP, Imiquimod cream USP) (5, 6). Apart from these five impurities, various suppliers of imiquimod enlist other impurities of imiquimod, as process impurities of their synthesis routes. Taro Pharmaceutical Industries Ltd. supplies imiquimod with two process impurities, impurity C and a benzyl impurity, which were not listed in the USP monograph. Sequent Pharmaceuticals Ltd. also listed imiquimod with three process impurities, impurity C, methoxy impurity and a hydroxy impurity, where methoxy and hydroxy impurities are not listed in the USP. The chemical structures of all these impurities are depicted in Figure 1. USP provides three analytical methods for determination of Imiquimod and its five impurities. The methods suggested by USP can only determine five of the eight known impurities. Donnelly et al. have developed an assay method for quantitative © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: 249 Development and Validation of Stability Indicating UPLC–PDA/MS for the Determination of Imiquimod and its Eight Related Substances: Application to Topical Cream 250 Balireddi et al. determination of imiquimod (7). Similarly, Daniel et al. have also developed an assay method for estimation of imiquimod in skin samples (8). These known analytical methods do not determine impurities of imiquimod. Developing a single analytical method instead of multiple methods for the determination of all compounds would find its use in a simplifying the process of routine analytical determinations in the product development. It would be compelling to adopt a single method, which can determine imiquimod and all its impurities at once. The method in USP uses long chain ion pair reagents which are not compatible to liquid chromatography in tandem with mass spectrometry due to its non-volatile nature and column saturation effects during analysis. Developing a mass compatible method could be useful to trace any unknown impurities which may raise during shelf life or clinical studies in the process of the product development (9–11). Herein, we developed a unique, stability indicating, mass compatible analytical method for determination of imiquimod and all its impurities. The newly developed method portrays its superiority over existing methods by its ability to determine the unlisted known impurities along with five impurities, and by proving its mass compatibility. Experimental Regents used Trifluoroacetic acid (TFA) extra pure was purchased from Acros organics (New Jersey, USA). Acetonitrile HPLC grade was purchased from Merck Specialties Pvt. Limited (Mumbai, India). Approximately 35% of hydrochloric acid (HCl), Emplura, was purchased from Finar Chemicals Limited (Ahmedabad, India). HPLC grade water was used from in-house water purification systems (Thermo Scientific TKA, Germany, and Mill-Q®; Millipore Private Limited, Bangalore, India). Other standards, excipients and samples were provided by Dr. Reddy’s Laboratories Ltd. (Hyderabad, India). Methods Instrumentation and chromatography conditions Acquity ultra-pressure liquid chromatography (UPLC) system (Waters Corporation, USA) equipped with a model ACQ-BSM (S.N: K10UPB207A) LC pump, an online degasser, model ACQ-SM (S.N: K10UPA060M) auto-sampler, and model ACQ-photo diode-array (PDA) (S.N:J10UPD130A) detector was used. The data was acquired via Empower 2 software Built 2154 from Waters Corporation. The 0.1% (v/v) TFA in water was used as mobile phase A, and 0.1% TFA in acetonitrile was used as mobile phase B. Over a 12-minute run time gradient elution, the ratio of time (minutes)/mobile phase B (% v/v) changes as 0/10, 0.5/10, 5.0/25, 8.0/ 55, 10.0/80, 10.5/10, and 12.0/10 with the constant flow rate of 0.4 mL/min. The injection volume was 3 μL, and the detection wavelength was set at 240 nm for imiquimod, related compound A, related compound B, related compound C, related compound D, benzyl impurity and methoxy impurity. Related compound E was detected at 253 nm. Samples were maintained at 22°C within the system and analysis was performed at 35°C using Acquity UPLC® CSH TM fluoro-phenyl column, 100 mm length, 2.1 mm inner diameter and with 1.7 μm partial size (part no:186005352) (Waters Corporation, USA). Mass spectrometry Analysis was c (...truncated)