An eco-friendly direct spectrofluorimetric method for the determination of irreversible tyrosine kinase inhibitors, neratinib and pelitinib: application to stability studies H.M. Maher, a,b * N.Z. Alzoman a and S.M. Shehata a ABSTRACT: A new rapid and simple stability-indicating spectrofluorimetric method has been developed for the determination of two irreversible tyrosine kinase inhibitors (TKIs), neratinib (NER) and pelitinib (PEL). The method is based upon measurement of the native fluorescence intensity of both drugs at λ ex 270 nm in aqueous borate buffer solutions (pH 10.5). The fluorescence in- tensity recorded at 545 nm (NER) and 465 nm (PEL) were rectilinear over the concentration range of 0.1–10 μg/mL for both drugs with a high correlation coefficient (r > 0.999). The proposed method provided low limits of detection and of quantitation of 0.07, 0.11 μg/mL (NER) and 0.02, 0.05 μg/mL (PEL), respectively. The method was successfully applied for the determination of NER and PEL in bulk powder. The proposed methods were fully validated as per the International Conference on Harmonisation (ICH) guidelines. The application of the method was extended to stability studies of both NER and PEL under different forced- degradation conditions (acidic-induced, base-induced, oxidative, wet heat, and photolytic degradation). Moreover, the kinetics of the base-induced and oxidative degradation of both drugs was investigated and the pseudo-first-order rate constants and half-lives were estimated at different temperatures. Also, an Arrhenius plot was applied to predict the stability behaviour of the two drugs at room temperature. Copyright © 2016 John Wiley & Sons, Ltd. Keywords: spectrofluorimetry; neratinib; pelitinib; stability studies; degradation kinetics Introduction Lung cancer is the main cause of cancer mortality worldwide, of which non-small cell lung cancer (NSCLC) is the most predominant of all the lung cancer cases. Generally, lung cancer is associated with poor prognosis (1,2). The human genome encodes for hun- dreds of tyrosine kinases that are responsible for the regulation of different physiological mechanisms, including cell proliferation, differentiation, migration, and metabolism. Dysfunction in kinase activity results in a disruption of the normal control of cellular phosphorylation signalling pathways with numerous abnormali- ties leading to cancer (3,4). This situation has encouraged the de- velopment of small molecule tyrosine kinase inhibitors (TKIs) as targeted therapy against tyrosine kinases, particularly epidermal growth factor receptors (EGFR). Most of these compounds target the ATP binding site across the human protein kinases. The first emerged class of TKIs (e.g. gefitinib, erlotinib) was designed to in- hibit protein catalytic activity in a reversible manner through bind- ing to the kinase domain of the target by weak interactions (hydrogen bonds, van der Waals forces and hydrophobic interac- tions). In the past decade, much progress has been made in the de- velopment of a new class of potent and selective TKIs that irreversibly inhibit their target protein by the formation of covalent bonds (1). These covalent irreversible TKIs constitute the second- generation TKIs. Several theoretical advantages of the second- generation irreversible EGFR TKIs over the first-generation revers- ible EGFR TKIs are that some of these have prolonged pharmaco- logical effect, long duration of action, and a higher affinity and selectivity for the EGFR kinase domain, and may allow a more com- plete blockade of the EGFR signalling pathway (1,5). Furthermore, covalent bond formation gives irreversible inhibitors the potential to overcome resistance, compared with the reversible inhibitors. The number of irreversible TKIs entering clinical trials studies is steadily increasing. Examples of these are neratinib (NER) and pelitinib (PEL) (Fig. 1) (1). NER has demonstrated antitumor activity and an acceptable safety profile in patients with breast cancer and other solid tumours (6–14), uterine serous carcinoma, and NSCLC (13). PEL is currently being tested in phase II clinical trials for its efficacy on NSCLC (13,15) and metastatic colorectal cancer (16). A review of the literature revealed the absence of reports deal- ing with the analysis of NER/PEL in pharmaceutical matrices and that only few liquid chromatography tandem mass spectrometry (LC–MS/MS) methods have been reported for the analysis of NER (10–12,14) or PEL (15,16) in biological samples. As the effective * Correspondence to: H. M. Maher, College of Pharmacy, Department of Phar- maceutical Chemistry, King Saud University, Riyadh 11495, P.O. Box 22452, Saudi Arabia. Tel: +966–118052929; Fax: +966–118054589; E-mail: hadirrona@yahoo.com a College of Pharmacy, Department of Pharmaceutical Chemistry, King Saud University, Riyadh 11495, Saudi Arabia b Faculty of Pharmacy, Department of Pharmaceutical Analytical Chemistry, University of Alexandria, El-Messalah, Alexandria 21521, Egypt Abbreviations: EGFR, epidermal growth factor receptor; HPLC, high pressure liquid chromatography; ICH, International Conference on Harmonisation; LOD, limit of detection; LOQ, limit of quantitation; NSCLC, non-small cell lung can- cer; PCT, Patent Cooperation Treaty; RSD, relative standard deviation; TKI, tyro- sine kinase inhibitors; UV, ultraviolet. Luminescence 2017; 32: 149–158 Copyright © 2016 John Wiley & Sons, Ltd. Research article Received: 12 March 2016, Revised: 10 April 2016, Accepted: 20 April 2016 Published online in Wiley Online Library: 1 June 2016 (wileyonlinelibrary.com) DOI 10.1002/bio.3160 149