Validated sensitive spectrofluorimetric method for determination of antihistaminic drug azelastine HCl in pure form and in pharmaceutical dosage forms: application to stability study Amal A. El-Masry, Mohammed E.A. Hammouda,* Dalia R. El-Wasseef and Saadia M. El-Ashry ABSTRACT: A highly sensitive, simple and rapid spectrofluorimetric method was developed for the determination of azelastine HCl (AZL) in either its pure state or pharmaceutical dosage form. The proposed method was based on measuring the native fluo- rescence of the studied drug in 0.2 M H 2 SO 4 at λ em = 364 nm after excitation at λ ex = 275 nm. Different experimental parameters were studied and optimized carefully to obtain the highest fluorescence intensity. The proposed method showed a linear depen- dence of the fluorescence intensity on drug concentration over a concentration range of 10–250 ng/mL, with a limit of detection of 1.52 ng/mL and limit of quantitation of 4.61 ng/mL. Moreover, the method was successfully applied to pharmaceutical prep- arations, with percent recovery values (± SD) of 99.96 (± 0.4) and 100.1 (± 0.52) for nasal spray and eye drops, respectively. The results were in good agreement with those obtained by the comparison method, as revealed by Student’s t-test and the variance ratio F-test. The method was extended to study the stability of AZL under stress conditions, where the drug was exposed to neutral, acidic, alkaline, oxidative and photolytic degradation according to International Conference on Harmonization (ICH) guidelines. Copyright © 2016 John Wiley & Sons, Ltd. Keywords: azelastine HCl (AZL); eye drops; nasal spray; native fluorescence; spectrofluorimetry; stability study Introduction Azelastine HCl (AZL), chemically (4-(4-chlorobenzyl)-2-[(4RS)-1- methylhexahydro-1H-azepin-4-yl]phthalazin-1(2H)-one hydro- chloride (1); (Fig. 1), is a potent second-generation selective antihistaminic drug. In addition to its H 1 -receptor blocking activity, it has an anti-inflammatory effect in which it appears to inhibit the release of inflammatory mediator cells. It is used topically in allergic conditions including rhinitis and conjuncti- vitis, and is also used in non-allergic rhinitis (2). A review of the literature revealed several analytical methods for the determination of AZL in either its pure or dos- age forms, including spectrophotometry (3–5), electrochemical methods (6,7), thin-layer chromatography (TLC) (8) high- performance liquid chromatography (HPLC) (9–12) and liquid chromatography–tandem mass spectrometry (LC/MS/MS) (13,14). However, all these methods use expensive instruments and tedious procedures. The effective and critical use of antihistaminic drugs all over the world confirms the need to establish a simple, sensitive, rapid and cost-effective method to help in the determination of AZL in both its pure and pharmaceutical dosage forms. To the best of our knowledge, there are no reports on the suc- cessful use of the spectrofluorimetric method for the determina- tion of the native fluorescence of AZL in either its pure or pharmaceutical dosage forms; this is due to the presence of a highly conjugated aromatic system. Spectrofluorimetry is one of the most sensitive, precise and spe- cific analytical techniques compared with other absorption methods, and has important applications in the analysis of food stuffs, pharmaceuticals, clinical samples and natural products. Moreover, our method is one of the most sensitive reported for the determination of AZL, permitting determination of the drug at the ng level and facilitating its quantitation in metered dose na- sal sprays. The proposed method has been found to be ~20–50 times more sensitive than the previously reported method (3–11); in addition, the proposed method does not require compli- cated set-up, unlike another method reported in the literature that needs a sophisticated LC–MS instrument (13,14). Experimental Apparatus Spectrofluorimetric analyses were carried out on a RF-1501 Shimadzu spectrofluorimeter with a xenon arc lamp, and a 1 cm quartz cell. A Consort NV P-901 pH meter (Belgium) was used to * Correspondence to:M. E. A. Hammouda, Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, 35516, Mansoura, Egypt. Email: drmo4u@yahoo.com Department of Medicinal Chemistry, University of Mansoura, Mansoura, Egypt Luminescence 2016 Copyright © 2016 John Wiley & Sons, Ltd. Research article Received: 30 September 2015, Revised: 06 January 2016, Accepted: 26 April 2016 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/bio.3164