RESEARCH ARTICLE Spectrofluorometric determination of clonazepam in dosage forms: Application to content uniformity testing and human plasma Fathalla Belal | Fawzia Ibrahim | Zainab Sheribah | Heba Alaa Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura, Egypt Correspondence Heba Alaa, Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, 35516, Mansoura, Egypt. Email: heba.alaa2@yahoo.com Abstract The present paper describes a developed and validated simple, highly sensitive and cost‐effective spectrofluorometric method for determination of clonazepam (CNP). The proposed method depends on forming a highly fluorescent product through the reduction of CNP with Zn/HCl. The produced fluorophore exhibits a strong fluorescence at λ em 350 nm after excitation at λ ex 250 nm. The use of carboxymethylcellulose (CMC) greatly enhanced the fluorescence intensity of the produced fluorophore to the extent of about 100%. Calibration curve showed good linear regression (r 2 > 0.9998) within test ranges of 20–400 ng ml -1 with a lower detection limit of 0.67 ng ml -1 and lower quantification limit of 2.22 ng ml -1 upon using CMC. The method was successfully applied to the analysis of CNP in its pharmaceutical formulations and the results were in agreement with those obtained using a reference method. Furthermore, the content uni- formity testing of the tablets was also performed. The application of the proposed method was extended to determine CNP in spiked human plasma sample as a preliminary investigation and the results were satisfactory. KEYWORDS carboxy methyl cellulose, clonazepam, pharmaceutical formulations and content uniformity testing, spectrofluorimetry 1 | INTRODUCTION Clonazepam (CNP), 5‐(2‐chlorophenyl)‐1,3‐dihydro‐7‐nitro‐1, 4‐ benzodiazepin‐2‐one (Figure 1). is a benzodiazepine derivative similar to diazepam, with marked antiepileptic properties. It is used in the treatment of all types of epilepsy and seizures, including status epilep- ticus, but its usefulness in chronic treatment is sometimes limited by the development of tolerance and sedation. [1] CNP is official in both the British Pharmacopoeia (BP) [2] and the United States Pharmaco- poeia (USP). [3] Various methods were reported for estimation of CNP; including spectrophotometric methods, [4,5] spectrofluorometric method based on reduction of nitro group to the corresponding amino group and then reacting the reduced form with 2‐cyanoacetamide in the pres- ence of ammonia (25%), [6] HPTLC, [7] HPLC, [8,9] LC/MS [10] and GC. [11] Most of these analytical methods are tedious and time consuming. [6] Others require expensive and complex equipment, [8,9] while others need labour‐intensive sample preparation. [10,11] The proposed method depends on the fact that CNP has no native fluorescence because of the powerful quenching effect of its nitro group. [12] However, when the nitro group was reduced to the amino group, this allowed CNP to retain its fluorescence. [13,14] Using fluorescence enhancers, such as CMC the fluorescence intensity of RCNP product increased by a factor of about 100%. [15] This encouraged us to develop a simple, sensitive and selective spec- trofluorometric method for the determination of CNP commercial tab- lets and drops. The proposed procedure was validated according to the ICH guidelines. [16] Novelty statement: The present work is the first report on the spectrofluoro- metric determination of clonazepam based on its reduction to amino derivative Abbreviations: CTAB, Cetyl trimethyl ammonium bromide; LOD, Limit of detection; LOQ, Limit of quantification; RFI, Relative fluorescence intensities; RSD, Relative standard deviations; SDS, Sodium dodecyl sulphate; USP, United States Pharmacopoeia. Received: 13 November 2016 Revised: 14 January 2017 Accepted: 1 March 2017 DOI: 10.1002/bio.3325 Luminescence. 2017;1–8. Copyright © 2017 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bio 1