RESEARCH ARTICLE Highly sensitive HPLC method for the determination of galantamine in human plasma and urine through derivatization with dansyl chloride using fluorescence detector Elif Özdemir 1 | Sevgi Tatar Ulu 2 1 Department of Analytical Chemistry, Istanbul Yeni Yuzyil University, Istanbul, Turkey 2 Department of Analytical Chemistry, Istanbul University, Istanbul, Turkey Correspondence Elif Özdemir, Department of Analytical Chemistry, Faculty of Pharmacy, Istanbul Yeni Yuzyil University, 34010, Istanbul, Turkey. Email: elifkel@live.com; elif.ozdemir@yeniyuzyil.edu.tr Funding Information Research Fund of Istanbul University, Grant/ Award Number: 55961 Abstract A new method based on fluorescence derivatization with 5‐(dimethylamino) naphthalene‐1‐ sulfonyl chloride (dansyl chloride) was developed for the quantitative determination of galanta- mine in human plasma and urine using high‐performance liquid chromatography. The reaction between galantamine and dansyl chloride was optimally realized in 30 min at room temperature and pH 10.5, with a reagent to galantamine molar ratio of 2.13. The derivative was extracted with dichloromethane, and the extract was dried under a nitrogen stream and dissolved in the mobile phase. Chromatographic analysis was performed with an Inertsil C 18 column and a mobile phase comprising 40% acetonitrile and 60% 10 mM o‐phosphoric acid, 1.2 ml/min. The injection volume was 20 μl. The derivatives were detected with a fluorescence detector (excitation 375 nm/emis- sion 537 nm). The retention time for the dansyl derivative of galantamine was 16.8 min. Linearity was observed between 125 and 2000 ng/ml in water, urine and plasma. The limit of detection and limit of quantification for the developed method were 6.27–70.99 and 18.81–212.97 ng/ml, respectively. Per cent recovery was calculated as 95.15 for urine and 95.78 for plasma. Interday repeatability values for urine and plasma samples (n = 6) at three different concentrations were calculated as a per cent relative standard deviation of 0.24–0.59 and 0.35–0.56. The corresponding per cent relative standard deviation values for intraday repeatability were 0.13–0.51 and 0.04–0.15, respectively. KEYWORDS dansyl chloride, derivatization, HPLC, plasma, urine, validation 1 | INTRODUCTION Galantamine hydrobromide is a reversible and competitive acetylcho- linesterase inhibitor. Its chemical expression is (4aS,6R,8aS)‐ 4a,5,9,10,11,12‐hexahydro‐3‐methoxy‐11‐methyl‐6H‐benzofuro[3a,3, 2‐ef][2]benzazepin‐6‐ol hydrobromide and its empirical formula is C 17 H 21 NO 3 .HBr. The molecular weight of galantamine hydrobromide is 368.27. Galantamine hydrobromide is used for the treatment of mild to moderate dementia of the Alzheimer’s type. Galantamine hydrobromide is a white to almost white powder and is faintly water soluble. [1] The mean maximum plasma drug concentration (C max ) and time to reach C max (t max ) after a single oral (tablet) 10 mg dose of galan- tamine are 49.2 μg/L and 0.88 h. [2] There are many methods for the determination of galantamine, including high‐performance liquid chromatography (HPLC). [3–15] The determination of galantamine in dosage form using the UV–photometric method has also been described. [7] This study examined a new spectrofluorimetric method for the determination of galantamine in plasma and urine. The derivatization of galantamine using dansyl chloride is the basis of this new method. Because highly fluorescent derivatives with primary and secondary amines, imidazoles and phenols are formed by dansyl chloride under the used reaction conditions, which are comparatively moderate, dansyl chloride was selected as a reagent used in the derivatiza- tion. [15–19] To the best of our knowledge, this is the first time that gal- antamine has been derivatized by a dansyl chloride and determined using HPLC with a fluorescence detector. In this study, a sensitive, accurate and precise HPLC method was developed and validated to identify galantamine. The stability of the Abbreviations used: HPLC, High‐performance liquid chromatography; LOD, Limit of detection; LOQ, Limit of quantification; RSD, Relative standard deviation; UV, Ultraviolet. Received: 30 November 2016 Revised: 23 January 2017 Accepted: 26 January 2017 DOI 10.1002/bio.3301 Luminescence. 2017;1–5. Copyright © 2017 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bio 1