Highly sensitive HPLC method for the determination of galantamine in human plasma and urine through derivatization with dansyl chloride using fluorescence detector

Abstract

A new method based on fluorescence derivatization with 5-(dimethylamino) naphthalene-1-sulfonyl chloride (dansyl chloride) was developed for the quantitative determination of galantamine in human plasma and urine using high-performance liquid chromatography. The reaction between galantamine and dansyl chloride was optimally realized in 30min at room temperature and pH10.5, 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% 10mM o-phosphoric acid, 1.2ml/min. The injection volume was 20l. The derivatives were detected with a fluorescence detector (excitation 375nm/emission 537nm). The retention time for the dansyl derivative of galantamine was 16.8min. Linearity was observed between 125 and 2000ng/ml in water, urine and plasma. The limit of detection and limit of quantification for the developed method were 6.27-70.99 and 18.81-212.97ng/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.