Determination of ABT-089 in human plasma by high performance liquid chromatography using in situ precolumn derivatization with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole

• Published in: Journal of pharmaceutical and biomedical analysis 1999-06, Vol.20 (1), p.49-63
• Main Authors: Bryan, Peter D., Emry, Michelle L., El-Shourbagy, Tawakol A.
• Format: Article
• Language: English
• Subjects: 4-Chloro-7-nitrobenzofurazan - analogs & derivatives; ABT-089; Analysis; Bioanalysis; Biological and medical sciences; Calibration; Cholinergic Agents - blood; Cholinergic channel modulator; Chromatography, High Pressure Liquid; Fluorescence detection; Fluorescent Dyes; Freezing; General pharmacology; Humans; Indicators and Reagents; Medical sciences; NBD-F; Pharmacology. Drug treatments; Pre-column derivatization; Pyridines - blood; Pyrrolidines - blood; Quality Control; Reference Standards; Reversed-phase HPLC; Spectrometry, Fluorescence; Time Factors
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/S0731-7085(98)00301-X

ABT-089 is a potent, selective neuronal cholinergic channel modulator with cognition enhancing activity in several animal paradigms. A simple and sensitive chromatographic method for the specific determination of ABT-089 in human plasma has been developed and validated. The method utilizes in situ precolumn fluorescence derivatization of the sample with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) prior to liquid-liquid extraction followed by reverse phase HPLC and fluorescence detection (λ ex 495 nm, λ em 533 nm). The described method significantly simplifies sample preparation. The derivatized product was separated from interference using a YMC ODS-AQ, 5 μm, 250×4.6 mm i.d. column using a mobile phase consisting of 30:5:65 (v:v:v) acetonitrile/methanol/aqueous buffer at a flow rate of 0.75 ml min −1. The aqueous buffer consisted of 0.01 M tetramethylammonium perchlorate, 0.1% (v:v) trifluoroacetic acid, pH 3.0. An Alltech Absorbosphere CN, 5 μm, cartridge guard column was also used before the analytical column. Plasma samples were alkalinized with 0.1 M NaHCO 3, 300 μl of a 1 mg ml −1 ethanolic solution of NBD-F was added and the samples were heated in a water bath for 10 min at 50°C. The samples were then extracted with tert-butylmethylether, evaporated to dryness and then reconstituted in mobile phase. For 1 ml of plasma, a limit of quantitation (LOQ) of 1.6 ng ml −1 was obtained. The method was linear from 1.6 to 836 ng ml −1. Inter and intra-day assay RSD ( n=6) were less than 9%. Accuracy determinations showed the quality control samples to range between 88–114% of the theoretical concentration.