HPLC of (±)-Fenfluramine and Phentermine in Plasma After Derivatization With Dansyl Chloride

Abstract

A high-performance liquid chromatographic method is described for the simultaneous determination of the dansyl derivatives of (±)-fenfluramine (Fen) and phentermine (Phen) in addition to four other sympathomimetic amines, namely, norephedrine (NE), ephedrine (E), 2-phenylethylamine (2-PEA), and 4-bromo-2,5-dimethoxyphenethylamine (2-CB), using fluoxetine (FLX) as an internal standard. The separation was performed on a reversedphase C 18 column, using a gradient system and fluorescence detection. The dansylation conditions were examined and the optimum ORDER REPRINTS 58KADDOUMI ET AL.derivatization was obtained at pH 9.0 with 4.7 mM dansyl chloride concentration. A preliminary study for the simultaneous determination of the dansyl derivatives in spiked human plasma was performed. The derivatives were well separated within 45 min and showed good linearities from 0.005 to 2µM for E, Phen, and Fen, from 0.002 to 0.8 µM for 2-CB, and from 0.025 to 2 µM for NE and 2-PEA with detection limits ranging from 16 to 255 fmol on the column. Extraction recovery of the compounds was greater than 90%. The applicability of the method was studied in rat plasma following an intraperitoneal administration of Fen and Phen.

Differential 12C-/13C-Isotope Dansylation Labeling and Fast Liquid Chromatography/Mass Spectrometry for Absolute and Relative Quantification of the Metabolome

“…In this work, we report a chemical derivatization strategy based on dansylation reaction for absolute and relative quantification of amine- and phenol-containing metabolites in a complex sample. Dansylation is simple, robust, and routinely performed for many years as precolumn derivatization for quantification of amino acids, biogenic amines and phenolic hydroxyls by thin layer chromatography (TLC) and HPLC separation followed by fluorescence or UV detection. − Dansylation has also been used to form derivatives of targeted analytes, followed by LC/MS analysis, for the detection of p -chlorophenol and amines, four phenol-containing metabolites of a drug, fenfluramine and phentermine, and β-estradiol and estrone . In a recent conference report, we illustrated that endogenous human metabolites in urine could be sensitively detected by dansylation derivatization and LC/MS .…”

“…Dansylation is simple, robust, and routinely performed for many years as precolumn derivatization for quantification of amino acids, biogenic amines and phenolic hydroxyls by thin layer chromatography (TLC) and HPLC separation followed by fluorescence or UV detection. [47][48][49][50][51][52] Dansylation has also been used to form derivatives of targeted analytes, followed by LC/MS analysis, for the detection of p-chlorophenol and amines, 53 four phenolcontaining metabolites of a drug, 54 fenfluramine and phentermine, 55 and β-estradiol and estrone. 56 In a recent conference report, we illustrated that endogenous human metabolites in urine could be sensitively detected by dansylation derivatization and LC/ MS. 57 Herein we report the synthesis of an isotope coded reagent for dansylation of amines and phenols for quantitative metabolome profiling and demonstrate that differential isotope labeling via dansylation, combined with fast LC separation of labeled metabolites and Fourier-transform (FT) ion cyclotron resonance (ICR) MS, can substantially enhance the ESI sensitivity, improve chromatographic retention, and facilitate MS-based quantification and identification of potentially hundreds of amine-and phenol-containing metabolites in a complex biological sample.…”

Analytical Studies on the Development of High-Performance Liquid Chromatographic Methods with Fluorescence or Chemiluminescence Detections and Their Practical Applications

“…At first, simultaneous determination of dansylated Fen and Phen, in addition to sympathomimetic amines including norephedrine, ephedrine, 2-phenylethylamine and 4-bromo-2,5-dimethoxyphenethylamine was performed by an HPLC-FL method. 60 The labels were well separated within 45 min with LOD ranging from 16 to 255 fmol (S/N = 3).…”

Analytical Techniques and Quality Control of Pharmaceuticals

“…HPLC-FL and HPLC-CL due to their excellent detection power and reactivity are very useful methods in forensic sciences to determine medicaments such as illicit drugs, for example, methamphetamine (MP) and amphetamine (AP) [20]. An example would suffice: obesity drugs like fenfluramine (Fen), phentermine (Phen), N-nitrosofenfluramine (N-Fen), mazindol (Maz), and phenylpropanolamine (PPA) which can be contained in diet products and can cause serious adverse effects, have amino groups in their molecules which can react with labeling reagents [21,22].…”