A high performance liquid chromatographic post-column fluorescent ion pair extraction system: application to physostigmine and its metabolite eseroline in human serum.
A high performance liquid chromatographic post-column fluorescent ion pair extraction system was developed for the analysis of quaternary ammonium and amine drugs in serum. A new fluorescent ion pair reagent, sodium alpha-(3,4-dimethoxyphenyl) cinnamonitrile-2'-sulfonate (DPS), was synthesized and characterized. The post-column extraction system consisted of a three-dimensional knitted teflon mixing coil and a membrane phase separator which was modified from an original literature design. Physostigmine and its metabolite eseroline were used as model cations. A solid phase extraction procedure using octadecylsilane columns was developed to extract the compounds and neostigmine bromide (internal standard) from human serum. The compounds were chromatographed on a diol column using a 80:20 aqueous phosphate buffer pH 4 absolute methanol mobile phase at a flow rate of 1 mL/min. Methylene chloride was used as the on-line extraction solvent for the DPS ion pairs formed. Fluorescence of the extracted ion pairs was measured using an excitation of 243 nm and an emission cut-off filter at 418 nm. Linearity was in the 2-100 ng/mL and 5-100 ng/mL ranges for physostigmine and eseroline, respectively. Detection limits based on a signal-to-noise ratio of 2, were 2 and 5 ng/mL, respectively. Precision of the method was found to be in the 1.5-3% range and percentage error in the 1.5-7% range for both compounds.
Quinn
K D
KD
Department of Medicinal Chemistry, College of Pharmacy, University of Georgia, Athens 30602.
Stewart
J T
JT
D001557:Benzenesulfonates; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007211:Indoles / Q000097:blood*; D009570:Nitriles; D010830:Physostigmine / Q000097:blood*
D001557:Benzenesulfonates; D005456:Fluorescent Dyes; D007211:Indoles; D009570:Nitriles; C068907:(3,4-dimethoxyphenyl)cinnamonitrile-2'-sulfonate; D010830:Physostigmine; C022594:eseroline