Determination of sparteine in human serum by column-switching HPLC with electrogenerated chemiluminescence detection using tris(2,2'-bipyridine) ruthenium (II)

Abstract

A sensitive column-switching high-performance liquid chromatography with a tris(2,2'-bipyridine) ruthenium(II) electrogenerated chemiluminescence detection method for the determination of sparteine (SP) in human control serum is described. The procedure is based on the enrichment of SP on column 1, followed by transfer of SP to column 2. Column 1 was a Shim-pack MAYI-ODS (10×4.6 mm, SHIMADZU, Tokyo, Japan). Column 2 was a CAPCELL PACK C18 (150×4.6 mm, SHISEIDO, Tokyo, Japan). The electrogenerated chemiluminescence detector was an ECR COMET-3000KANAGAWA (Comet, Kawasaki, Japan). The mobile phase of column 1 was composed of a 15 mM KH2PO4 buffer at pH 6.0 containing 10 mM octanesulfonate-CH3CN (98:2, v/v) and at a flow rate of 0.5 ml/min. The mobile phase of column 2 was composed of 150 mM KH2PO4 buffer at pH 4.0 containing 10 mM 1-octanesulfonate-CH3CN (73:27, v/v) and at a flow rate of 1.0 ml/min. The reagent solution was 0.3 mM Ru(bpy)32+ in 10 mM H2SO4 and at a flow rate of 0.45 ml/min. The sample in the control serum was directly injected to the HPLC. The linear dynamic range was 125 fmol-2 nmol/ml. The mean recovery for 1 nmol/ml was 98%. The limit of quantitation was 125 fmol/ml and the intermediate precision (CV) was 1.8% (1 nmol/ml, n=5).