Ultrasensitive determination of pyrroloquinoline quinone in human plasma by HPLC with chemiluminescence detection using the redox cycle of quinone.

ABSTRACT

A fast, accurate, and ultrasensitive high-performance liquid chromatography method with chemiluminescence detection (HPLC-CL) was optimized and validated for the determination of pyrroloquinoline quinone (PQQ) concentration in human plasma following solid-phase extraction (SPE). This method is based on the redox cycle of the reaction between PQQ and dithiothreitol, which generates reactive oxygen species that can be detected using luminol as a CL probe. The isocratic HPLC system comprised an ODS column and 4.0mM tetra-n-butylammonium bromide in Tris-HNO3 buffer (pH 8.8; 50mM)-acetonitrile (73, v/v) as mobile phase. A novel, rapid, and simple SPE method was also developed providing excellent %recovery (≥95.2%) for PQQ from human plasma samples. The proposed method was linear over the range of 4.0-400nmol/L plasma of PQQ with a lower detection limit (S/N=3) of 1.08 nmol/L plasma (0.27nM). The method was successfully implemented to determine PQQ concentration in the plasma of healthy individuals after administration of PQQ supplements.