Determination of Pyrroloquinoline Quinone in DNA Binding by LC

Abstract

A simple and repeatable liquid chromatography method with UV detection is described for the determination of pyrroloquinoline quinone in DNA binding studies first. Binding of pyrroloquinoline quinone to DNA was measured indirectly by determining unbound pyrroloquinoline quinone. Pyrroloquinoline quinone was chromatographed on a reversed-phase column using gradient elution with mobile phases of 0.1% trifluoroacetic acid (TFA) in deionized water and 0.1% TFA in acetonitrile at a flow-rate of 0.8 mL min⁻¹ and detected at 249 nm. The mean content of unbound pyrroloquinoline quinone to three DNA in (hydroxymethyl) aminoethane(Tris–HCl) media were dropped from 99.3 to 80.3% in 250 min, and than to 64.8% at 1200 min, but the binding remained constant in deionized water. The method precision were validated with RSD 1.05 and 0.69% for peak areas and retention times. Validation of the method showed that the assay was linear from 25 to 2,000 μM and 5 to 2,000 μM pyrroloquinoline quinone in Tris–HCl and deionized water, respectively. The accuracy for three concentrations of pyrroloquinoline quinone with different types DNA in two media were within the given range of 80.9–113.6%.