Retention behaviour and fluorimetric detection of procaine hydrochloride using carboxymethyl-β-cyclodextrin as an additive in reversed-phase liquid chromatography

The retention behaviour of procaine hydrochloride on an Alltima octadecyl silica (C₁₈) column, with a mobile phase containing negatively charged carboxymethyl-β-cyclodextrin (CM-β-CD), influenced by a combination of hydrophobic and electrostatic interactions was systematically investigated. Various factors affecting the retention of procaine on the C₁₈ column such as the concentration of CM-β-CD, pH and the methanol percentage in the mobile phase, were studied. An equation was applied to estimate the apparent binding constant of the CM-β-CD-procaine inclusion complex as an aid for understanding the retention mechanism. The first analytical application of CM-β-CD as a mobile phase additive for the determination of procaine was developed. The calibration curve was linear in the range 22-1360 ng ml^-1 with an RSD of 2.1%. The detection limit based on 3σ was 1 ng ml^-1 with fluorimetric detection at the excitation and emission wavelengths of 305 nm and 350 nm, respectively. The limit of quantitation based on 10σ was 22 ng ml^-1. The proposed method has been successfully applied to real sample analysis.