New protamine quantification method in microtiter plates using o-phthaldialdehyde/ N-acetyl- l-cysteine reagent

Abstract

Protamine is a well-known excipient in pharmaceutics. It represents a peptide consisting of exclusive aliphatic amino acids, hence it cannot be quantified by UV-spectroscopy ( λ max 280 nm). A new and sensitive quantification method based on the derivatisation of protamine with ortho-phthaldialdehyde (OPA) in the presents of 2-mercaptoethanol (ME) or N-acetyl- l-cysteine (NAC) in basic aqueous solution using 96-well microtiter plates are introduced in this report. The resulting isoindol derivatives reveal a fluorescence excitation (maximum λ ex 345 nm) and emission (maximum λ em 450 nm) spectra. Derivatives of OPA/NAC reagent were found to be useful for protamine quantification in pharmaceutical nanoparticle preparation containing DNA. A sufficient stability of the isoindol derivatives was shown. It was possible to determine protamine free base, protamine sulphate and protamine chloride with limits of detection less than 1.1 μg/ml.