New protamine quantification method in microtiter plates using o-phthaldialdehyde/N-acetyl-L-cysteine reagent.
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 (lambdamax 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 lambdaex 345 nm) and emission (maximum lambdaem 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 microg/ml.
Lochmann
Dirk
D
Institute for Pharmaceutical Technology, Johann Wolfgang Goethe University, Marie Curie Strasse 9, D-60439 Frankfurt am Main, Germany.
Stadlhofer
Sylvia
S
Weyermann
Jörg
J
Zimmer
Andreas
A
D000111:Acetylcysteine; D006494:Heparin Antagonists / Q000737:chemistry*; D007202:Indicators and Reagents; D011479:Protamines / Q000737:chemistry*; D013678:Technology, Pharmaceutical; D009764:o-Phthalaldehyde
D006494:Heparin Antagonists; D007202:Indicators and Reagents; D011479:Protamines; D009764:o-Phthalaldehyde; D000111:Acetylcysteine