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

Area of Science:

• Pharmaceutical Science
• Analytical Chemistry
• Biochemistry

Background:

• Protamine is a peptide excipient crucial in pharmaceutical formulations.
• Standard UV-spectroscopy at 280 nm is unsuitable for protamine quantification due to its aliphatic amino acid composition.

Purpose of the Study:

• To develop a sensitive and novel method for protamine quantification.
• To enable accurate measurement of protamine in pharmaceutical preparations, particularly nanoparticle formulations.

Main Methods:

• Protamine derivatization using ortho-phthaldialdehyde (OPA) in the presence of 2-mercaptoethanol (ME) or N-acetyl-L-cysteine (NAC) under basic aqueous conditions.
• Utilized 96-well microtiter plates for the reaction and fluorescence detection of resulting isoindole derivatives.
• Measured fluorescence excitation at 345 nm and emission at 450 nm.

Main Results:

• A sensitive fluorescence-based assay for protamine was established.
• The method demonstrated effectiveness for quantifying protamine in DNA-containing pharmaceutical nanoparticle preparations.
• The detection limit for protamine (free base, sulfate, and chloride) was found to be less than 1.1 µg/ml.
• The resulting isoindole derivatives exhibited sufficient stability for analysis.

Conclusions:

• The OPA derivatization method provides a sensitive and reliable means for protamine quantification.
• This fluorescence assay overcomes the limitations of UV-spectroscopy for analyzing protamine.
• The method is suitable for quality control and formulation development involving protamine-based pharmaceutical products.