Determination of Proteins, Phosphatidylethanolamine, and Phosphatidylserine in Lipid-Rich Materials by Analysis of Phenylthiocarbamyl Derivatives

Abstract

In classical, ninhydrin-based amino acid analysis (1,2), the ion-exchange matrix used for separation becomes contaminated upon consecutive analyzes of extremely lipid-rich samples; in our experience, already after approx 20–30 samples. Therefore, in analysis of lipid-rich material we focused on amino acid analysis involving reversed phase (RP) chromatography, because lipids are soluble in the organic solvents commonly used for elution and regeneration of such columns (e.g., acetonitrile and 2-propanol). Precolumn derivatization with phenylisothiocyanate (PITC) of protein hydrolysates from physiological samples followed by RP-HPLC is equivalent in analytical quality to the classical ion-exchange chromatography/ninhydrin method (3), and is sensitive down to at least 10 pmol (4). PITC reacts both with primary and secondary amines, the reproducibility is high and the phenylthiocarbamyl (PTC) amino acid derivatives are stable for months when stored dry at −20°C, or for days in solution at ambient temperature (3,4). This approach can be used conveniently for analysis of lipid-rich material, where at least 300 samples can be analyzed with the same column, i.e., the column lifetime is comparable to those encountered during analysis of lipid-free samples (5).