Measurement of plasma free choline by high performance liquid chromatography with fluorescence detection following derivatization with 1-naphthyl isocyanate.

Choline is an essential nutrient which is difficult to measure because it has no native absorbance or fluorescence and only relatively unreactive functional groups. The method described here uses the reaction of the hydroxyl group on choline with 1-naphthyl isocyanate to form a stable cationic aromatic urethane that can be measured by high performance liquid chromatography (HPLC) on a cation exchange column, followed by fluorescence detection. The sample was directly added to acetonitrile and mixed with magnesium oxide and 1-naphthyl isocyanate. The 1-naphthylurethane choline derivative was separated by HPLC using a strong cation exchange column with a tetramethylammonium glycolate buffer in the mobile phase, and measured by fluorescence detection. The recoveries from blood plasma were over 94%. In this study an internal standard was not used, and quantification was achieved by calibration using standards containing known choline concentrations. The within batch and between batch coefficients of variation (CVs) were below 6%. The response was linear over the biological range investigated (8.9-58.9 micromol L(-1), r2=0.998). This is a technically simple method that can be carried out with an inexpensive HPLC system with fluorescence detection. It has sufficient sensitivity to measure choline in biological materials such as human plasma, and is suitable for processing batches of samples.