Determination of γ-carboxyglutamic acid by paired-ion reverse-phase high performance liquid chromatography

A rapid, simple, and sensitive method for determination of free γ-carboxyglutamic acid (Gla) in urine and alkaline hydrolysates of proteins was developed. The method involves purification of Gla on an anion exchange column, pre-column formation of o-phthaldehyde (OPA) derivatives of Gla, and quantitation by high performance liquid chromatography (HPLC). Neutralized alkaline hydrolysates of protein or deproteinized urine samples were applied to a small anion exchange column (Pasteur pipet with glass fiber support, 1 cm height of AG1X8 resin) and washed with phosphate buffer (0.01M, pH 7.2) and 0.2N acetic acid, and eluted with 0.1N hydrochloric acid. Over 85% of the Gla was recovered in the HC1 fraction. Chromatographic analysis was performed using paired-ion reverse-phase HPLC with a fluorescence detector. The mobile phase was methanol:phosphate buffer 40:60 with PIC-A reagent. The same HPLC system was also suitable for decarboxylation studies of Gla. The standard addition technique showed linearity is excellent from 1 μg to 5 μg Gla in urine. A 'Gla-doublet' phenomenon similar to that observed in many laboratories using amino acid analyzers was also detected in the HPLC system. The classic OPA reaction buffer, sodium tetraborate appears to be involved in the splitting of Gla. Therefore, a sodium bicarbonate buffer was used for OPA derivatization.