A simultaneous assay of the differentiating agents, phenylacetic acid and phenylbutyric acid, and one of their metabolites, phenylacetyl-glutamine, by reversed-phase, high performance liquid chromatography

This paper describes a reversed-phase, high performance liquid chromatographic (HPLC) method for the isolation, detection and quantification of phenylacetic acid, phenylbutyric acid and one of their metabolites, phenylacetylglutamine, from serum. The compounds are initially extracted from serum after protein precipitation with perchloric acid. The solution is neutralized with potassium bicarbonate and injected onto a C-18 column. The compounds are then differentially eluted using an increasing gradient of acetonitrile. Ionization of the two acids is suppressed by adding phosphoric acid to the mobile phase. The compounds are detected by UV absorbance at 208 nm. The assay yields a lower limit of detection of 2 pg/ml and is linear to concentrations as high as 2,000 μg/ml. Between 20 and 1,000 μg/ml the interassay percent coefficient of variation is less than 10%.