Analysis of plasma amino acids by HPLC with photodiode array and fluorescence detection.

摘要

BACKGROUND: Plasma amino acids are usually analyzed by ion-exchange chromatography (IEC), a reproducible but time consuming method. Here, we test whether plasma amino acids can be analyzed using reverse-phase high performance liquid chromatography (HPLC). METHODS: Filtered plasma, with S-carboxymethyl-l-cysteine as the internal standard, was derivatized and analyzed by an Agilent 1100 HPLC system. Primary amino acids were derivatized with o-phthalaldehyde 3-mercaptopropionic acid (OPA) and detected by a diode array detector. Secondary amino acids were derivatized with 9-fluorenylmethyl chloroformate (FMOC) and detected fluorometrically. Chromatographic separation is achieved by two gradient elutions (two injections per sample), starting at different pHs, on a reverse phase Agilent Zorbax Eclipse C(18) column AAA (4.6x150 mm). RESULTS: The HPLC method evaluated correlated well with IEC (0.89