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

Abstract

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 (4.6×150 mm). Results The HPLC method evaluated correlated well with IEC (0.89≤ r≤1.00) with linearity up to 2500 μmol/l. The between- and within-run CVs were <6.0%. In addition, this method is able to separate argininosuccinic acid, homocystine and allo-isoleucine, rare but clinically significant amino acids. Conclusion This HPLC method was comparable to IEC and could represent an alternative for amino acid analysis.