Optimal Conditions for the Direct RP-HPLC Determination of Underivatized Amino Acids with Online Multiple Detection

The combined use of a dual-UV detector as well as a fluorimetric and a multielectrode electrochemical detector (equipped with a dual electrode, consisting of a conventional size 3 mm diameter glassy carbon electrode (GCE) and of a pair of 30 μm thick carbon microfibers) is proposed for the detection of the following 15 underivatized amino acids: L-histidine (His), L-cysteine (Cys), creatine (Crn), S-methyl-L-cysteine (Me-Cys), DL-homocysteine (Hcy), L-methionine (Met), beta-(3,4-dihydroxyphenyl)-L-alanine (DOPA), L-tyrosine (Tyr), DL-m-tyrosine (m-Tyr), L-a-methyl-DOPA (Me-DOPA), L-phenylalanine (Phe), DL-alpha-methyltyrosine (Me-Tyr), 5-hydroxy-tryptophan (5-HTP), 3-nitro-L-tyrosine (NO₂Tyr), and L-tryptophan (Trp), as well as of 2 dipeptides L-cystathionine (Cysta) and L-carnosine (Car), and of creatinine (Cre). A multilinear solvent (acetonitrile) gradient elution program, determined by a simple optimization algorithm, is required for the efficient reversed-phase separation of the above mixture of 18 solutes within 27 min at a flow rate of 1.0 mL/min and at 25 °C.