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: -phenylanine (phe), DL: -alpha-methyltyrosine (me-tyr), 5-hydroxy-tryptophan (5htp), 3-nitro-L: -tyrosine (NO(2)Tyr) and L: -tryptophan (trp), as well as of two dipeptides: L: -cystathionine (cysta), 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.