Retention/quantitation properties of the o-phthaldialdehyde-3-mercaptopropionic acid and the o-phthaldialdehyde-N-acetyl-L-cysteine amino acid derivatives in reversed-phase high-performance liquid chromatography

The separation/identification of 25 amino acids as their o-phthaldialdehyde-3-mercaptopropionic acid (OPA/MPA) and o-phthaldialdehyde-N-acetyl-L-cysteine (OPA/NAC) derivatives have been optimized [paying particular attention to those amino acids which elute with more than one derivative (glycine, histidine, gamma-aminobutyric acid, beta-alanine, ornithine, lysine) and that are expected to be present in apples in their free form]. Optimum separation conditions are reported on six reversed-phase columns: Nucleosil 3 and 5 microm, 150(+20 guard)x4.0 mm; Gromsil 3 microm, 150(+10 guard)x4.0 mm; Hypersil 5 microm, 150(+20 guard)x4.0 mm and 200(+20 guard)x4.0 mm; and Hypersil 3 microm, 150(+20 guard)x4.0 mm. Elutions were followed, simultaneously, with photodiode array and fluorescence detectors connected in line. Optimization studies carried out in model solutions as a function of temperature (30-55 degrees C) and eluent flow-rate (0.8-2.5 mL/min) demonstrated that optimum resolutions are obtained with the highest flow-rate applicable (remaining on the safe side with a column pressure of << 3500 p.s.i.; 1 p.s.i.=6894.76 Pa) in the temperature range 30-50 degrees C. Twenty-five amino acids, eluting in 31 separate, characteristic derivatives, were determined on all six columns (the main component, asparagine, present in overwhelming excess, together with the minor constituents glutamine, beta-alanine, gamma-aminobutyric acid, homoserine, and homoarginine). Optimum conditions in the case of both derivatives were obtained on the same type of column (Hypersil, 5 microm), as follows: for the OPA/MPA amino acids with programmed flow-rate [1.3-2.3 ml/min; column, 200(+20 guard)x4 mm], at 50 degrees C, while, for the OPA/NAC amino acids at 2.1 ml/min flow rate, at 30 degrees C [column, 150(+20 guard)x4 mm], with 40 and 37 min run times, including equilibration. Responses of the corresponding amino acids proved to be independent of the column used; reproducibility in the concentration range 6-12,000 pmol, related to the injected amount of amino acids, was