Analytical approach

Amino acid enantiomers are separated by reverse phase HPLC (method of Kaufman & Manley, 1998). Samples are derivitised with o-phthaldialdehyde and thiol N-isobutyryl-L-cysteine automatically prior to injection. The resulting diastereomeric derivatives are then separated on Hypersil C18 BDS column (sphere d. 5 µm; 250 x 3mm) using a linear gradient of a sodium acetate buffer, methanol and acetonitrile on an integrated HP1100 liquid chromatograph (Hewlett-Packard, USA). The fluorescence intensity of derivitised amino acids are measured (Ex=230nm, Em=445nm) and normalised to the internal standard. All samples and a blank extracts - subjected to identical preparation procedures - are run in duplicate. To assess recovery and induced racemization a standard mixture of amino acids (25 nM; AA-S-18, Sigma, UK) is subjected to the procedures as the samples. Percentage recovery for each amino acids ranges from 75%-85% and the amount of racemization induced is small but significant.

Example HPLC trace