High performance liquid chromatographic assay of Amicar, ε‐aminocaproic acid, in plasma and urine after pre‐column derivatization with o ‐phthalaldehyde for fluorescence detection

A simple, reliable and highly sensitive procedure was devised for measuring the levels of Amicar in blood and urine. 100 μL of serum or urine sample was added to 10 μL of a 10% w/v zinc sulfate solution and 100 μL of methanol, as previously described (Lam et al. , 1980) for the removal of proteins by precipitation. 50 μL of the supernatant was then mixed with 300 μL of 1 M borate buffer containing D ‐valine as the internal standard before derivatization with o ‐phthalaldehyde. The amino acids were then separated by a stereoselective reversed‐phase system using a mobile phase containing 10% of acetonitrile in 2.5 mM Cu(II) complexes of L‐proline. The chromatography is highly selective, resolving Amicar from L‐valine which in turn is resolved from its unnatural D‐antipode, the internal standard. The procedure including sample preparation and separation required a total of 15 min. As little as 50 ng/mL of Amicar in body fluids could be detected as the o ‐phthalaldehyde derivative by fluorescence.