General procedure for preparing a reference mixture and determining the amount of dipeptide contaminant in N‐alkoxycarbonylamino acids by high‐performance liquid chromatography

Abstract
The amount of N‐alkoxycarbonyl‐dipeptide contaminant in a sample of N‐alkoxycarbonylamino acid is determined by reversed phase high‐performance liquid chromatography on a μBondapak‐C18 column using aqueous methanol or acetonitrile as solvent and u.v.‐absorbance monitoring. Reference mixtures of the N‐substituted monomer and dipeptide are prepared by reaction of the symmetrical anhydride of the N‐alkoxycarbonylamino acid with the sodium salt of the amino acid in aqueous dimethylformamide. The proportions of the two components in the reference mixture are established by 1H‐n.m.r. spectroscopy. N‐Protected‐dipeptide was detected (0.1–0.7%) in seven out of eight commercial benzyloxycarbonylamino acids examined.

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