Determination of the purity of valine by isocratic liquid chromatography coupled with charged aerosol detection (CAD)

摘要

A liquid chromatography coupled with charged aerosol detection (LC-CAD) procedure; capable of separating and quantifying the most common impurities of valine at levels as low as 0.05 % (m/m), has been developed. The procedure is simple (isocratic), rapid, linear, sensitive and repeatable. It employs a widely available and inexpensive stationary phase (C18). As L-amino acids are produced in large amounts and are ingredients in medicinal, nutritional and food products, the control of their purity is essential for consumer safety. Commonly used procedures to analyse L-amino acids include thin-layer chromatography (TLC) after spraying with reagents such as ninhydrin [1]. Although relatively simple, TLC offers only a limited degree of separation and the results are usually only semi-quantitative, which makes TLC more suitable for identification than for the quantitative control of purity. Although liquid chromatography (LC) in general provides good separation with quantitative responses, ultraviolet (UV) direct detection is not viable with valine, because its chromophores (NH2 and carbonyl moieties) cause weak and unspecific UV absorption. The problem can be circumvented by reaction with a chromophoric derivatising agent [2,3,4]. LC with derivatisation is more selective and sensitive and this is why it was recently introduced in the European Pharmacopoeia [2]. However derivatisation may come with limitations, such as: i) only the impurities capable of reacting with the derivatising agent respond and ii) accuracy is affected by the degree of reaction of the impurity with the derivatising agent.