HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC TECHNIQUE FOR NON-DERIVATIZED LEUCINE PURIFICATION - EVIDENCE FOR CARBON ISOTOPE FRACTIONATION

A high-performance liquid chromatographic (HPLC) method, utilizing an ion-pairing agent with a reversed-phase column, isocratic elution and ultraviolet detection, was developed for the separation of non-derivatized leucine from a mixture of amino acids. Fractionation of leucine isotopes during HPLC separation was observed, creating potential problems for collection of leucine plasma or tissue protein hydrolyzates for subsequent isotopic determinations. It was demonstrated that [C-13]leucine and [C-12]leucine have different retention times (p < 0.01). It is concluded from this study that partial collection of leucine eluted from an HPLC column will result in erroneous estimation of the isotopic enrichment of leucine.