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 [13C]leucine and [12C]leucine have different retention times (p less than 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.
Baumann
P Q
PQ
Department of Medicine, University of Vermont, Burlington 05405.
Ebenstein
D B
DB
O'Rourke
B D
BD
Nair
K S
KS
D000596:Amino Acids / Q000032:analysis; D002247:Carbon Isotopes; D002250:Carbon Radioisotopes / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007930:Leucine / Q000097:blood / Q000302:isolation & purification*; D013058:Mass Spectrometry; D009555:Ninhydrin; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D002247:Carbon Isotopes; D002250:Carbon Radioisotopes; D007930:Leucine; D009555:Ninhydrin