Determination of citrulline and the prominent amino acids involved in small intestine diseases by liquid chromatography after derivatization. Comparison of separation by a monolithic and a core-shell column

ACQUAVIVA, A.; ROMERO, L. M.; CASTELLS, C. B.; RAMIS RAMOS, G.; HERRERO MARTINEZ, J. M.

Analytical Methods

RSC, Royal Society of Chemistry

Año: 2014 vol. 6 p. 5830 - 5837

1759-9660

We describe the development of a high-performance liquid chromatography (HPLC) method for the determination of citrulline and other amino acids relevant to intestinal diseases. The amino acids were derivatized with 9-fluorenylmethylchloroformate (FMOC-Cl) and their derivatives separated on two different columns, a core-shell column (Halo C18) and a silica-based monolith (Chromolith Performance RP-18). The derivatization reaction was optimized with respect to pH, buffer concentration and reproducibility. The optimal derivatization conditions were achieved with 0.4 M borate buffer at pH 9.20, a constant ratio FMOC-Cl/total amino acids (10:1) and 75 mM tyramine after 1 min (quenching reaction). The separation conditions with both chromatographic supports were also optimized. The chromatographic performance of these two columns was compared. This proposed HPLC-UV method was satisfactorily applied to the analysis of a real plasma sample.