Determination of citrulline in human plasma samples using a RP-HPLC method

ACQUAVIVA, A.; ROMERO, L. M.; CASTELLS, C. B.

Budapest

Simposio; 36th. Internacional Symposium on HPLC and Related Techniques (HPLC2011); 2011

HPLC Symposium

Citrulline is a nonessential amino acid critical to the detoxification and elimination of unwanted ammonia within cells; it is a precursor of arginine during metabolism and its blood level is an important biomarker of several pathologies prognostics [1]. The amino acids concentration can be measured by reverse phase liquid chromatography using pre-column derivatization agents. Among several derivatization agents available we choose 9-fluorenylmethyl chloroformate (FMOC) because the reaction kinetics is very fast at room temperature and the derivatives are very stable [2]. These features make the automatization a straightforward step. The pre-column reaction was carried out at pH 9.2 conditioned by borate buffer; the reaction was stopped with β-phenylethylamine or tyramine after 1 minute of vortex shaking. Variables from this pre-column reaction were optimized.  Very good separation of FMOC-citrulline from other FMOC-amino acids usually present in plasma was achieved by using an octadecylsilice column. The conditions of separation were optimized by using an elution gradient between A solution of ACN:MeOH:Acetate buffer (pH 4.2, 50 mM), and B solution of ACN:Acetate buffer. The proposed methodology was applied to a human plasma sample. The method established presents a short time-consuming, it is friendly green and feasible of automation. Besides, this method applied to citrulline concentration determination can be used as a diagnostic or monitoring method for a wide range of diseases fundamentally those related to intestine failure. [1] Hui-ming Mao, Wei Wei, Wu-jun Xiong, Ying Lu, Bing-guan Chen, Zhongmin Liu. Clinical Biochemistry 43 (2010) 1141?1147 [2] Einarsson S., Josefsson B., Lagerkvist S. J. Chromatog., 282 (1983) 609-618