HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY INTERCOMPARATIVE STUDY FOR AMINO ACID ANALYSIS IN TWO TISSUES BY PITC- AND OPA-DERIVATIZATIONS

Abstract

In this paper, we have extensively investigated the pre-column derivatization method with o-phthaldialdehyde (OPA) to analyze cysteine, cystine, among with other 18 amino acids, using reversed-phase high-performance liquid chromatography analysis (RP-HPLC), and after we have compared it with an other method involving phenyl-isothiocyanate (PITC) in order to have a two sensitive methods to use accordingly the matrix. We have studied the analytical conditions for the OPA-reaction together with precision, linearity range, correlation coefficient (>0.998) and limit of detection (between 14 fmol and 1 pmol) for each amino acid. Then, we have applied the two methods to the analysis of larynx and kidney tissues: between the two methods, the amino acid recoveries (between 80% and 100%) and the correspondence of the OPA and PTC results are satisfactory considering the different involved derivatization procedures. PTC- and OPA-chromatograms obtained in the analysis of actual samples are reported together with the amino acid level, with particular regard to the cysteine/cystine determination.

Keywords:

• Amino acids
• Comparison
• Cysteine
• Phenylisothiocyanate
• o-Phthaldialdehyde

ACKNOWLEDGMENTS

The authors wish to thank dr. M. Santulli of “Policlinico Umberto I” of the University of Rome “La Sapienza” and dr. V. Anatra of the University of Rome “La Sapienza” for their kind help during the sample collection and the HPLC analyses.