Modified RP-LC of Phenylthiocarbamyl Amino Acid Adducts in Plasma Acetonitrile Extracts Using Multiple Internal Standards and Photo-Diode UV Detection

摘要

Among the techniques available for quantitative analysis of physiological amino acids, systems using optical detection are of low specificity because of possible interference at the analytical wavelength. Another disadvantage is problems of sample extraction from complex biological matrices, for example plasma. This paper describes reversed-phase LC of phenylthiocarbamyl (PTC) amino acids in plasma deproteinated by addition of acetonitrile. Specificity was monitored by photo-diode UV detection and accuracy was assessed by a plasma spiking procedure with more than one internal standard. Dual-wavelength spectrophotometry (254 and 283 nm) was also used for separate measurement of co-eluting adducts of tryptophan and ornithine. This method enables the quantification, with high reproducibility, of a total of twenty-three plasma amino acids from fasting healthy subjects. LOQ values are satisfactory for all the amino acids (average 6 mu mol L-1). However, the method does not enable analysis of aspartate and overall homocystine, present at very low concentrations, in all plasma samples. This PTC-amino acid chromatographic method is inexpensive, reliable, and suitable for clinical research and therapeutic drug monitoring, but adaptation to dual on-line detection is required to improve its sensitivity.