Evaluation of conditions for the HPLC determination of plasma amino acids using precolumn derivatization

Abstract

SummaryFor the determination of free amino acids in plasma, the conditions for precolumn derivatization of the amino acids and the chromatographic separation were examined. The isoindole products, formed by the reaction of the primary amino acids with orthophthalaldehyde (OPA), were readily separated by RPLC and detected spectrofluorometrically using an excitation wavelength of 300 nm and an emission cut-off filter of 440 nm. Since the sensitivity of this method permits determination of amino acids in the femtomole range, the analysis can be performed on samples as small as 10 pI of filtered plasma or serum. The separation is achieved in approximately 35 minutes with good precision for the majority of the amino acids.

Advancement in the derivatizations of the amino groups with the o-phthaldehyde-thiol and with the 9-fluorenylmethyloxycarbonyl chloride reagents

“…The increased methyl alcohol concentration of the reagent improved the stability of the OPA/MCE- [41] and OPA/ET-AAs [42].…”

The high performance liquid chromatography of enzyme systems relating to purine and pyrimidine metabolism: an overview

“…By the correct choice of buffer pH and percent organic modifier, rapid separations of small numbers of compounds can be easily developed empirically. Halfpenny and Brown (1986) used simplex optimization to develop methods for PNP and HGPRTase. *…”