Analysis of amino acids in human serum by isocratic reversed-phase high-performance liquid chromatography with electrochemical detection.

A simple, sensitive and reproducible isocratic high-performance liquid chromatography (HPLC) method has been developed for the determination of amino acids in human serum. The method involves precipitation of the serum proteins with methanol followed by pre-column derivatization of amino acids with o-phthalaldehyde-2-mercaptoethanol or o-phthalaldehyde-sodium sulfite. HPLC separation of the derivatives was performed using an ODS column with an isocratic mobile phase system and electrochemical detection (+0.75 V). The response was linear over the range 5-300 microM for all amino acids. The method allows quantitative determination of glutamic acid, asparagine, serine, glutamine, histidine, taurine, alanine, arginine, methionine, isoleucine, ornithine, leucine, phenylalanine, lysine and tryptophan at concentrations as low as 0.5-5.0 pmol (signal-to-noise ratio=2). Using this method, the levels of amino acids in serum from healthy donors and patients with ischemic stroke were determined.

Simultaneous determination of several amino acids, including homocysteine, cysteine and glutamic acid, in human plasma by isocratic reversed-phase high-performance liquid chromatography with fluorimetric detection.

A method for the simultaneous measurement of two biologically important thiol compounds cysteine and homocysteine and five amino acids including neurotransmitters aspartate and glutamate is reported. This method utilized derivatization of compounds with o-phthalaldehyde in the presence of 2-mercaptoethanol following alkylation of the free sulfydryl group with iodoacetic acid followed by separation using reversed-phase high-performance liquid chromatography. These o-phthalaldehyde-2-mercaptoethanol-labeled compounds were separated within 30 min on a Spherisorb ODS-2 column with isocratic elution using 17% methanol, 0.04 M sodium phosphate buffer (pH 7.0), 0.002 M Na2EDTA and detected fluorimetrically (excitation 340 nm, emission 450 nm). Using this method, the concentrations of homocysteine, cysteine, glutamic acid. aspartic acid, asparagine, serine and glutamine in human plasma were determined.