Simple plasma work-up for a fast chromatographic analysis of homocysteine, cysteine, methionine and aromatic amino acids.
Simplified sample workup obviating protein precipitation and eluent evaporation commonly employed in earlier reports using chloroformate-mediated derivatization of aminothiols prior to mass spectrometric (MS) detection is presented. The reduction of disulfides in plasma is accomplished with dithiothreitol within minutes. A simultaneous derivatization with ethyl chloroformate (ECF) and extraction of derivatives into organic phase takes place within seconds. Along with S-amino acids, also aromatic amino acids can be determined during a 5-min run. Gas chromatography with flame ionization detection (GC-FID) proved to be sensitive enough to reach plasma homocysteine levels. A prerequisite for a reliable quantitation was fulfilled under the given conditions. Intra-assay precision was <5%, recoveries from spiked plasma complete (101.2%), detection and quantitation limits for homocysteine came to <1 and 3 micro mol/l. Our results were in full agreement with those obtained by liquid chromatography (r=0.999 for homocysteine and 0.987 for cysteine), and were close to two homocysteine immunoassays (r=0.991 and 0.939, respectively).
Institute of Endocrinology, Národní Ave 8, 116 94 1, Prague, Czech Republic. phusek@undo.cz
Petr
P
Husek
Petr
P
Matucha
Alice
A
Vránková
Petr
P
Simek
D000596:Amino Acids / Q000097:blood*; D002849:Chromatography, Gas / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000596:Amino Acids