Assays for total homocysteine and other thiols by capillary electrophoresis-laser-induced fluorescence detection. I. Preanalytical condition studies.
In recent papers, we presented a new analytical method for thiol quantification in serum. It is based on the use of capillary electrophoresis and laser-induced fluorescence to analyze thiol 6-iodoacetamidofluoresceine (IAF) derivatives. Quantitative results of homocysteine, glutathione, cysteine-glycin, and cysteine were shown (Clin. Chem. 45 (1999) 412). A comprehensive comparison of the quantitation of homocysteine in serum, using high-performance liquid chromatography/conventional fluorescence detection and fluorescence polarization immunoassay was also used (E. Caussé et al., Electrophoresis 21 (2000) 2074). Sample preparation prior to derivatization with IAF had never been investigated. In this work we present the results of quantitation of thiols in serum and plasma with three different anticoagulants widely used: ethylenediaminetetraacetic acid (EDTA), heparin, and sodium citrate. We show that serum and EDTA plasma gave the same results. Then serum protein precipitations by acetonitrile, acetone, sulfosalicylic acid, perchloric acid and trichloracetic acid, prior to derivatization by IAF, were also investigated. Their influence on the concentrations of the thiols were determined. Sulfosalicylic acid and acetonitrile precipitations are well adapted, whereas acetone cannot be used.
Caussé
E
E
Laboratoire de Biochimie/lNSERM U466, Centre Hospitalier de Rangueil, Toulouse, France. causse.e@chu-toulouse.fr
Issac
C
C
Malatray
P
P
Bayle
C
C
Valdiguié
P
P
Salvayre
R
R
Couderc
F
F
D000328:Adult; D000925:Anticoagulants / Q000494:pharmacology; D019075:Electrophoresis, Capillary / Q000379:methods*; D005260:Female; D016231:Fluorescence Polarization Immunoassay; D006710:Homocysteine / Q000097:blood*; D006801:Humans; D007834:Lasers; D008297:Male; D008875:Middle Aged; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000097:blood*
D000925:Anticoagulants; D013438:Sulfhydryl Compounds; D006710:Homocysteine