Determination of homocysteine and other thiols in human plasma by capillary electrophoresis.

A new capillary electrophoresis (CE) assay for thiols in human plasma, including homocysteine, which is an indicator of several clinical states has been developed. The thiols were derivatized quantitatively at 50 degrees C, pH 8.0 with a fluorogenic reagent, ADB-F (4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole), which is about 30 times faster compared to the other fluorogenic reagent, SBD-F (ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate). The separation of ABD-thiols was performed in a 50 mM sodium phosphate buffer (pH 2.1) using a bare fused silica capillary (27 cm x 50 microns i.d.) at 25 degrees C. With the electric field of 560 V cm-1, the time needed for the separation of homocysteine, glutathione and cysteine was less than 8 min. A filter-type ultraviolet detector and a 512-channel diode-array detector (DAD) were employed for ABD-thiol analysis. DAD was used to confirm the ABD-thiol peaks. The limits of detection (S/N = 3) for homocysteine, glutathione, and cysteine were 0.5, 1 and 2 microM at 220 nm, respectively.