Capillary electrophoresis coupled with chloroform-acetonitrile extraction for rapid and highly selective determination of cysteine and homocysteine levels in human blood plasma and urine.

A rapid and selective method has been developed for highly sensitive determination of total cysteine and homocysteine levels in human blood plasma and urine by capillary electrophoresis (CE) coupled with liquid-liquid extraction. Analytes were first derivatized with 1,1'-thiocarbonyldiimidazole and then samples were purified by chloroform-ACN extraction. Electrophoretic separation was performed using 0.1 M phosphate with 30 mM triethanolamine, pH 2, containing 25 µM CTAB, 2.5 µM SDS, and 2.5% polyethylene glycol 600. Samples were injected into the capillary (with total length 32 cm and 50 µm id) at 2250 mbar*s and subsequent injection was performed for 30 s with 0.5 M KОН. The total analysis time was less than 9 min, accuracy was 98%, and precision was <2.6%. The LOD was 0.2 µM for homocysteine and 0.5 µM for cysteine. The use of liquid-liquid extraction allowed the precision and sensitivity of the CE method to be significantly increased. The validated method was applied to determine total cysteine and homocysteine content in human blood plasma and urine samples obtained from healthy volunteers and patients with kidney disorders.

Plasma low-molecular-weight thiol/disulphide homeostasis as an early indicator of global and focal cerebral ischaemia.

OBJECTIVE: Recent studies have shown that cerebral ischaemia causes not only local, but also systemic oxidative stress. This leads to oxidation of thiol-containing compounds, including low-molecular-weight thiols (cysteine, glutathione, homocysteine and others). Therefore, the aim of this work was to verify the hypothesis that the thiol/disulphide homeostasis of low-molecular-weight thiols is disturbed in the early stages of cerebral ischaemia. METHODS: Two experimental rat models of ischaemia were used: a global model of vascular ischaemia (clamping the common carotid arteries + haemorrhage) and focal ischaemia (middle cerebral artery occlusion). The total levels of thiols and their reduced forms were measured before surgery and after 40 minutes of reperfusion (global) or 3 hours (focal) ischaemia. RESULTS: The global ischaemia model caused a marked (2.5-4 times, P < 0.01) decrease in the plasma thiol/disulphide redox state, and focal ischaemia caused an even larger decrease (30-80 times, P < 0.001). DISCUSSION: These results suggest that plasma low-molecular-weight thiols are actively involved in oxidation reactions at early stages of cerebral ischaemia; therefore, their reduced forms or redox state may serve as a sensitive indicator of acute cerebrovascular insufficiency.

Capillary electrophoresis and phenylboronic acid solid phase extraction for the determination of S-adenosylmethionine/S-adenosylhomocysteine ratio in human urine.

An approach that allows direct analysis of the ratio of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) by using CE is presented. The analytes were extracted on phenylboronic acid phase and eluted with 100 mmol/L HCl. CE separation of the analytes took place in the transient isotachophoresis mode with addition of NaCl and meglumine to the samples. The sensitivity (S/N = 3) and quantification limit (S/N = 10) of the method were 0.07 and 0.2 µmol/L, respectively, using a silica capillary with 50 µm internal diameter and 30.5 cm total length. The BGE was 0.02 mol/L Tris with 1 mol/L HCOOH (pH 2.2), and the separation voltage was 15-17 kV. Accuracy of SAM and SAH analysis in urine was 96 and 105%, respectively; interday precision for the SAM/SAH ratio was within 6%. The theoretical plate number exceeded a million. Total analysis time was 8.5 min.

Capillary electrophoresis coupled with 1,1'-thiocarbonyldiimidazole derivatization for the rapid detection of total homocysteine and cysteine in human plasma.

A simple and rapid approach is described for the determination of total plasma cysteine and homocysteine using capillary electrophoresis. Human plasma samples were reduced with dithiothreitol and then processed with 1,1'-thiocarbonyldiimidazole in acetonitrile. After centrifugation, the sample supernatant was injected directly into a capillary by applying negative voltage and analytes were stacked after alkaline post-injection. Using a 50µm i.d. silica capillary of 35cm total length, filled with 0.1M triethanolamine, 0.15M formic acid, and 50µM hexadecyltrimethylammonium bromide (pH 3.9), we reached a limit of quantification of 2.5µM for homocysteine. Accuracy was 94.7-105.1%, intra- and inter-day imprecisions were <2.5 and <3%, respectively. The total analysis time was 6min. Furthermore, liquid-phase extraction with isopropanol led to a fourfold increase in sensitivity.