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.
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.
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.
This work proposes an approach to the direct analysis of S‐adenosylhomocysteine (SAH) and the methylation index in blood using CE with UV detection (CE‐UV). After application of meglumine postinjection, we achieved SAH in‐capillary preconcentration in the HClO4 extracts of erythrocytes, which improved the detection limit (S/N = 3) of SAH up to 3 fmol or 180 nmol/L at the injection volume of 50 nL, taking into account the sample dilution rate. CE‐UV was carried out in 30 mM glycine and 45 mmol/L HCl (pH ∼1.8) at 17 kV in a capillary 48 cm in length and 50 μm id. Accuracy of the technique was 101% and reproducibility was about 12%.
Fraction analysis of homocysteine in biological fluids is important for the diagnosis and studies of cardiovascular, nervous, urological, and other diseases. Measurements of total, free, and reduced homocysteine by mass spectrometry with HPLC by means of its modification with N-ethylmaleimide are proposed. The linearity and detection threshold were 0.025-10 and 0.001 μM, respectively, for reduced homocysteine fraction and 0.2-100 and 0.1 μM, respectively, for the rest fractions. The accuracy and reproducibility of the method were within 12%.
The proposed method matches the basic requirements of all methods used to analyze drugs or metabolites in an antidoping laboratory, i.e., sensitivity, selectivity, and specificity. The acquisition of full-scan mass spectra with accurate masses can be a valuable tool in the retrospective evaluation of analyzed samples for anabolic steroids recently added to the prohibited list.
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