Determination Of Thiols By High-performance Liquid Chromatography Combined With Solid-phase Extraction

Thiol compounds such as glutathione (GSH), cysteine (Cys) widely occur in biological tissues and fluids and are known to play important roles in living systems. Their altered levels in plasma have been linked to specific pathological conditions including muscular dystrophy, chronic renal failure and cataractogenesis. In view of the possible harmful effects of their altered levels for human health, the development of sensitive and routine methods for their analysis is of great importance.Current analytical methods for thiol compounds were usually accomplished by high performance liquid chromatography (HPLC). For thiol compounds analysis, sample pretreatment can be used as enrichment process to improve detection limits. Solid-phase microextraction (SPE) is a solvent-free and miniaturizable extraction technique, which has gained widespread acceptance for analyte matrix separation and preconcentration. In this dissertation, SPE is used as the sample pretreatment technique to improve sensitivity of analytical methods and reduce the interference from the complex sample. The main content is listed as follows:1. SPE coupled with HPLC-fluorescence detection (FL) was developed for the extraction and determination of three thiol compounds including glutathione (GSH), cysteine (Cys) and acetylcysteine (NAc) in human plasma samples. Fluorescence probe 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) was applied for the derivatization of thiol compounds. The LODs of thiol compounds were in the range of 0.005-0.02μmol/L. The intra-day precisions ranged from 1.9 to 3.4% and the inter-day precisions ranged from 3.2 to 4.7%. The recoveries of the three thiol compounds from human plasma samples were in the range between 86% and 112.9%. The proposed method has been successfully applied to the analysis of the three target thiol compounds in human plasma samples.2. SPE coupled with HPLC-FL detection was developed for the extraction and determination of three thiol compounds (GSH, Cys and NAc) in human urine samples. N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl)iodoacetamide (BODIPY Fl-C1-IA) was used as pre-column derivatization reagent. The limits of detection ranged from 0.2 to 4 nmol/L (S/N=3). The utility of the proposed method has been validated by measuring thiol-containing compounds in human urine samples from healthy persons, with recoveries of 92.5-110%. The fluorescence labeling reagent (BODIPY Fl-C1-IA) not only reacts readily with sulfydryl with good selectivity in aqueous solution, but also provides the advantages of few by-products and mild reaction conditions.3. Multi-walled carbon nanotubes (MWCNTs) as SPE stationary phases was proposed for the determination of thiol compounds including glutathione (GSH), cysteine (Cys) and acetylcysteine (NAc) in human urine and plasma samples. Fluorescence probe (BODIPY Fl-C1-IA) was applied for derivatization of thiols and HPLC-FL detection was used for the determination of the derivatives. The limits of detection were 0.004-0.08 nmol/L. The proposed method was applied successfully to the analysis of thiols in human urine and plasma samples and spiked recoveries ranged from 85 to 113.1%. The results confirmed that MWCNTs-based SPE was a simple and efficient enrichment method and could be used for the determination of trace amount of thiol compounds.