Application Investigation Of The Chromatographic Analysis Of Thiol Compounds

The thiol compounds widely occur in living organisms, and play a key role in scavengingfree radicals, heavy metal detoxification, antagonism of ionizing radiation, and so on. They areclosely associated with body growth and development, anti-aging and several diseasesincluding leukemia, arthritis and Parkinson’s disease. Furthermore, the thiol drugs have beenused in the treatment of various illnesses for many years. Therefore, the establishment of simple,fast, and sensitive determination of the sulfydryl-type substances is of great significance. In thispaper, the capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) andhigh performance liquid chromatography with fluorescence detection (HPLC-FL) are employedto assay the thiol compounds in biological samples. Our research mainly includes the followingthree chapters:(1) A capillary electrophoresis method coupled with laser-induced fluorescence detection for thesimultaneous measurement of homocysteine, cysteine and glutathione is established. Themethod involves a precolumn derivatization of the thiol compounds with a BODIPY fluorescentdye (1,3,5,7-tetramethyl-8-bromomethyl-difluoroboradiaza-s-indacene, TMMB-Br). Theeffects of species, concentration and pH of background electrolyte, operating voltage andinjection time on the separation of three derivatives were studied in detail. Several factorsinfluencing the derivatization efficiency such as the type and pH of buffer solution, TMMB-Brconcentration, reaction temperature and time were also investigated. Under the optimizedconditions, the detection limits for homocysteine, cysteine and glutathione were0.73nmol/L,0.72nmol/L and0.36nmol/L (S/N=3), respectively. The applicability of the CE-LIF methodwas demonstrated by analyzing homocysteine, cysteine and glutathione in human serum andurine samples with recoveries of92.6%-109.1%. In addition, we tried using TMMB-Br for cellimaging of thiol compounds in living cells.(2) Two rapid and sensitive analytical methods are developed for the determination ofD-Penicillamine and tiopronin using high performance liquid chromatography withfluorescence detection and capillary electrophoresis with laser-induced fluorescence detection.A fluorescence labeling reagent, TMMB-Br was perfectly applied to derivatize these two thioldrugs. The electrophoresis and chromatographic separation conditions, derivatization conditions were discussed carefully. Under the optimum conditions, the high performance liquidchromatography and capillary electrophoresis separation of D-Penicillamine and tiopronincould be achieved within12min, and the limits of detection were5.0nmol/L,2.0nmol/L forfluorescence detection and0.88nmol/L，0.47nmol/L for laser-induced fluorescence detection.The drugs in human urine and plasma samples were determined successfully, and the recoverieswere95.0%-106.7%and95.2%-104.3%, respectively.(3) A simple and highly sensitive capillary electrophoresis with laser-induced fluorescencedetection method for captopril and N-Acetylcysteine is presented. Fluorescein was used as aninternal standard (I.S.). At room temperature, the I.S.and derivatives of captopril andN-Acetylcysteine are baseline separated within15min using25mmol/L boric acid solution (pH9.5) as the running buffer, with a separation voltage of11.0kV. Under the optimum conditions,excellent linearity was obtained for the analytes over their concentration ranges of6–600nmol/L (R2=0.998), and detection limits of the thiol derivatives were0.65nmol/L and0.76nmol/L, respectively. We tested the proposed method by analyzing the drugs in human plasmaand urine samples with recoveries of97.0%-105.7%. Advantages of the method were simple,reproducible, and highly sensitive.