Study On The Separation And Detection Of Branched Chain Amino Acids By Capillary Electrophoresis And Its Application

The branched chain amino acids(BCAAs) have a pivotal role in the sports nutrition. They stimulate protein synthesis, prevent sports fatigue and strengthen immune function. The quantitative of BCAAs in sports nutritional supplements and urine samples from athletes could not only achieve the purpose of quality control and efficiency evaluation, but also provide data support for the scientific use of sports nutrition supplements by the comparison of intake and excretes volume of BCAAs. Besides, the elevation of BCAAs in urine could also act as biomarker for some diseases(such as maple syrup disease). Among the numerous amino acids separation methods, capillary electrophoresis(CE) is widely used owing to its short analysis time, high resolving power and less reagent consumption. UV detector has the advantages of low cost and widely application. This article was aimed at the development of convenient methods for the determination of BCAAs by CE-UV.Chapter one introduced the principle of CE, and elaborated the important role of BCAAs to the athletes and the potential hazards when excessive BCAAs(or proteins) were consumed and the determination methods for the tolerable upper intake levels. A good prospect for the application of CE on BCAAs analysis and the development of sports nutrients usage policies was put forward.Chapter two investigated the ultraviolet absorption properties of six background UV-absorbing probes and the electrophoretic mobility of the probes and amino acids. Considered this properties and combined with the stability, p-aminosalicylic acid(PAS) was selected as the most desirable background UV-absorbing probe. With the addition of EDTA, leucine(Leu) andisoleucine(Ile) could be partly separated, whereas the peak of L-Ile and Leu was still overlapped.Hence, a proper additive was necessary for the complete separation of Leu and Ile.Chapter three developed a CE-indirect UV method for the analysis of BCAAs with the optimization of pH value, concentration of additive and background UV-absorbing probes. The discussion of their influencing mechanism was also proposed. The optimal conditions were 2.0mM Na2HPO4,10.0 mM PAS,40.0 m M β-cyclodextrin(β-CD), at pH=12.2. The resolution of Leu and L-Ile was 1.0(sample concentration was 0.5 mM). The reproducibility of the method was desirable(peak area RSD<2.1%, n=5). Even though the sensibility was lower than the detector like fluorescence and mass, the tedious and troublesome derivation process could be avoid and the cost was low due to the UV detector was standard configuration for most CE system.Chapter four developed a CE-direct UV detection method based on the results that BCAAs have UV absorption at the UV region lower than 220 nm. At the conditions of 40.0 mM borate,40.0 mM β-CD, pH=10.2, the BCAAs could be well separated, the background electrolytes was more simple than indirect UV detection method, and the sensitivity was higher than a similar work reported by others.Chapter five was focus on the applications of the present methods onto the BCAAs analysis in sports nutrition supplements and athletes urine. The quantitative data of BCAAs in supplements obtained by CE-indirect UV method was close to some reports in literature, whereas the peak of L-Ile in direct UV method was interfered by an unknown peak, and the BCAAs in urine could not be detected by this methods, The reason might be the low concentration of BCAAs in human urine and the consumed BCAAs did not exceed the safe upper limit. In order to solve the above problems, a standard extraction method was established.