| Alpha-fetoprotein, leptin and cardiac troponin are the important protein secreted by human cells. They participate in many important physiological regulations of human body and their changes of serum content are closely related to human health. Based on plenty of investigation, they have been identified as important biological markers, and therefore they have a wide range of applications in clinical testing. At present commonly used assays for the detection of AFP, LEP and cTnI are immune analyses, such as enzyme-linked immunosorbent assay (ELISA), chemiluminesent immunoassay (CILA), radioimmunoassay (RIA). However, these methods mentioned above are time-consuming and lack of comparability beyond time and space, therefore they are restricted in practical clinical applications. In order to fulfill the requirements of clinincal testing, ensure the accurate, comparable and traceable results, it is urgent to develop standard reference methods for protein quantification and find the related reference materials.Isotope dilution mass spectrometry with a wide dynamic range, high sensitivity, available absolute quantification and the results being able to directly traceable to the International System of Units, has been widely used, as a reference method for the quantitative analysis of protein standard materials inrecent years. As far as we know, however, nothing has been reported about absolute quantification of human AFP, LEP and cTnI by isotope dilution mass spectrometry.In this dissertation,the quantitative method of HPLC-IDMS/MS has been developed for the absolute quantification of the human AFP, human LEP and recombinant human cardiac troponin I, respectively. The main research findings are described as the following:(1) The method for the quantification of human AFP by HPLC-IDMS/MS has been established. Both of the hydrolysis and tryptic digestion conditions were optimized. The contents of the human AFP were determined to be6.371μmol/g and5.464μmol/g with the relative expanded uncertainty of0.505μmol/g (k=2) and0.495μmol/g (k=2). Both of the linear correlation coefficients of the two methods were acceptable (R2≥0.998), through measurement the products form the hydrolysis and tryptic digestion procedures, respectively. The RSD were1.2%and2.1%, the results were satisfactory. It was shown good consistency between the results of the two methods (hydrolysis and tryptic digestion). (2) The method for the quantification of human LEP by HPLC-IDMS/MS has been established. The hydrolysis condition was optimized.The content of Human Leptin was calculated to be0.582g/g with the uncertainty of0.014g/g (k=2), through the determination of the products (proline, valine and leucine) from hydrolhysis. The LOD and LOQ were0.8x10-4g/g and2.7×10-4g/g respectively. The RSD was satisfactory (1.7%). The calculated results were furither validated by the purity deduction assay.(3)The method for the quantification of recombinant cTnI by HPLC-IDMS/MS has been established. The hydrolysis condition was optimized.The content of Human cTnI was calculated to be0.22g/g with the relative expanded uncertainty of0.012g/g (k=2). The LOD and LOQ were1.6x10-4g/g and5.2×10-4g/g, respectively. The RSD was satisfactory (4.4%).In this study, a quantitative method of HPLC-IDMS/MS for the absolute quantification for the human AFP, human LEP and recombinant human cardiac troponin I has been successfully developed. The conditions of hydrolysis and tryptic digestion were optimized. The quantitative results of the biomarker proteins were accurate and credible. Compared with other quantitative assays, the developed HPLC-IDMS/MS method was superior in easier sample pretreatment, excellent repeatability and can availability to trace to the Standard Systems of units. The findings in this study also laid the groundwork for the future research of reference materials of AFP, LEP and cTnl. |
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