Study Of Accurate Quantification Method For Cardiovascular Disease Risk Factor--Ⅰ. Accurate Quantification For Human C-reactive Protein (Liquid Chromatography-isotope Dilution Tandem Mass Spectrometry Amino Acid Analysis Method)--Ⅱ. Determination Of T

Detection of cardiovascular disease risk factors has been an important issue in the world. In recent years, with the development of detection technology, homocysteine and C-reactive protein have become the new indicators in clinical laboratory. To achieve standardization of the measurement and ensure comparability, it is necessary to establish accurate and reliable reference methods,develop relevant reference materials for clinical testing.High-sensitivity C-reactive protein (hsCRP) as a sensitive indicator of cardiovascular inflammation is gradually acknowleged. The determination method of human C-reactive protein with amino acid hydrolysis analysis, leucine and valine measurement by ID-LC-MS/MS was established in this paper. The CRP samples were spiked with isotope labelled valine and leucine solution, vacuum drying, after hydrolysis of48hours in HCl,dried and dissolved for HPLC-ID-MS/MS analysis. The signal was detected under MRM mode of using Agilent6410LC/MS/MS system, monitoring of ion pairm/z=118.1->72.1(valine), 123.1->75.1(13C5-valine),132.1->86.1(leucine),142.1->96.1(D10-leucine). Bracketing method was used for amino acids quantification and protein concentrations were converted from amino acids amount calculated. Hydrolysis time was optimized. We have participated in the co-validation for C-reactive protein organized by National Metrology Institute of Japan for method validation, and the results were well agreed with the results of both national metrology institute of Japan and Korea, which lay an important foundation for our study of CRP reference material.Homocysteine (Hcy) is an independent risk factor for cardiovascular disease. In this paper, the determination method of total homocysteine in human serum by ID-LC-MS/MS was established. D8-homocystine was used as an internal standard, dithiothreitol as the reduction agent, and acetonitrile as the protein precipitating agent.A triple quadrupole mass spectrometry with electrospray ion source was used for monitoring the target ions (m/z140.1->94.1,136.1->90.1) with MRM mode.Bracketing method was used for quantification.Optimization of pre-treatment conditions,liquid chromatography separation,mass spectrometry detection and measurement conditions for the determination of total homocysteine in human serum were discussed.The results of three concentrations (from low to high) of homocysteine in human serum samples were determined by ID-LC/MS/MS method, the relative standard deviation (RSD) ranged from0.58%to2.86%. Recoveries of the method ranged between96.54%～100.05%. The detection limit is about0.07ng/g (3times the signal to noise ratio),the expanded uncertainty of this method is4.0%. The accuracy was validated by using NIST standard reference material SRM1955Level Ⅰ,Ⅱ,Ⅲ. The measurement results within the reference range.This ID-LC/MS/MS method for homocysteine in human serum, which demonstrates reliable, simple, good accuracy and precision, can be used as the candidate reference method for the determination of homocysteine in serum.