Determination Of Serum Total Cholesterol And Triglycerides By Isotope Dilution Liquid Chromatography Mass Spectrometry

The determination of serum total cholesterol (TC) and triglycerides (TG) have long been the important clinical measurements in regard to identifying individuals with hyperlipidemia and at risk for cardiovascular diseases. There are mainly two forms of cholesterol in human body:free cholesterol and cholesterol ester, which are both fat-soluble. TG is the main components of serum total glycerol (TTG), about 90～95%, followed by free glycerol (FG) and a small amount of monoglycerides and diglycerides. It's generally recognized the difference of TTG and FG as TG. In fact, the determination of serum TG means determination of TTG in most clinical tests. Several analytical techniques have been employed for the determination of TC and TG in serum, in which enzymatic methods are the most widely used. These methods offer rapid sample throughput, the capability for automation and simplicity of use. But there is a need to establish the reference measurement procedures for critically evaluation the trueness of the routine methods.The aim of our study is to develop the candidate reference measurement procedures for the determination of TC and TG. Reference measurement procedures need to be analytically specific and precise and have the uncertainty of measurement commensurate with their use in assessing the accuracy of routine methods and characterizing reference materials. Isotope dilution mass spectrometry (ID-MS) is analytically more specific and less susceptible to systematic effects and has been recognized as the preferred principle for reference measurement in clinical chemistry. In ID-MS, an isotopeically labelled analogue of the analyte is used as the internal standard and the analyte is quantified by measuring the ratio of the analyte to the internal standard with mass spectrometry. Since the same properties between the analyte and the isotopeically labelled analogue, the internal standard behaves very similarly to the analyte during the analysis. So the absolute recovery of the analyte is not essential to the accuracy of the analysis. Also, mass spectrometry is highly specific, a necessity of isotope dilution analysis and offers the second assurance for the accurate and specific determination. ID-MS was thus selected as the measurement principle in our study.Isotope dilution liquid chromatography mass spectrometric (ID-LC/MS) and isotope dilution liquid chromatography tandem mass spectrometric (ID-LC/MS/MS) methods for serum cholesterol were developed. Serum samples were supplemented by addition of [3,4-13C2] cholesterol, hydrolyzed with alcoholic potassium hydroxide and oxidized into cholest-4-en-3,6-dione by chromic acid. The oxidized products of cholesterol and its isotopically labelled analogue were detected by atmospheric pressure chemical ionization source and multiply reaction monitoring mode. By using a polynomial calibration, a precolumn conversion and aqueous calibrators, the method needed no pre-measurement, allowed automatic sampling. The relative expanded uncertainty of individual determination of TC in serum was about 0.7%.An ID-LC/MS/MS method for serum total glycerol was developed. Serum samples were spiked by addition of [13C3] glycerol, the serum protein was precipitated with acidic ethanol. The glycerides were hydrolyzed with alcoholic potassium, then benzoylated with the Schotten-Baumann reaction and the benzoates were analyzed by LC/MS/MS at the multiply reaction monitoring mode using electrospray ionization source. By using a calibration curve, a precolumn conversion and aqueous calibrators, the method allowed automatic sampling. The relative expanded uncertainty of individual determination of serum was about 1%. At the same time, an ID-LC/MS/MS method for serum free glycerol was developed, which had a bias of-0.11%with the HPLC method of free glycerol.The conditions of sample preparation, the selection of calibration solution, internal standard and quantitative method had been investigated in detail in the determination of serum TC and TTG. Various liquid chromatographic and mass spectrometric conditions were also tested and optimized for best chromatographic separation and mass spectrometric performances. With the established methods of TC and TTG, the results of determination of the certified reference materials were within the uncertainty of the certified values and the results were consistent with other participants and achieved the limits of equivalence in international reference methods comparison launched by IFCC. In conclusion, the ID-LC/MS or ID-LC/MS/MS methods for accurate measurement of serum TC, TTG and FG have been developed. The method for serum TC and TTG demonstrated good accuracy and precision, low susceptibility to interferences and performed with a short runtime and showed measurement uncertainties comparable to the current ID-MS methods. It maybe used as reference methods in future. At last, the reference measurement procedures of serum TC and TTG have been compiled. The established methods have been well applied into standard plans of TC and TTG in clinical laboratories.