Comparison and bias estimation of isotope dilution mass spectrometry and HPLC for cholesterol: methodological comparison of two internationally accepted reference methods listed in JCTLM

Abstract

Objective To evaluate the consistency and comparability of two kinds of cholesterol reference methods listed in the Joint Committee for Traceability in Laboratory Medicine (JCTLM). Methods 52 fresh frozen sera with cholesterol concentrations among 3-10 mmol/L were tested by isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) and high performance liquid chromatography (HPLC). The precision and accuracy of the two methods were calculated, and then a series of analysis were conducted including plotting scatter plots and deviation graphs, testing outliers, selecting the best fitting regression models and calculating the regression equations and parameters, and so on. The results of the two methods were statistically analyzed to estimate the expected deviation at the level of medical decision of cholesterol and the 95% confidence range. Results For HPLC method, the CV of instrument measurement was 0.22% (0-0.49%), the total CV of samples measurement was 0.36% (0.02%-0.83%), and the average bias of the reference materials was 0.37% (-1.31%-0.14%). For ID-LC/MS/MS method, the CV of instrument measurement was 0.50% (0-1.51%), the total CV of samples measurement was 0.55% (0.03%-1.17%), and the average bias of the reference materials was 0.24% (-0.53%-0.14%). No outliers were found from the scatter plots and the statistical analysis and the linear regression were fitted to analyze the results of the two methods. The linear regression parameters of two methods for 52 fresh frozen human sera were as follows: the slope was 0.989 5, the standard error of slope was 0.003 4, the intercept was 0.063 4 mmol/L, the standard error of intercept was 0.019 9 mmol/L, the standard error of Y-estimate was 0.034 8 mmol/L, and the correlation coefficient was 0.999 7. Compared with the ID-LC/MS/MS method, the absolute deviation of fresh sera by HPLC method was 0.000 mmol/L (-0.083-0.076 mmol/L), with a relative deviation of 0.07% (-1.22-1.24%). T-test results showed no statistically significant difference between the two methods. The expected deviations at the cholesterol medicine decision level were within the range 95% confidence range, and the expected deviations were far less than the allowable error. Conclusions The HPLC method of cholesterol has good consistency and comparability with ID/MS method using the primary measurement principle. Because of more advantages of HPLC method such as less cost, more simple, requirement, and better precision, HPLC method is expected to play an important role in the process of standardization and traceability of serum cholesterol.(Chin J Lab Med, 2017, 40: 780-786) Key words: Cholesterol; Indicator dilution techniques; Chromatography, liquid; Tandem Mass spectrometry; Chromatography, high pressure liquid; Isotopes