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Objective:To determine the analytical performance of a candidate reference measurement procedure for 17α-hydroxyprogesterone in human serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS).Methods:The serum spiked with a deuterium-labeled internal standard was extracted from serum from individual undergoing physical examination by liquid-liquid extraction with n-hexane/ethyl acetate (3∶2, v/v), separated by C18 reversed-phase chromatography and detected by positive electrospray ionization mass spectrometry. According to the Clinical and Laboratory Standards Institute (CLSI) C62-A documents, the analytical performance including linearity, limit of detection,limit of quantitation,relative matrix effect,precision and trueness,carry-over and specificity was evaluated.Results:The linear range of 17α-hydroxyprogesterone by LC-MS/MS was 0.21-119.67 μg/L. The limit of detection and limit of quantitation were 5.218 ng/L and 0.116 μg/L. The relative matrix effects were -0.02%, -0.40% and -0.90% for sera and solution mixtures in 3 different ratios (50∶50, 80:20 and 20∶80). The coefficients of variation ( CVs) of intra-assay were 1.73%-2.11%, 0.98%-1.71%, 0.47%-0.87% at 0.164 μg/L, 14.81 μg/L, 81.63 μg/L and the CVs of inter-assay were 1.82%, 1.03%, 0.80% at above three concentrations. The average recovery rates of 3 levels (0.5, 20 and 100 μg/L) were 100.4%, 101.7%, 102.8%, respectively. The measured values of GBW09829 of National Institute of Metrology were within the specified uncertainty range. Conclusion:The candidate reference measurement procedure for 17α-hydroxyprogesterone in human by LC-MS/MS is established with good accuracy and precision, which can be clinically used for measurement traceability.
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Objective To establish an isotope dilution liquid chromatography tandem mass spectrometry method (ID-LC-MS) for quantification of serum apolipoprotein E and phenotyping. Methods Method establishment. Samples underwent denaturing, alkylation and trypsin digestion with addition of internal standards as isotope labelling arginine. SB-C18 column was used for the liquid chromatographic separation and mass spectrometry positive ion mode and multiple reaction monitoring were employed for quantification and phenotyping. Precision, accuracy and linearity were investigated for method evaluation. 40 serum samples from Shanghai Dongfang Hospital during Oct. to Dec., 2018 were used for method comparison between ID-LC-MS and immunoassay. Deming regression and Bland-Altman were used for method comparison analysis and SPSS 24 for linearity. Results Target peptides reached their releasing maximum within 4 hours and SE did at 3 hours. 3 phenotyping of ApoE were observed, such as E3/E3, E2/E3 and E3 / E4. The imprecision of IQC was 5.2 ％ . The relative bias for low and high levels of accuracy-based samples was 7.6 ％ and 3.6 ％, respectively. Deming regression showed the intercept with 95 ％ confidence interval (CI) was 6.44-11.44 (P<0.05 and the 95％ confidence interval for the slopewas 0.77-0.89 (P<0.05). The coefficient was r=0.97. The mean difference was - 2.95 mg / L with 95 ％ CI-4.26--1.65 mg/L. The linearity covered from 16.9 to 58.5 mg/L. Conclusion ID-LC-MS can be used to quantify serum apolipoprotein E and simultaneously detect its phenotyping.
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Objective@#To establish a reference measurement procedure for the determination of human serum homocysteine by isotope dilution high performance liquid chromatography-tandem mass spectrometry (ID-LC/MS/MS), and to apply it to establish sample target values for external quality assessment (EQA) in clinical laboratories.@*Methods@#The reference method of Hcyquantification in our laboratory was establishedaccording to the method recommended by the Joint Committee for Traceability in Laboratory Medicine (JCTLM). The precision, trueness, specificity, residue and matrix effect of the method were evaluated. The reference method was applied to establish Hcy target values for samples of the second EQA in Shanghai of 2018.@*Results@#The method detects 12.5μmol/L and 37.4μmol/L samples in three batches in three days, and the CV between batches is 1.03% and 2.10%, respectively. The measured values of Standard reference material (SRM) 1955 of National Institute of Standards and Technology (NIST) were within the specified uncertainty range. No matrix effect and carryover were observed. The second EQA data in 2018 showed that the average value of domestic reagent group was lower than that of reference method, and that of imported reagent group was higher than that of reference method.@*Conclusion@#Thereference measurement procedure of ID-LC/MS/MS was successfully established to determine the human serum homocysteine. It is expected to play a role in tracing the quantities of Hcy in clinical laboratories.
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Objective To establish a reference measurement procedure for the determination of human serum homocysteine by isotope dilution high performance liquid chromatography-tandem mass spectrometry (ID-LC/MS/MS), and to apply it to establish sample target values for external quality assessment (EQA) in clinical laboratories. Methods The reference method of Hcyquantification in our laboratory was establishedaccording to the method recommended by the Joint Committee for Traceability in Laboratory Medicine (JCTLM). The precision, trueness, specificity, residue and matrix effect of the method were evaluated. The reference method was applied to establish Hcy target values for samples of the second EQA in Shanghai of 2018. Results The method detects 12.5μmol/L and 37.4μmol/L samples in three batches in three days, and the CV between batches is 1.03％and 2.10％, respectively. The measured values of Standard reference material (SRM) 1955 of National Institute of Standards and Technology (NIST) were within the specified uncertainty range. No matrix effect and carryover were observed. The second EQA data in 2018 showed that the average value of domestic reagent group was lower than that of reference method, and that of imported reagent group was higher than that of reference method. Conclusion Thereference measurement procedure of ID-LC/MS/MS was successfully established to determine the human serum homocysteine. It is expected to play a role in tracing the quantities of Hcy in clinical laboratories.
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Objective To investigate the digestion kinetics of Apolipoprotein A-I and B by ID-LC-MS method for accurate quantification of proteins .Methods Methodological research .The target peptides of ApoA-I and B were determined .The ApoA-I and B from 5 human serum samples on market with levels from 0.90-2.54 g/L and 0.54-1.39 g/L separately , were measured in terms of target peptides by isotope dilution liquid chromatography mass spectrometry method .The releasing amount and rate of peptides were analyzed and plotted according to different time points .The correlation coefficient R2 was calculated among peptide releasing amount between samples .Results Most peptides reached their peaks within 4 hours.The peptides VQ , DY and VS from Apo A-I, TR and FP from Apo B were released relatively slowly .After getting to their peak stage , the ratio between TEV and SIL-TEV, AK and SIL-AK, VQ and SIL-VQ presented stable state.As for Apo A-I the correlations among peptides are high , from 0.904 to 0.999.Some peptides from Apo B show lower correlations , such as TG-SV with R20.543 (3 h).Conclusions Peptides from Apo A-I and Apo B present different releasing properties after trypsin digestion .Proper selection of representative peptides and enzymatic conditions can benefit accurate quantification of target proteins .
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The establishment of laboratory medicine reference system is an important way for routine detection results to achieve traceability.The complete reference system consists of reference measurement procedures,reference materials and reference laboratories.The reference measurement procedure is usually characterized by a thorough study with a small measurement uncertainty.Based on the advantages of high specificity and high sensitivity in quantitative analysis, mass spectrometry plays an important role in the research of the reference measurement procedures in laboratory medicine.In the past 40 years, mass spectrometry technology has been more and more widely applied in medicine, electrolytes, metabolites and substrates, non-electrolyte metals, non-peptide hormones, protein and vitamin.It provides a powerful guarantee for the precise detection of these well-defined measureds.