Analysis of blood methylmalonic acid with a liquid chromatography-tandem mass spectrometry method and its application / 中华检验医学杂志

ABSTRACT

Objective To establish a LC-MS/MS method for the determination of MMA in serum,and provide a assay for the diagnosis and screening of methylmalonic academia in the clinic. Methods MMA was extracted from 205 serum samples from healthy controls and 146 serum samples from patients with liquidliquid extraction method with MTBE as the extraction solvent. The supernatant was transferred to a tube and dried with nitrogen gas. Then the residual was derived with HCI-BuOH mixed agent to give a product, which was analyzed directly by LC-MS-MS system with a gradient elution, selective reaction monitor, a Discovery C18 column (50 mm × 2. 1 mm ,5 μm) as the isolation column and a mobile phase consisting of methanol and water (0. 1% formic acid, V/V), respectively. The concentration of MMA was detected with the isotope internal standard method. The stand curve was employed with a series of calibrators. The recovery was estimated with the 3 serum samples with the concentrations of 2, 25, 80 μg/L respectively. The accuracy,precision and stability were also tested with quality control samples. Moreover, the range of concentrations in healthy people were detected to investigate the influence of hemolysis on the detection results. Thirteen samples were randomly tested according to the digital chart. The testing results were compared with the results provided by Medical Diagnositic institution (MDI) in Germany. The paired t-test was applied to statistical analysis. Results The linear range of this method was 2-100 μg/L, and the correlation coefficient (R2 ) was more than 0. 995. The retention time of MMA derivative was 10. 5 min. Succinic acid and MMA were not found to interfere with each other. The within-run RSD was less than 6. 4%, and the between-run RSD was less than 5.0%. The recovery rates were from 96. 42% to 103. 33%. The limit of quantification was 1 μg/L.The accuracies of the method were form 94. 2% to 108. 2%. The samples were stable for 6 h at room temperature and stable for 70 d even keep at - 20 ℃. The samples were stable after 10 freeze-thaw cycles. The derivatives of MMA were found to be stable at least for 5 d at 4 ℃. The medians of the hemolysis group and the normal group were 102.53 (13.84-302.33) μg/L and 39.52 (11.94-203.08) μg/L,respectively. There was significant difference between the 2 groups ( T = 8, P ＜ 0. 05 ). The medians of comparison test in our laboratory and the MDI were 32. 82(24. 50-100. 42) μg/L and 32. 20(26. 65-93. 30)μg/L There was no significant difference between the 2 groups( T=7 ,P ＞0. 05 ). The mean value (-x± s)of 158 healthy adults( 18-58 years old) and 47 healthy teenages( 1-17 years old)were ( 18.46 ± 10.49 )μg/L and (22. 38 ± 11.45) μg/L, respectively. Conclusions A LC-MS/MS method for analysis of MMA in serum is established successfully. The quantitative method is simple and accurate with good sensitivity,specificity and repeatability. The method can be applied for diagnosis, screening and monitoring of methylmalonic acidemia.