期刊
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
卷 1061, 期 -, 页码 89-96出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jchromb.2017.07.010
关键词
Endogenous compounds; ATP; 2,3-DPG; Quantitative; Blood; Biomarker
Many hemolytic anemias results in major metabolic abnormalities: two common metabolite abnormalities include increased levels of 2,3-diphosphoglycerate (2,3-DPG) and decreased levels of adenosine triphosphate (ATP). To better monitor the concentration changes of these metabolites, the development of a reliable LC-MS/MS method to quantitatively profile the concentrations of 2, 3-DPG and ATP in whole blood is essential to understand the effects of investigational therapeutics. Accurate quantification of both compounds imposes great challenges to bioanalytical scientists due to their polar, ionic and endogenous nature. Here we present an LC-MS/MS method for the reliable quantification of 2,3-DPG and ATP from K(2)EDTA human whole blood (WB) simultaneously. Whole blood samples were spiked with stable isotope labeled internal standards, processed by protein precipitation extraction, and analyzed using zwitterionic ion chromatography-hydrophilic interaction chromatography (ZIC-HILIC) coupled with tandem mass spectrometry. The linear analytical range of the assay was 50-3000 mu g/mL. The fit-for-purpose method demonstrated excellent accuracy and precision. The overall accuracy was within 10.5% (%RE) for both analytes and the intra- and inter-assay precision (%CV) were less than 6.7% and 6.2% for both analytes, respectively. ATP and 2,3-DPG were found to be stable in human K(2)EDTA blood for at least 8 h at 4 degrees C, 96 days when stored at -70 degrees C and after three freeze/thaw cycles. The assay has been successfully applied to K(2)EDTA human whole blood samples to support clinical studies.
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