Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
7612375 | Journal of Chromatography A | 2014 | 10 Pages |
Abstract
Metabolic profiling has become an important tool in biological research, and the chromatographic separation of metabolites coupled with mass spectrometric detection is the most frequently used approach for such studies. The establishment of robust chromatographic methods for comprehensive coverage of the anionic metabolite pool is especially challenging. In this study, the development of a capillary ion exchange chromatography (capIC) - negative ESI tandem mass spectrometry (MS/MS) workflow for the quantitative profiling of the phosphometabolome (e.g., sugar phosphates and nucleotides) is presented. The chromatographic separation and MS/MS conditions were optimized, and the precision of repetitive injections and accuracy in terms of error percentage to true concentration were assessed. The precision is excellent for a capillary flow system with an average CV% of 8.5% for a 50-fmol standard injection and in the lower 2.4-4.4% range for higher concentrations (500-7500Â fmol). The limit of detection (LOD) ranges from 1 to 100Â nM (5-500Â fmol injected on column), and the limit of quantitation (LOQ) ranges from 1 to 500Â nM (5-2500Â fmol injected on column). A fast gradient method with the injection of 50% methanol in water between analytical samples is needed to eliminate carry-over and ensure optimal re-equilibration of the column. Finally, the quantitative applicability of the system was tested on real biological matrices using the constant-volume standard addition method (SAM). Extracts of the human kidney Hek293 cell line were spiked with increasing concentrations of standards to determine the concentration of each metabolite in the sample. Forty-four metabolites were detected with an average uncertainty of 4.1%. Thus, the capIC-MS/MS method exhibits excellent selectivity, sensitivity and precision for the quantitative profiling of the phosphometabolome.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Hans F.N. Kvitvang, KÃ¥re A. Kristiansen, Per Bruheim,