期刊
JOURNAL OF CHROMATOGRAPHY A
卷 1172, 期 2, 页码 127-134出版社
ELSEVIER
DOI: 10.1016/j.chroma.2007.09.075
关键词
metabolomics; mass spectrometry; nano-LC
资金
- NIDDK NIH HHS [R37 DK046960, R37 DK046960-16, R37 DK 46960] Funding Source: Medline
Capillary liquid chromatography coupled with electrospray ionization to a quadrupole ion trap mass spectrometer was explored as a method for the analysis of polar anionic compounds in complex metabolome mixtures. A ternary mobile phase gradient, consisting of aqueous acidic, aqueous neutral and organic phases in combination with an aqueous compatible reversed-phase stationary phase allowed metabolites with a wide range of polarities to be resolved and detected. Detection limits in the full scan mode for glycolysis and tricarboxylic acid cycle intermediates were from 0.9 to 36 fmol. Using this system, 111 +/- 9 (n = 3) metabolites were detected in Escherichia coli lysate samples. Reducing column I.D. from 50 to 25 mu m increased the number of metabolites detected to 156 +/- 17 (n = 3). The improvement in number of metabolites detected was attributed to an increase in separation efficiency, an increase in sensitivity, and a decrease in adduct formation. Implementation of a second separation mode, strong anion exchange, to fractionate the sample prior to capillary RPLC increased the number of metabolites detected to 244 21 (n = 3). This improvement was attributed to the increased peak capacity which decreased co-elution of molecules enabling more sensitive detection by mass spectrometry. This system was also applied to islets of Langerhans where more significant improvements in metabolite detection were observed. In islets, 391 +/- 33 small molecules were detected using the two-dimensional separation. The results demonstrate that column miniaturization and use of two-dimensional separations can yield a significant improvement in the coverage of the metabolome. (c) 2007 Elsevier B.V. All rights reserved.
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