Simultaneous quantitative analysis of metabolites using ion-pair liquid chromatography-electrospray ionization mass spectrometry.
We have developed an analytical method, consisting of ion-pair liquid chromatography coupled to electrospray ionization mass spectrometry (IP-LC-ESI-MS), for the simultaneous quantitative analysis of several key classes of polar metabolites, like nucleotides, coenzyme A esters, sugar nucleotides, and sugar bisphosphates. The use of the ion-pair agent hexylamine and optimization of the pH of the mobile phases were critical parameters in obtaining good retention and peak shapes of many of the above-mentioned polar and acidic metabolites that are impossible to analyze using standard reversed-phase LC/MS. Optimum conditions were found when using a gradient from 5 mM hexylamine in water (pH 6.3) to 90% methanol/10% 10 mM ammonium acetate (pH 8.5). The IP-LC-ESI-MS method was extensively validated by determining the linearity (R2 > 0.995), sensitivity (limit of detection 0.1-1 ng), repeatability, and reproducibility (relative standard deviation <10%). The IP-LC-ESI-MS method was shown to be a useful tool for microbial metabolomics, i.e., the comprehensive quantitative analysis of metabolites in extracts of microorganisms, and for the determination of the energy charge, i.e., the cellular energy status, as an overall quality measure for the sample workup and analytical protocols.
Coulier
Leon
L
Analytical Research Department, TNO Quality of Life, Utrechtseweg 48, 3700 AJ, Zeist, The Netherlands. leon.coulier@tno.nl
Bas
Richard
R
Jespersen
Sonja
S
Verheij
Elwin
E
van der Werf
Mariët J
MJ
Hankemeier
Thomas
T
D000588:Amines / Q000737:chemistry; D001412:Bacillus subtilis / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D003065:Coenzyme A / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D004952:Esters / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009711:Nucleotides / Q000378:metabolism*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013403:Sugar Phosphates / Q000378:metabolism*
D000588:Amines; D004952:Esters; D009711:Nucleotides; D013403:Sugar Phosphates; C007940:hexylamine; D003065:Coenzyme A