Simultaneous Quantitative Analysis of Metabolites Using Ion-Pair Liquid Chromatography−Electrospray Ionization Mass Spectrometry

Abstract

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.

Structure and mechanism of a canonical poly(ADP-ribose) glycohydrolase

“…The poly(ADPribosyl)ated PARP1 was treated with PARG, and the mixture filtered using centricons (30 kDa cutoff). The analysis of the filtrate was performed using a modified procedure by Coulier et al 26 , which employed ion-pair chromatography for the separation of nucleotides and negative electrospray ionization for the mass spectrometric detection. All analyses were performed using a Waters Acquity UPLC system (Waters Corp., Milford, MA, USA) coupled to a QTOF Premier mass spectrometer (Waters Micromass, Manchester, UK).…”

Quantification of Glucuronide Metabolites in Biological Matrices by LC-MS/MS

“…A successful and robust application of hexylamine as an ion pairing reagent together with a careful control of mobile phase pH has been described for acidic metabolites retention and detection with LC-negative-ESI-MS. In this case, no interference was observed from hexylamine as it is a volatile reagent and ionizes only in the positive mode (Coulier, Bas et al 2006). With a similar ion pairing reagent, dimethylhexylamine, a switch from the negative to the positive ESI is possible, which increases the sensitivity and selectivity of the method (Pruvost, Théodoro et al 2008).…”

Optimization of the quenching method for metabolomics analysis of Lactobacillus bulgaricus

“…Recently, rapidly growing reports demonstrated that microbial metabolomics could provide more data and describe higher complex metabolite profiles (Oldiges et al, 2007;Wu et al, 2008;Tang et al, 2009). Typical detection methods in metabolome analysis include gas chromatography-mass spectrometry (GC-MS), liquid chromatography (LC)-MS, and nuclear magnetic resonance spectroscopy (NMR), which offer higher sensitivity and the ability to detect a larger number of low-molecular weight metabolites (van Dam et al, 2002;Coulier et al, 2006;Koek et al, 2006). Accordingly, further studies on metabolomics could contribute to a better understanding on the necessary role of L. bulgaricus in fermentation.…”