Derivatization Methods for Improved Metabolome Analysis by LC-MS/MS

[摘要] Metabolomics, the study and characterization of small molecule metabolites, faces challenges due to the size and diversity of the metabolome.No current instrumentation is capable of measuring the entire metabolome.Liquid chromatography with mass spectrometry detection is widely used in metabolomics for its sensitivity and selectivity.Detection is limited to metabolites which are compatible with the selected column chemistry.Many small molecule neurochemicals are polar and thus not well retained with the popular reversed phase chromatography.Hydrophilic interaction liquid chromatography is better suited for this application, but the observed limits of detection are not sufficient for trace neurochemicals.One way to overcome this limitation is through the use of chemical derivatization.Polar metabolites are labeled with a hydrophobic moiety, increasing retention and ionization efficiency in reversed phase chromatography, allowing for lower limits of detection.Additionally, internal standards are easily produced using stable isotope labeled derivatization reagents, allowing for improved quantification.Previous work in the Kennedy lab has been performed with benzoyl chloride, which reacts primarily with amines and phenols.This reaction is virtually instantaneous at room temperature and increases sensitivity by up to 1,000 fold.Benzoyl chloride derivatization was previously described for analysis of 17 neurochemicals in microdialysate with reversed phase liquid chromatography and triple quadrupole mass spectrometry.In this work, benzoyl chloride derivatization is expanded upon in both metabolome coverage and applicability.To allow benzoyl chloride derivatization of proteinaceous samples such as plasma or tissue, a sample preparation method was developed and optimized.The resulting solvent precipitation method is fast, simple, and compatible with very small sample volumes.This was demonstrated in two brief applications monitoring epinephrine in mouse plasma and serotonin in Drosophila tissue.With sample preparation optimized, we then aimed to expand metabolome coverage and subsequently developed a method for the analysis of 70 benzoylated neurochemicals.With nanomolar limits of detection and internal standards for each metabolite, this method has many advantages over other methods of comparable scale.We make use of this expanded coverage by investigating plasma metabolic changes in Parkinson;;s disease as well as wine analysis.Thirteen metabolites were found to be linked to Parkinson;;s disease or disease severity, though current evidence suggests L-DOPA treatment plays a major role in these findings.Using a 56-metabolite panel, we were able to distinguish wines based on their varietal and area of production.Finally, we move away from benzoyl chloride and introduce a novel derivatization reagent, benzylamine.Benzylamine derivatization is complementary to benzoyl chloride, reacting with carboxylic acids, carbonyls, and phosphates.Moreover, the two reagents are compatible so derivatized samples can be mixed for analysis within a single run.Two short methods are introduced, using benzylamine as a standalone reagent to monitor energy metabolites, or in conjunction with benzoyl chloride to monitor phenylalanine metabolism.Together, the two reagents have the potential to cover the majority of the metabolome.

[发布机构] University of Michigan

[效力级别] derivatization

[关键词] metabolomics;derivatization;liquid chromatography;mass spectrometry;LC-MS;Chemistry;Science;Chemistry