High precision measurement and fragmentation analysis for metabolite identification.

The degree of precision in measuring accurate masses in LC MS/MS-based metabolomics experiments is a determinant in the successful identification of the metabolites present in the original extract. Using the methods described here, complex broccoli extracts containing hundreds of small-molecule compounds (mass range 100-1,400 Da) can be profiled at resolutions up to 100,000 (full width half maximum, FWHM), useful for accurate and sensitive relative quantification experiments. Using external instrument calibration, analyte masses can be measured with high (sub-ppm to a maximum of 2 ppm) accuracy, leading to compound identifications based on elemental composition analysis. Unambiguous identification of four analytes (citric acid, chlorogenic acid, phenylalanine, and UDP-D: -glucose) is used to validate the performance of the different MS/MS fragmentation regimes. Identifications are carried out either via resonance excitation collision induced dissociation (CID) or via higher energy collision dissociation (HCD) experiments, and validated by infrared multiphoton dissociation (IRMPD) fragmentation of standards. Such results, obtained on both hybrid and non-hybrid systems from metabolite profiling and identification experiments, provide evidence that the strategies selected can be successfully applied to other LC-MS based projects for plant metabolomic studies.

Quantitative-qualitative data acquisition using a benchtop Orbitrap mass spectrometer.

Current approaches to discovery-stage drug metabolism studies (pharmacokinetics, microsomal stability, etc.) typically use triple-quadrupole-based approaches for quantitative analysis. This necessitates the optimization of parameters such as Q1 and Q3 m/z values, collision energy, and interface voltages. These studies detect only the specified compound and information about other components, such as metabolites, is lost. The ability to perform full-scan acquisition for quantitative analysis would eliminate the need for compound optimization while enabling the detection of metabolites and other non-drug-related endogenous components. Such an instrument would have to provide sensitivity, selectivity, dynamic range, and scan speed suitable for discovery-stage quantitative studies. In this study, a prototype benchtop Orbitrap-based mass analyzer was used to collect both quantitative and qualitative data from human microsomal incubation samples as well as rat plasma from pharmacokinetic studies. Instrumental parameters such as scan speed, resolution, and mass accuracy are discussed in relation to the requirements for a quantitative-qualitative workflow. The ability to perform highly selective quantitative analysis while simultaneously characterizing metabolites from both in vitro and in vivo studies is discussed.