Comprehensive Analysis of Human Plasma Using Gas Chromatography–High Resolution Time-of-Flight Mass Spectrometry: A Workflow to Leverage Electron and Chemical Ionization - - Spectroscopy
 Home   Mass Spectrometry   ICP-MS   Infrared   FT-IR   UV-Vis   Raman   NMR   X-Ray   Fluorescence  
Home
Magazine
Issue Archive
Subscribe/Renew
Special Issues
Reprints
The Application Notebook
Current Issue
Archive
Submission Guidelines
Training
SpecAcademy
E-solutions
Digital Edition
Subscribe to the Digital Edition
The Wavelength
Subcribe to The Wavelength
Subscribe to the MS E-news
Resources
Market Profiles
Information for Authors
SpecTube
Webcasts
Advertiser services
Contact Us
Columns
Atomic Perspectives
Chemometrics in Spectroscopy
Focus on Quality
Laser and Optics Interface
Mass Spectrometry Forum
The Baseline
Molecular Spectroscopy Workbench

Comprehensive Analysis of Human Plasma Using Gas Chromatography–High Resolution Time-of-Flight Mass Spectrometry: A Workflow to Leverage Electron and Chemical Ionization


Special Issues
Volume 28, Issue 10, pp. 34-37

Gas chromatography with electron ionization and mass spectrometry (GC–EI-MS) detection is a workhorse among analytical techniques in metabolomics. A major challenge in the utilization of GC–EI-MS in metabolomics is the identification of unknowns. The recent availability of high resolution, accurate mass, time-of-flight mass spectrometry (TOF-MS) systems provides great potential for identifying unknowns by reducing uncertainty in possible formulas and enhancing detection continuity between sample sets. A workflow using GC–EI-MS spectra for library identification, with molecular formula information for unknowns provided by chemical ionization-mass spectrometry (CI-MS) and accurate mass analysis was used for the analysis of blood plasma.

Metabolomics provides a foundation for quantitative, comparative biology and is indispensable for the comprehensive characterization of molecules and differential analysis of populations. It entails instrumental detection, characterization, and quantification of small molecules (molecular weight < 1500 Da) produced, or transformed in the cells of living organisms (1,2). Although no single instrument is capable of fully profiling the metabolome, the unique capabilities of time-of-flight mass spectrometry (TOF-MS) make it an ideal tool for metabolomic studies. Gas chromatography–time-of-fight-mass spectrometry (GC–TOF-MS) is a powerful approach for unbiased metabolic profiling and quantitation of a diverse array of metabolites (3,4). High-resolution TOF-MS provides additional benefits such as mass accuracies below 1 ppm for robust formula determinations and resolving power in excess of 50,000 for resolution of isobars and minimization of background interferences. A workflow that combines accurate mass electron ionization (EI) and chemical ionization (CI) high-resolution TOF-MS for confident characterization of different compound classes was used for the analysis of human blood plasma. It includes recommendations for sample preparation, analytical conditions, and data analysis.

General Workflow


Figure 1: (a) Extracted ion chromatogram showing one major product for the two-step derivatization of glucose. (b) Products for the one-step MSTFA derivatization of glucose.
The majority of metabolites in blood plasma are labile and exhibit low volatility making their analysis by GC difficult. This is addressed by using a two step derivatization procedure involving the treatment of samples with O-methylhydroxylamine hydrochloride in pyridine (MEOX) followed by silylation with N-methyl-N-trifluoroacetamide/1% trimethylchlorosilane (MSTFA) (5). This two-step procedure minimizes the number of possible forms of reducing sugars as shown for glucose in Figure 1. Treatment of glucose with MSTFA (Figure 1b) results in four different forms (α- and β-furanose and pyranose trimethylsilyl derivatives), while the two-step procedure typically produces two geometric isomers of the open chain structure of the monosaccharide (Figure 1a). Note that one isomer is produced preferentially in this two-step derivatization of glucose. This not only simplifies the analysis of blood samples, but also increases the likelihood of compound detection.


Figure 2: General workflow.
GC–MS compound characterization in metabolomics relies primarily on retention time and spectral similarity. A higher confidence for feature identification can be achieved through the addition of accurate mass formula determinations for molecular ions, quasimolecular ions, and fragments. Key to the success of analyte identification was a workflow that included both EI and CI high-resolution TOF-MS data acquisition (Figure 2) (6). CI high-resolution TOF-MS data were critical in characterizing compounds that produced poor spectral matches in relation to commercially available libraries or did not exhibit molecular ions in their EI high-resolution TOF-MS mass spectra.


Rate This Article
Your original vote has been tallied and is included in the ratings results.
View our top pages
Average rating for this page is: 10
Headlines from LCGC North America and Chromatography Online
Thermo Fisher Scientific NA - Major Improvement in Throughput and Resolution of Xanthones in Mangosteen Pericarp
Thermo Fisher Scientific EU - Major Improvement in Throughput and Resolution of Xanthones in Mangosteen Pericarp
Postnova Analytics Expands Into Asia-Pacific Regions
Pharma Focus: Developing bioanalytical methods for pharma and clinical research
Detecting Pharmaceuticals in Irrigated Crops
Source: Special Issues,
Click here