Gas chromatographytandem mass spectrometry using chemical ionization for analysis of blood metabolites

Abstract

Introduction: Gas chromatography-mass spectrometry (GC-MS) is a powerful tool when
used in metabolomics, the study of small molecules in the body, and investigating new modes
of using it might lead to even better tools for diagnostics and research.

Background: Chemical ionization (CI) for GC-tandem MS (GC-MS/MS) is relatively rarely
used compared to the more commonplace electron ionization (EI). By developing such a
method and conducting a comparison with an established and optimized electron ionization
method a deeper understanding of chemical ionization and its role in metabolomics analyses
can be gained.

Aims: This study aims to develop an analytical method using GC-CI-MS/MS followed by a
partial validation and comparison with a an established and optimized EI method.
Methods: Using previously developed sample preparation protocols and GC methods the
mass spectrometric method was developed by identification or multiple reaction monitoring
transitions for a number of metabolites followed by optimization of ion source conditions
using design of experiment and a partial validation. The same partial validation was
performed for the same samples using EI and the results from the two ionization modes were
compared.

Results: A list of MRM transitions for 54 metabolites was generated, ion source optimization
for chemical ionization generally favored the highest available pressure of methane gas in ion
source and a low ionization voltage of 42 V with a temperature close to the center point at 200
°C. CI performed relatively poorly compared to EI with regards to limits of detection and
quantification as well as sensitivity and precision. Recovery lay within 80-120 % for a
slightly higher number of analytes when using CI than when using EI.

Conclusion: CI tends to perform less well than EI. However, there are aspects, such as
recovery, where it seemingly performs similar or better.

Popular Abstract

In the body of any living thing, a massive number of small biological molecules, metabolites,
are constantly formed and consumed in order to generate energy or create the building blocks
of all life. In humans, measuring these metabolites, together called the metabolome, can yield
valuable information when trying to understand or diagnose certain diseases. However, the
large number of molecules with vastly different character in e.g. human blood makes analysis
difficult as it is very hard to measure only one thing and not many at the same time.

One popular method is gas chromatography-mass spectrometry. In gas chromatography, a
sample containing many different molecules in injected into an instrument before the
temperature is gradually increased, causing these molecules to travel through a column and
exit the instrument at different times, which correlate strongly with boiling point, limiting the
number of molecules measured at the same time by separating them over several minutes.
The molecules are then measured in a mass spectrometer, an instrument which can determine
the mass of an molecules. The chromatographic separation of molecules in time might not be
enough to avoid measuring many different components in a sample that is as complex as
blood. For even higher selectivity, the mass spectrometer can be used as a filter, only
permitting molecules of one certain mass to be detected. If this is still not selective enough,
tandem mass spectrometry can be used. This technique is based on the first filtering out
molecules of one single mass, colliding them with gas molecules causing them to fragment
and again using a mass filter to only measure fragments with one specific mass. This means
that to distinguish two molecules they only need to be different in regards to one of three
parameters, time spent in the chromatographic system, original mass, and resulting fragment
mass, but it comes at a cost of having to identify fragments and energies used to fragment the
ions for each analyte. The first part of this work describes generating such a transition.

As mentioned above, the mass spectrometer can identify and separate molecules based on
mass, but this requires making every molecule charged. The most common way to do this for
gas chromatography is by electron ionization which itself causes the molecules to fragment at
the very beginning of mass spectrometry. Another, promising method is chemical ionization.
The bulk of this work describes an investigation of how to best use chemical ionization and is
followed by a comparison between electron ionization and chemical ionization.

• author: Palmer, Simon
• supervisor: Peter Spégel
• organization: Department of Chemistry
• course: KEMR30 20221
• year: 2022
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: Chemical ionization, gas chromatography, mass spectrometry, multiple reaction monitoring, analytical chemistry
• language: English
• id: 9095280
• date added to LUP: 2022-08-02 09:56:31
• date last changed: 2022-08-02 09:56:31