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Unlike GC-MS, most molecules do not require derivatization to be detected by LC-MS, allowing for faster sample preparation and more stable extractions. Another key

difference is that the LC and MS segments are manufactured independently in LC-MS, and therefore mixing segments from different vendor is common. The run times for LC- MS can also be quite short in comparison to GC-MS (5 min vs 60 min respectively for methods used in this thesis). This is due to in part to advancements in HPLC/UHPLC. By

combining small particle (< 2µm) packing material with high pressure (> 200 bar),

superior resolution can be achieved in a short amount of time73. HPLC columns are

coated with a packing material or stationary phase, the most widely used being octadecyl, also known as “C18”. C18 is a non-polar stationary phase, and is best suited to detect non-polar compounds, although detection of polar compounds is also possible. This is conventionally referred to as “reverse-phase” chromatography. In reverse phase, a polar mobile phase such as water is applied, followed by an organic phase, for example acetonitrile. In contrast, polar stationary phases such as HILIC (hydrophilic interaction liquid chromatography) are referred to as normal phase, but are less commonly used. Once molecules have been separated by HPLC, they enter the source where they are ionized by electrospray ionization (ESI). Ions are formed by applying high voltage to

liquid passing through a capillary tube69. The resulting charged droplet will eventually

break, creating the ‘electrospray’. By changing the voltage of the applied charge, positively or negatively charged ions are created. In contrast to EI, ESI is a known as a ‘soft’ ionization technique because it does not usually induce fragmentation of the molecular ion.

The most common form of ionization are protonation [M+H] and deprotonation [M-H], however chemicals may ionize in a number of different ways, sometimes in an

unpredictable manor. For positive ESI, the most common form of alternative ions are salt adducts such as [M+Na], [M+NH4] or [M+K]. For negative ESI, acids present in the mobile phase, such as formic acid, form the most common adducts ([M-HCOO]), as well as [M-Cl]. Additionally, molecules can be multiply charged if they possess multiple ionizable sites, such is the case with most peptides. In this case the observed ion will be

smaller than the parent, as only the mass-to-charge ratio (m/z) of an ion can be measured

by the mass spectrometer.

Once the electrospray has been created, ions enter the mass spectrometer. A schematic of the Thermo Q-Exactive Orbitrap mass spectrometer, the model used throughout this thesis, is shown in Figure 1-6. Briefly, ions are guided into the quadrupole by a bent

flatapole. The quadrupole then selects the range of m/z to be analyzed in the same fashion

as was described for GC-MS. After mass selection, ions enter the C-trap which guides the ions into the orbitrap for mass detection. The orbitrap is a relatively new form of mass

analyzer, having first been described in the year 2000 by Alexander Makarov74. It is

composed of a barrel-shaped outer electrode and an inner spindle-shaped electrode. The

ions oscillate around this spindle at a frequency that is proportional to their m/z, enabling

mass detection with high accuracy. The orbitrap’s superior resolution and mass accuracy make it an ideal choice for untargeted metabolomics.

The Q Exactive is capable of performing both ‘full MS’ and/or ‘MS/MS’ analyses, the choice of which is pre-set by the user. In full MS analysis, molecules are ionized and detected intact without fragmentation. This mode maximizes the sensitivity of the instrument and is therefore most useful for relative quantification of ions, but gives little information about the structure of compounds. Alternatively, MS/MS, also known as tandem mass spectrometry, provides information about the structure of the molecule at the expense of sensitivity. For MS/MS, ions selected by the C-Trap are sent into a collision cell (HCD cell) where they collide with neutral molecules of nitrogen gas,

inducing fragmentation of the molecular ion69. These fragments can then be used the piece together the structure of the parent molecule.

Figure 1-6. Thermo Q-Exactive Orbitrap configuration75.

1.4.2.2

Software

As with GC-MS, a number of vendor-derived and open source software have been developed to view and analyze LC-MS metabolomics data. To view raw chromatograms and spectra, a vendor provided software is most commonly used. The Thermo Scientific software for LC-MS spectral viewing is called Xcaliber. This program allows one to view all ions in a given mass spectrum, as well as MS/MS of metabolites of interest. It is also useful for calculating molecular formulas, as well as adduct and neutral loss

determination. Xcaliber is a low throughput program and is best suited to viewing a single or at most a handful of samples at a time. For this reason, other programs have been created to align and integrate multiple LC-MS files into a feature list which can then be used for multivariate modelling and statistical analysis. One of the most widely used

tools for LC-MS feature alignment is XCMS76,77. XCMS is an open source software, and

is compatible with a wide range of LC-MS platforms. One of the main advantages of XCMS over other software is that it is available as both an online point and click

interface as well as a command line R package. The R version allows for rapid processing of a large number of samples and is highly adaptable compared to the online version.

XCMS also excels at identifying signal from noise in comparison to other available programs.

1.4.2.3

Compound identification

Compound identification remains the most challenging and time consuming aspect of untargeted LC-MS analysis. Even if a molecular formula can be assigned based on the

m/z alone, there can be hundreds if not thousands of molecular species with the same

formula. For this reason, the mass alone provides little information about the structure of the molecule. To confidently identify a metabolite, an MS/MS spectra must be acquired. This spectra can then be searched against an MS/MS databases, the largest being

METLIN61. Although these databases can be extremely useful, they are far from

complete, and an MS/MS spectra for the vast majority of known molecules cannot be

found in any database. In these cases, molecules must be identified ‘de novo’ from the

fragments present in the mass spectra. To help address this issue, fragmentation

prediction tools such as CFM-ID78 have been created. These programs use machine

learning algorithms to predict the MS/MS spectra of a known compound, or alternatively to predict the compound identity from an experimental MS/MS spectra. In many cases, neither of these approaches yield plausible identities and more advanced methods such as neutral loss and fragment matching must be used to piece together the putative structure. Ultimately, an MS/MS spectra of an authentic standard must be run to confirm the predicted identity, only then can one be confident in the annotation.