Search Research Equipment

Description

Introduction to UHPLC Tribrid Orbitrap MS
Ultra-high performance liquid chromatography (UHPLC) coupled to mass spectrometry (MS) is a powerful technique to analyse and identify many different compounds like phytochemicals, lipids, peptides, and carbohydrates as present in complex mixtures. The UHPLC system is used for initial separation of analytes and is equipped with photodiode array (PDA) detector to assist with identification and quantification. Prior to entry in the MS, analytes need to be ionised which is most commonly done via electrospray ionization (ESI). However, ESI is not suitable for all types of analytes, which is why the IQ-X is also able to utilize another ionisation technique, namely atmospheric pressure chemical ionisation (APCI). After entry in the MS, the mass-to-charge ratio (m/z) of analytes is determined by a mass analyser. In the case of the IQ-X, three mass analysers are present: a quadrupole for mass filtering, a linear ion trap for fast and sensitive m/z determination (nominal mass, i.e. 0 decimals), and a high-field Orbitrap for high resolution mass analysis (accurate mass, i.e. 5 decimals). The IQ-X is equipped with a sensitive high-field Orbitrap that can achieve resolutions up to 500,000 FWHM (mass accuracy < 1 ppm). At this high resolution, the molecular formulae of analytes can be confidently determined based on the measured m/z.  Besides m/z determination of the intact parent ions generated by ionisation, analyte ions can be fragmented, which provides information on the molecules’ structural features and the fragmentation spectrum functions as a sort of molecular fingerprint that can be used for identification. The flexible setup of the IQ-X allows detection of parent and fragment ions to be detected in either the Orbitrap or the ion trap, depending on the desired resolution, speed, and sensitivity.

Principles of fragmentation in the IQ-X
The IQ-X features three different techniques for fragmentation, namely collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and ultraviolet photodissociation (UVPD). CID is the most widely used of the three fragmentation techniques, which allows for comparison of CID fragmentation spectra with references spectra reported in databases or scientific publications, however, CID provides only limited specific structural information. HCD provides more extensive fragmentation and therefore more (sub)structural information, but is less widely used and fewer reference spectra are available for comparison. In contrast with CID and HCD, UVPD is a non-collisional fragmentation technique that is used because it can provide specific fragments that can differentiate specific closely structurally related structures such as isomers. These three types of fragmentation are complimentary, as they each provide different pieces of the puzzle for structure identification. The IQ-X can be used for design of intricate MSⁿ (up to MS¹⁰) strategies, in which the three types of fragmentation can be used and combined freely at each stage. After fragmentation, the fragments can be analysed either in the ion trap for relatively fast nominal mass determination, or in the Orbitrap for accurate mass determination.

Data acquisition and processing
Acquisition of data on the IQ-X is supported by Met-IQ and AcquireX software packages, that help to set up workflows to obtain valuable in-depth spectral information for each analyte in the sample. This is enabled, amongst others, by real-time library searches to direct the MSⁿ acquisition towards metabolites of (potential) interest. The obtained data can then be processed using Compound Discoverer, which is a toolbox that helps in identification of the detected analytes based on the spectral data obtained using MSⁿ acquisition. Compound Discoverer uses user-defined workflows to align chromatograms, detect peaks, extract spectral data, and compare spectral data with those present in in-house or publicly available databases. Data processing with Compound Discoverer is further supported by mzCloud, other spectral databases, and Mass Frontier.

Technical Details

Specifications: Thermo Vanquish UHPLC system

• Binary UHPLC pump with flow rates of 0.001-5 ml/min
• Split-loop autosampler for injection volumes of 0.01-1000 μL
• Temperature controlled sample compartment (4-40 ⁰C) for up to 216 samples
• Temperature controlled column compartment (5–120 ⁰C) with post-column cooler
• System pressure up to 1500 bar (22,000 psi)
• Compatibility with pH range 2-12
• Photodiode array detector (wavelength range 190-750 nm) with data collection rates of up to 5 Hz (multi-channel) or 125 Hz (single-channel)

Specifications: Thermo Orbitrap IQ-X Tribrid Mass Spectrometer

• Orbitrap mass range: m/z 50 – 2000 (in Full MS), m/z 40 – 2000 (in MSn)
• Quadrupole mass filter: m/z 50 – 2000 (Isolation width ± 0.4)
• Acquisition rate: Up to 40 Hz (Orbitrap) or 45 (ion trap)
• Orbitrap mass resolution: 500,000 FWHM at m/z 200
• Mass accuracy: < 1 ppm (internal mass calibration)
• Auto-Ready and EASY-IC ion sources for (automated) external and internal mass calibration
• Ionisation with OptaMax NG ion source: Interchangeable heated ESI or APCI probe
• Fragmentation: CID, HCD, and/or UVPD with MSn (n up to 10)
• AcquireX software for design of acquisition workflows
• Real-Time Library Search during acquisition

Applications

• Profiling, identification, and quantification of diverse food(-related) molecules:
  o Phytochemicals (e.g. from crude plant extracts)
  o Bioactive or biofunctional small molecules
  o Peptides (e.g. protein digests, modified peptides)
  o Lipid mixtures from (processed) fats or oils
  o Charged and neutral saccharides
• Reaction monitoring, including characterisation of molecular changes upon biological, enzymatic, or chemical treatment of food and related materials
• Compositional analysis of novel food sources or ingredients, plant tissues, and biobased materials
• Proteomics
• Metabolomics
• Lipidomics

Complementary Techniques

- Flash chromatography and preparative LC-MS for purification of compounds of interest

- MALDI-ToF-MS for characterization of oligo- and polymeric food(-related) compounds

- LC-ESI-cIMS-MS for separation of complex mixtures of (isomeric) compounds