6.2. Mass spectrometry

This section summarises practical analysis aspects of mass spectrometry, and guidelines on choosing a suitable ion source and mass analyser combination for investigating different materials are given.

Practical aspects of direct MS

Mass spectrometry is a powerful technique that provides valuable molecular information about the components of complex materials. It is often combined with gas chromatography (GC) or liquid chromatography (LC). It is also applicable as a stand-alone (direct) technique allowing to combine different ionisation methods (electrospray ionisation (ESI), atmospheric pressure chemical ionisation (APCI), matrix-assisted laser desorption ionisation (MALDI), etc.) and mass analysers (quadrupole (Q), ion trap (IT), Fourier transform ion cyclotron resonance (FT-ICR), time of flight (ToF), etc.).

Please see Table 1 and the videos below for more detailed information. The information in Table 1 is a very broad generalisation. Exceptions can be found in almost all statements presented in the table.

Table 1. Comparison of some mass spectrometric techniques based on the ion source and their suitability for the analysis of cultural heritage materials. Under the ion source (in bold), in brackets are the more common mass analysers that are combined with the source. [1,6]

	EI and CI (+ Q, IT)	MALDI (+ToF, FT-ICR, also Q, QQQ, IT and combinations)	ESI and APCI (+QQQ, IT, FT-OT, FT-ICR, also ToF and combinations)
Sample preparation	− Gas-phase ion source → analytes are introduced in gas phase. − Mainly used (a) in hyphenation with gas chromatography, as detector; (b) with direct temperature MS techniques: dissolved/extracted or solid sample is inserted into the ion source (through a specific inlet) and gradually heated → compounds are desorbed/pyrolyzed. − Suitable solvents: methanol, ethanol, toluene, DCM, etc.	− The sample is co-crystallized with a matrix material: sample (solution) is mixed with matrix solution and placed on MALDI target plate, solvent(s) is (are) evaporated, and dried crystals are analysed. − The sample does not need to be fully soluble (the solutions can be hazy). − Matrix substances: *positive ion mode: DHB, CHCA, SA, etc. *negative ion mode: 9-AA, 3-AA, etc. − Suitable solvents: water, methanol, toluene, DCM, DMSO, etc.	− Liquid phase ion source → analytes are introduced in liquid phase, most often in combination with liquid chromatography in MS detector. − Sample is dissolved/extracted with solvent(s). Sample solution must be filtered (or centrifuged)! − Suitable solvents: ESI: most commonly methanol, acetonitrile, water, and their mixtures; less often toluene, ethyl acetate, dichloromethane, tetrahydrofuran, etc. APCI: methanol, hexane, acetonitrile, dichloromethane, etc.
Sample size	Less than 1 µg, usually only few ng of a compound in a mixture.	Less than 1 µg (per mL of sample solution).	ESI and APCI: approx. 1 mg per mL of sample solution. Nano-ESI: few dozen µg per mL of sample solution.
Material types that can be analysed	Compounds with sufficient vapor pressure (boiling points below ca 600 °C) directly; non-volatile compounds, such as resinous materials (resins, varnishes, tars), oils/fats, waxes, saccharides, proteins/peptides, dyes, polymers can be analysed using derivatization and/or pyrolysis.	Resinous materials (resins, varnishes, tars), oils/fats, waxes, saccharides, proteins/peptides, dyes, polymers, inorganic compounds.	ESI: Resinous materials (resins, varnishes, tars), oils/fats, waxes, saccharides, proteins/peptides, dyes, polymers. APCI: Resinous materials (resins, varnishes, tars), oils/fats, waxes, saccharides, dyes.
Information in mass spectra (more info)	EI: − Highly fragmenting → fragment ions are detected, sometimes also the molecular ion → characteristic fragmentation pattern for compounds. − Only positive ions are generated. CI: − Less fragmenting → molecular ions and fragment ions → simple mass spectrum − Adduct ions with the reagent gas may form. − Positive and negative ions can be generated.	− Positive and negative ions can be generated → mainly adduct ions with H+, Na+, K+, Cl–, etc. → characteristic to specific materials. − Mainly singly charged ions. − Low fragmentation. − Macromolecules can be ionised.	− Positive and negative ions. − Low fragmentation. ESI: − Singly and multiply charged ions → multiply charged ions: analysis of compounds with very high mass (approx. 100000 Da), singly charged ions: information about small molecules. − Generates mainly adduct ions with H+, Na+, K+, Cl–, etc. − Preferably more polar compounds are ionised. APCI: − Mainly singly charged ions. − Mainly protonated or deprotonated ions. − Preferably less polar to nonpolar small (up to 2000 Da) compounds are ionised.
Advantages	− Very small sample can be analysed → very sensitive. − Very good ionisation efficiency. − Fragmentation leads to characteristic spectra (“fingerprints”) and gives information about the structure of the compounds. − Extensive and well accessible spectral libraries exist that are help with identification. − Qualitative and quantitative analysis.	− Can be used for partially dissolved samples (hazy solution) or directly on the solid samples without extraction/decomposing − Easy sample preparation. − Aged, oxidised, degraded samples can be analysed, incl. materials that are (partly) polymeric and non-volatile. − No extensive fragmentation → typically ions corresponding to original compounds and their derivatives are detected − Good ionization efficiency.	− Soft ionisation. Qualitative and quantitative analysis. ESI: − Suitable for the analysis of most organic compounds. − Suitable for analytes with low concentration (in case of nano-ESI). APCI: − Less matrix effects compared to ESI.
Disadvantages	− Molecules can be extensively fragmented (e.g., aliphatic compounds) and spectra may be insufficiently characteristic for identification. In particular, molecular ions are not always observed. − Instrument needs constant monitoring and maintenance. − Skilled operator is needed. − CI → limited amount of structural information.	− Selection of matrix substance and suitable solvents is crucial → the matrix and solvent(s) must be compatible with sample. − Poorly reproducible spectra. − Not suitable for quantitative analysis. − Interpretation relies on comparison to standard materials and literature. − Instrument needs constant monitoring and maintenance. − Skilled operator is needed.	− Liquid environment for the ionisation needed → Sample has to be dissolved (only the dissolved/extracted part of the sample can be analysed). − Higher sample amount needed. − Instrument needs constant monitoring and maintenance. − Skilled operator is needed. ESI: − Matrix effects − Spray stability depends on the solvent. APCI: − Only suitable for small molecules (under 2000 Da). − System needs constant cleaning → contamination of the corona needle and MS inlet.

As demonstrated in Table 1, the information obtained with different MS instruments can vary because the ionisation method is different. As an example, in Fig. 1, mass spectra of shellac resin, colophony resin (both in positive mode) and extract of dyer’s madder plant (negative mode) obtained with FT-ICR-MS coupled to MALDI, APCI and ESI sources, respectively are presented. The selection of ion mode – positive or negative depends on the analysed material (e.g., natural resins preferably give positive ions while dyes give negative ions). In positive mode, ESI and APCI give mostly protonated ions, sometimes also adducts with Na⁺. With MALDI, preferably adducts with Na⁺, sometimes also K⁺, Al⁺, etc., are observed (such adducts are common for compounds with a higher amount of oxygen in them). In negative ion mode, mainly deprotonated ions are observed but also the addition of Cl^– or other anions may be possible (see Fig.1c). With ESI, more polar compounds can be detected (reins, dyes, oils/fats, etc.), MALDI and APCI are also suitable for less polar compounds (waxes, hydrocarbons).

Interpretation of the obtained mass spectra is based on the identification of detected ions/fragments and also on the isotope pattern, which helps to determine the charge of the ion (by the spacing between the satellite peaks), as well as the presence of some elements with characteristic isotope patterns (such as Cl, Br, S). If a HRMS instrument is used, like FT-ICR-MS, Orbitrap, or high-end ToF, then the obtained m/z values are very accurate (four to six decimals) and often a single mass analyser is enough for assigning correct ion formulas and, hence, identifying the corresponding compounds, as demonstrated on Fig.1 (reference mass spectra are still very helpful). With LRMS instruments only nominal m/z values are obtained. This makes m/z-based identifications difficult or impossible because the number of potential ion formulas corresponding to a nominal value can be very high. In the case of LRMS, tandem instruments (see section 5.2) are helpful. This technique allows to select specific m/z values, fragment them, and analyse the obtained fragments. In this case, identification is mostly based on fragmentation patterns – different compounds (even with the same nominal m/z value) can fragment differently.

Analysis with MALDI-, ESI- and APCI-FT-ICR-MS

In the following video, Dr Anu Teearu-Ojakäär introduces the principles of MALDI-FT-ICR-MS, sample preparation and how to perform analysis with this instrument.

In the following video, Dr Anu Teearu-Ojakäär introduces the FT-ICR-MS with APCI and ESI ionisation sources, sample preparation and how to perform analysis with ESI-FT-ICR-MS.