molecules Review An Organic Chemist’s Guide to Electrospray Mass Spectrometric Structure Elucidation Arnold Steckel 1 and Gitta Schlosser 2,* 1 Hevesy György PhD School of Chemistry, ELTE Eötvös Loránd University, Pázmány Péter sétány 1/A, 1117 Budapest, Hungary; [email protected] 2 Department of Analytical Chemistry, ELTE Eötvös Loránd University, Pázmány Péter sétány 1/A, 1117 Budapest, Hungary * Correspondence: [email protected] Received: 16 January 2019; Accepted: 8 February 2019; Published: 10 February 2019 Abstract: Tandem mass spectrometry is an important tool for structure elucidation of natural and synthetic organic products. Fragmentation of odd electron ions (OE+) generated by electron ionization (EI) was extensively studied in the last few decades, however there are only a few systematic reviews available concerning the fragmentation of even-electron ions (EE+/EE−) produced by the currently most common ionization techniques, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). This review summarizes the most important features of tandem mass spectra generated by collision-induced dissociation fragmentation and presents didactic examples for the unexperienced users. Keywords: tandem mass spectrometry; MS/MS fragmentation; collision-induced dissociation; CID; ESI; structure elucidation 1. Introduction Electron ionization (EI), a hard ionization technique, is the method of choice for analyses of small (<1000 Da), nonpolar, volatile compounds. As its name implies, the technique involves ionization by electrons with ~70 eV energy. This energy is high enough to yield very reproducible mass spectra with a large number of fragments. However, these spectra frequently lack the radical type molecular ions (M+) due to the high internal energy transferred to the precursors [1]. This possible lack of molecular weight information is one of the greatest limitations of this ionization method. Soft ionization techniques, including electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are nowadays routinely used to ionize thermally labile and at least moderately polar organic analytes. These methods usually yield ions with no unpaired electrons (even-electron ions, EE) and the resulting [M + H]+ species are referred to as protonated molecules. Note that use of the term quasi-molecular ions is discouraged [2]. These ions possess low internal energy, therefore fragmentation in a single stage MS experiment is negligible [3–8]. This way, the intact accurate mass for sensitive, fragile compounds and large biomolecules as well as their detailed structures could be determined, even when high pressure liquid chromatography is used as a prefractionation method. As the application of electrospray ionization is far more widespread than any other ionization type, only this technique will be reviewed herein. 2. Choosing the Precursor Ion for Fragmentation ESI methodology can be used in either positive (ES+) and negative ion mode (ES−), depending on the proton affinity of the compound. In ES+, only analytes with at least one positive elementary charge, and in ES−, only analytes with at least one negative elementary charge, can be detected. Molecules Molecules 2019, 24, 611; doi:10.3390/molecules24030611 www.mdpi.com/journal/molecules Molecules 2019, 24, x FOR PEER REVIEW 2 of 11 Molecules 2019, 24, 611 2 of 11 2. Choosing the Precursor Ion for Fragmentation possessingESI basic methodology characteristics can be couldused in beeither easily positive ionised (ES+) in and ES+ negative via making ion mode adducts (ES−), depending with proton(s), while moleculeson the proton having affinity acidic of the functional compound. groups—and In ES+, only lackinganalytes basicwith at ones—typically least one positive give elementary better spectra charge, and in ES−, only analytes with at least one negative elementary charge, can be detected. in ES−. Optimal ionization mode selection can be based on the Brønsted-Lowry acid-base theory [9]. Molecules possessing basic characteristics could be easily ionised in ES+ via making adducts with It shouldproton(s), be emphasized while molecules that only having electrospray-compatible acidic functional groups—and solvents lacking (e.g., basic acetonitrile, ones—typically water, give methanol, ethanol)—containingbetter spectra in ES 0.1%−. Optimal formic ionization or acetic mode acid toselection enhance can protonation—or be based on the Brønsted-Lowry solvent mixtures acid- can be used forbase dissolving theory [9]. It samples should be during emphasized sample that preparation. only electrospray-compatible Solvents are highly solvents recommended (e.g., acetonitrile, to be of LC-MSwater, grade. methanol, Doubly ethanol)—containing distilled water should 0.1% be formic used or for acetic aqueous acid to solutions enhance protonation—or to effectively minimize solvent the intensitymixtures of sodium can be adducts used for in thedissolving spectra. samples It is also during advisable sample to usepreparation. low-binding, Solvents high-quality are highly plastic samplerecommended tubes to decrease to be of the LC-MS otherwise grade. Doubly always distilled present water plasticizer should contamination.be used for aqueous solutions to effectively minimize the intensity of sodium adducts in the spectra. It is also advisable to use low- The type of EE ions generated in the source are usually singly or multiply protonated [M + zH]z+ binding, high-quality plastic sample tubes to decrease the otherwise always present plasticizer − − − ions incontamination. ES+, while singly deprotonated [M H] ions are dominant in ES . Multiply protonated species areThe usually type of formed EE ions fromgenerated large in molecules the source are (e.g., usually polymers), singly or or multiply ones having protonated multiple [M + z favorableH]z+ sites forions protonation. in ES+, while Thesesingly deprotonated ions are fragmented [M − H]− ions more are easily dominant due in to ES charge−. Multiply repulsion; protonated however, fragmentsspecies are are often usually not formed too informative. from large molecules If the molecule (e.g., polymers), is large enough,or ones having it will multiple be represented favorable by an envelope-shapedsites for protonation. distribution These of ions peaks. are fragmented more easily due to charge repulsion; however, Chargefragments distributions are often not can too informative. be altered byIf the using molecu buffers.le is large Adduct enough, formation it will be represented could also by take an place envelope-shaped distribution of peaks. depending on the Lewis basicity of the compound in the presence of NH +, Na+ and K+ or if Li+ Charge distributions can be altered by using buffers. Adduct formation could4 also take place or Ag+ is intentionally added to the sample [10,11]. Note that these ions should be referred to as depending on the Lewis basicity of the compound in the presence of NH4+, Na+ and K+ or if Li+ or Ag+ cationizedis intentionally molecules added as the toterms the sample quasi-molecular [10,11]. Note th ionat andthese pseudo-molecular ions should be referred ion to are as discouragedcationized [2]. Thesemolecules adduct ions as the usually terms quasi-molecular give no fragmentation ion and pseudo-molecular other than the lossion are of thediscouraged cation, but[2]. These for special compoundsadduct (e.g.,ions usually carbohydrates), give no fragmentation their fragmentation other than the could loss of be the also cation, significantly but for special different compounds from that of [M(e.g., + H] +carbohydrates),ions, giving their useful fragmentation structural informationcould be also [significantly12]. Ammonium different acetate from that or of bicarbonate [M + H]+ are usuallyions, used giving for useful adduct structural formation information typically [12]. in Ammonium 5 mM concentration acetate or bicarbonate (Figure1). are It usually is worth used noting for adduct formation typically in 5 mM concentration (Figure 1). It is worth noting that additives with that additives with high ionization efficiency (e.g., tertiary or quaternary amines) and ones causing high ionization efficiency (e.g., tertiary or quaternary amines) and ones causing significant significant suppression of signal intensity (e.g., phosphate buffers, sodium chloride, trifluoroacetate) suppression of signal intensity (e.g., phosphate buffers, sodium chloride, trifluoroacetate) should be shouldavoided. be avoided. In ES− In, adduct ES−, adductformation formation could also could happen; also anionized happen; anionizedmolecules may molecules be useful may in bethe useful in thedetection of of some some compounds compounds (e.g., (e.g., carbohydrates, carbohydrates, flavonoids). flavonoids). FigureFigure 1. Single 1. Single stage stage mass mass spectrum spectrum of of trehalose, trehalose, aa disaccharide found found e.g. e.g.,, in insects. in insects. Due Dueto the to lack the lack of basicof residuesbasic residues in the in the molecule, molecule, [M [M + + H]H]+ ionsions could could not not be bedetected detected if the if sample the sample was dissolved was dissolved in in acetonitrile-wateracetonitrile-water 1:1 1:1 (v(v//v) solventsolvent mixture, mixture, containing containing 0.1% 0.1 acetic% acetic acidacid (spectrum (spectrum not shown). not shown). However, using 5 mM of ammonium acetate in water resulted in the above spectrum. Although absolute intensity is still low, the ammonium adduct of trehalose and its cationized