Studying the chemistry of cationized triacylglycerols using electrospray ionization mass spectrometry and density functional theory computations.

B American Society for Mass Spectrometry, 2014

J. Am. Soc. Mass Spectrom. (2014) DOI: 10.1007/s13361-014-0917-9

RESEARCH ARTICLE

Studying the Chemistry of Cationized Triacylglycerols Using Electrospray Ionization Mass Spectrometry and Density Functional Theory Computations J. Stuart Grossert,1,2 Lisandra Cubero Herrera,1,4 Louis Ramaley,1,2 Jeremy E. Melanson1,3 1

National Research Council Canada, Halifax, Nova Scotia B3H 3Z1, Canada Department of Chemistry, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada 3 Measurement Science and Standards, National Research Council Canada, Ottawa, Ontario K1A 0R6, Canada 4 Canadian Food Inspection Agency, 1992 Agency Drive, Dartmouth, Nova Scotia B3B 1Y9, Canada 2

Abstract. Analysis of triacylglycerols (TAGs), found as complex mixtures in living organisms, is typically accomplished using liquid chromatography, often coupled to mass spectrometry. TAGs, weak bases not protonated using electrospray ionization, are usually ionized +by adduct formation with a cation, including those present in the solvent (e.g., Na ). There are relatively few reports on the binding of TAGs with cations or on the mechanisms by which cationized TAGs fragment. + + and + This work examines binding efficiencies, determined by mass spectrometry + + + computations, for the complexation of TAGs to a range of cations (Na , Li , K , Ag , NH4 ). While most cations bind to oxygen, Ag binding to unsaturation in the+ acid side chains is significant. The importance of dimer formation, [2TAG + M] was demonstrated using several different types of mass spectrometers. From breakdown curves, it became apparent that two or three acid side chains must be attached to glycerol for strong cationization. Possible mechanisms for fragmentation of lithiated TAGs were modeled by computations on tripropionylglycerol. Viable pathways were found for losses of neutral acids and lithium salts of acids from different positions on the glycerol moiety. Novel lactone structures were proposed for the loss of a neutral acid from one position of the glycerol moiety. These were studied further using triple-stage mass spectrometry (MS3). These lactones can account for all the major product ions in the MS3 spectra in both this work and the literature, which should allow for new insights into the challenging analytical methods needed for naturally occurring TAGs. Key words: Lithiated triglycerides, Binding efficiencies of cations to triacylglycerols, Breakdown curves, MS/ MS/MS product ions, Solvent–cation interactions in ESI Received: 4 February 2014/Revised: 1 April 2014/Accepted: 2 April 2014

Introduction

T

riacylglycerols (TAGs) are an important class of lipids that consist of three fatty acids esterified to a glycerol backbone. TAGs can be differentiated by the total carbon number, by the number, position, and geometrical configuration (cis or trans) of double bonds in the fatty acyl chains, and by the stereospecific positions (sn-1, sn-2 and sn-3) of fatty acids on the glycerol core. While a TAG having three

Electronic supplementary material The online version of this article (doi:10.1007/s13361-014-0917-9) contains supplementary material, which is available to authorized users. Correspondence to: J. Grossert; e-mail: [email protected]

identical acyl groups has a symmetry plane passing through the central carbon atom of the glycerol moiety, stereochemical considerations become important when the acyl groups are different. A convention for stereospecific numbering in which the pro-S ester group is defined as sn-1 has been adopted to describe the different substituent positions of a TAG [1]. The geometry of the structure is shown in Figure 1. If the groups R1 and R2 are the same, but different from R3, the C(2) atom of the glycerol moiety is chiral and the molecule can be described as AAB, which is enantiomeric with BAA. If all three groups are different, the molecule (ABC) is obviously chiral. For an ABA-type molecule, the sn-1 position can also be referred to as the pro-S position. Knowledge of the type and position of the fatty acyl groups in TAGs is

J. S. Grossert et al.: Studies of Triacylglycerol Adduct Ions

Figure 1. The stereospecific numbering convention for triacylglycerols

important from a biological point of view, as the fatty acid at the sn-2 position is reported to be more bioavailable to rats [2] and to humans [3]. Therefore, for health and nutritional purposes, the differentiation between the sn-2 and sn-1/sn-3 positions is becoming increasingly important and has been the subject of several studies [4–7]. The analysis of TAGs is challenging because of the wide variety of naturally occurring fatty acids and, therefore, a multidimensional approach is normally required. Comprehensive TAG analysis can be performed by silver-ion highperformance liquid chromatography [5, 8], usually with MS detection (Ag-HPLC-MS) or reversed-phase high-performance liquid chromatography with MS detection (RPHPLC-MS) [9–13]. Among HPLC-MS interfaces, atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) are the most commonly employed ionization methods. APCI of TAGs produces mainly protonated molecules, [TAG + H]+, and diacylglycerol-like (DAG-like) fragment ions of the type [TAG + H – RCOOH]+ formed by the loss of a neutral carboxylic acid moiety. The [TAG + H]+ ions provide molecular weight information, and the relative intensities of the DAG-like ions provide information on the location of fatty acids on the glycerol backbone, since losses from the sn-1 and sn-3 positions are generally more likely and equally favored [10–12]. However, tandem MS on the [TAG + H]+ ions generated by APCI provides no differentiation of positional isomers [11]. Therefore, positional analysis of TAGs using APCI is generally done using single-stage MS rather than dual-stage MS (MS2). TAGs are weakly basic esters and in contrast to APCIMS, which leads to protonated molecules, the conditions of ESI, in the absence of added cationization reagents, readily lead to the formation of sodiated TAGs, [TAG + Na]+, which in many cases can be fragmented to yield structural information. As a result, ESI is commonly used in combination with tandem MS for structural characterization of TAGs [6, 7, 10, 11, 13–18]. Salts can be added to the LC eluent to produce adduct ions [TAG + M]+ where most commonly M = Li+, Na+, NH4+, or Ag+ [10, 11, 13–16]. ESI-MS2 of ammoniated species generate uniquely DAGlike ions of the type [TAG + NH4 – NH3 – RCOOH]+, presumably by the loss of ammonia and a neutral fatty acid [10, 11, 13, 19], whereas adducts of TAGs with lithium [14, 15], sodium [16], and silver ions [6, 7] produce two major DAG-like ions, [TAG + M – RCOOH]+ and [TAG + M – RCOOM]+.

As seen in the APCI-MS of [TAG + H]+ ions, the abundance of DAG-like ions produced by ESI-MS2 of [TAG + M]+ adducts is dependent on the positions of the fatty acids on the glycerol backbone, with the sn-1 or sn-3 acyl groups generally being lost more readily than the sn-2 fatty acid. The preferential loss of the fatty acid at positions sn-1/3 seems to be a general phenomenon for TAG molecules since it has been observed in low-energy [13–16] and high-energy [20–23] collision-induced dissociation (CID) spectra, and is independent of the instrumentation employed. However, the opposite trend has been reported for the [TAG + Ag]+ adducts of TAG molecules PPO and POP (P=16:0 and O=18:1), in which the fatty acid at sn-2 is lost more favorably than the sn-1 or sn-3 fatty acids [16]. This indicates that fragmentation of TAG adducts can be dependent not only on the location of the fatty acids on the glycerol backbone (sn-1/3 or sn-2) but also on the nature of the cation and on the degree of unsaturation in the fatty acyl chain. Recent work describing high-energy CID spectra on [TAG + Na]+ ions using a MALDI-TOF/TOF system not only gave information about the position of the fatty acids on the glycerol skeleton but, because of a full range of charge-remote product ions, gave details of the location of unsaturation in the chains [23]. Although there is a significant body of literature on the use of mass spectrometry for the analysis of TAGs, very few studies on understanding the fragmentation mechanisms of TAG adducts have been carried out to date. Using TAGs that have been labeled with deuterium atoms, Hsu and Turk have demonstrated [14] that elimination of a neutral fatty acid from [TAG + Li] + adducts of TAGs involves abstraction of a hydrogen atom from the α-methylene group of one of the fatty acyl chains. Based on these findings, the authors have proposed fragmentation pathways for the losses of both sn-1/3 and sn-2 RCOOH and RCOOLi moieties leading to DAG-like product ions in ESI-MS2 [14]. These authors have recently updated their work with results of MSn spectra from a linear ion trap mass spectrometer but retaining the same basic mechanistic schemes [15]. Using deuterium labeling in a series of experiments, McAnoy et al. described fragmentation pathways for [TAG + NH4]+ ions, which typically result in the loss of a neutral acid molecule plus ammonia [24]. The authors additionally used results from MS3 spectra to propose mechanistic schemes to describe these fragmentations. This and a handful of other studies have been highlighted in a recent review [25]. Very recently, Renaud et al. have published detailed work on the energetics of fragmentation of ammoniated TAGs in ESIMS2, comparing losses from the sn-1/3 and sn-2 positions and proposing structures for both the precursor and product ions [19]. Apart from these studies, no information appears to exist as to how different cations actually interact with TAGs having different levels of unsaturation and how the resulting cationized TAGs can be fragmented. We have approached the problem of gaining a detailed, general understanding of the mechanism(s) by which product ions are formed in tandem mass spectra by studying


the geometries of cationized TAGs and the preferred cation binding sites using a combination of MS and density functional theory (DFT) methods. We have measured the binding efficiencies of Li+, Na+, and Ag+ ions with TAGs having both saturated and unsaturated chains and have compared these results with computed binding energies from DFT methods in both the gas phase [26] and using a Polarizable Continuum Model universal force field [27] set to the dielectric constant of methanol. We show also that dimer ions such as [2TAG + Li]+ can be expected to form with ease and contribute significantly to the total ion current. These studies of geometries and binding energies of TAG adducts have enabled conclusions to be drawn on the preferred binding sites between cations and TAGs, as knowledge about complexation sites of cations and geometries of [TAG + M]+ adducts is an important initial step towards understanding the fragmentation behavior of cationized TAGs. In this regard, we have obtained fragmentation data that were used to prepare breakdown curves for the fragmentation of sodiated glycerol, monopalmitoylglycerol, and dipalmitoylglycerol, as well as a series of cationized TAGs. These data, together with those from the literature and extensive computations have enabled us to propose novel detailed mechanistic schemes for the fragmentations of lithiated TAGs and to extend these to provide a logical explanation for the structures of the major product ions in the MS3 spectra of ions produced after the MS2 loss of an sn-1/3 neutral acid from a lithiated TAG.

were not applied [37]. Additionally, saddle-point structures were recognized as having one imaginary frequency, and these structures were linked to minima on both sides of the energy surface by freezing atom motion towards both extremities of the imaginary frequency and reoptimizing the resulting geometry to an energy minimum. In some cases, these minima were checked by intrinsic reaction coordinate (IRC) computations. Gas phase binding energies (GBE) at 298 K and 1 atm. were calculated at the B3LYP/6-31G(d) or B3LYP/DZVP levels as GBE=ΔEelec + ΔEZPVE, with ΔEelec and ΔEZPVE being the changes in electronic energies and zero-point vibrational energies, respectively, between the products and the reactant in the dissociation reaction, Equation 1 ½TAG þ Mþ →TAG þ Mþ

ð1Þ

where TAG = tripropionylglycerol or 1,3-diundecanoyl-2(9Z-undecenoyl)-glycerol; M+ = H+, NH4+, Li+, Na+, or Ag+. Ion energies were corrected for basis set superposition errors (BSSE) using the counterpoise method, as parameterized in G09. In addition, a set of ion affinities was calculated from the results of full optimizations using the polarizable continuum model (PCM) in a self-consistent reaction field (SCRF) with a universal force field set for the dielectric constant of methanol (G09) [27].

Materials and Solutions

Experimental Computational Details Density functional theory (DFT) computations were performed using the Gaussian 09 Revision A.02 suite of programs [28] on the ACEnet Placentia Cluster located at Memorial University, St. John’s, NL, Canada. The structures of two groups of species were fully optimized in the ground state at 298 K and 1.0 atm. Computations for the first group consisting of tripropionylglycerols, cationized by lithium or sodium, were performed using the B3LYP functional [29, 30] and the 6-31G(d) [31, 32] basis set. The second group consisted of monounsaturated triundecanoylglycerols, which were ionized by complexation with a range of cations including Ag+. For these cases, the B3LYP level with the DFT-optimized double-zeta plus valence polarization (DZVP) basis set [33] was applied to all atoms. The B3LYP/6-31G(d) system was selected because it yields reasonable geometries at acceptable computational costs for molecules of the size studied [34], and the DZVP basis set has been reported to produce Ag+ binding energies that agree with published experimental data [35, 36]. Vibrational frequencies were computed at the same levels of theory to confirm that the optimized structures corresponded to local minima on the potential energy surface and had no imaginary frequencies. Vibrational scaling factors for the infrared frequencies

Abbreviations for fatty acids are La = lauric acid (12:0), M = myristic acid (14:0), P = palmitic acid (16:0), S = stearic acid (18:0), O = oleic acid (18:1 cis-9), L = linoleic acid (18:2), Ln = linolenic acid (18:3), E = eicosapentaenoic acid (20:5), and D = docosahexaenoic acid (22:6). TAG standards LaOP, MMP, MEM, PSP, POP, PPO, PLP, OPO, LOL, PEP, PDP, and DPD were purchased from Larodan Fine Chemicals AB (Malmö, Sweden); monopalmitin (monoP), dipalmitin (diP), PPP, OOO, and LnLnLn were acquired from Nu-Chek-Prep (Elysian, MN, USA). Methanol, 2-propanol, and dichloromethane (distilled in glass or HPLC grade) were purchased from Caledon Laboratories Ltd. (Georgetown, ON, Canada). Glycerol, sodium carbonate (ACS reagent), trifluoroacetic acid (ReagentPlus), trifluoromethanesulfonic acid (ReagentPlus), and silver trifluoromethane sulfonate (≥ 99%) were obtained from Sigma-Aldrich (Oakville, ON, Canada). Lithium-7 carbonate (99.9% 7Li) was obtained from Cambridge Isotope Laboratories, Inc., (Andover, MA, USA). This study did not require isotopically enriched Li+, but the reagent was available in our laboratory and enabled simpler spectra. Lithium-7 trifluoroacetate was prepared by mixing stoichiometric amounts of lithium-7 carbonate and trifluoroacetic acid. All chemicals and solvents were used without further purification. Nitrogen (UHP) and argon for the mass spectrometers was obtained from Praxair (Halifax, NS, Canada).


Stock solutions of TAGs (~1.0×10–3 mol L–1) were prepared in a dichloromethane/2-propanol/methanol solvent system (2:1:7, v/v/v), and further diluted to 1.0×10–6 mol L–1 in methanol for all working solutions. Stock solutions of approximately 1.0×10–2 M of sodium, lithium, and silver trifluoromethanesulfonate were prepared directly or by mixing stoichiometric amounts of the metal carbonate and trifluoromethanesulfonic acid. These were diluted with methanol appropriately to form working solutions for binding efficiency studies. Dimer formation was examined using [LaOP] or [MMP] = 1.0 × 10–6 mol L–1 and complexed with either [Li+] or [Na+] = 1.0×10–4 mol L–1 singly, or with a cation mixture having [Li+] = 1.0×10–4 mol L–1 and [Na+] = 1.0×10–5 mol L–1. Breakdown curves for sodiated glycerol, monoP and diP were obtained using 1.0×10–5 mol L–1 solutions in methanol without addition of any sodium salt.

skimmer 18 V, heater 350°C). Full-spectrum scans were collected at a resolution of 50,000 FWHM over a mass range of 100-2,000m/z. Some spectra for the breakdown curves were obtained on a Waters Quattro Premier XE tandem quadrupole spectrometer on which the second resolving quadrupole was scanned over a range of m/z=15 to (TAG+43); source parameters were similar to those on the QTOF, for monoP: cone = 35 V, CE = 18–24 eV; for diP: cone = 50 V, CE = 18–30 eV; for POP: cone = 80 V, CE = 22–30 eV; collision gas was at a pressure to reduce the intensity of the precursor ion by ca 50%; solutions (MeOH) introduced by flow injection, flow rate = 20 μL min–1.

Results and Discussion Structures of TAGs Structures of Neutral Triacylglycerols in the Gas Phase

Instrumentation Binding efficiency spectra were obtained with an ABSCIEX Qtrap 2000 QqLIT spectrometer (Vaughan ON, Canada) in positive ion mode using Analyst v.1.4 software under the following conditions: all gases = N2, curtain gas = 40 arbitrary units (au), ion source gas 1=15 au, ion source gas 2=35 au, ion source = 100°C, ESI needle voltage = 5 kV, resolution = unit mass, declustering potential = 100 V, collision energy (CE) = 50 eV, fixed LIT fill time = 40 ms. Solutions (MeOH) were introduced by flow injection, rate = 10 μL min–1. Each mass spectrum from which peak intensities were determined was acquired by integrating the instrument response over a period of 4.0 min, and all measurements were made in triplicate. Other mass spectra were obtained with a Waters Q-TOF Premier mass spectrometer (Waters, Brossard PQ, Canada) running under MassLynx v.4.1 software and equipped with an ESI probe. The mass spectrometer was used in positiveion mode with nitrogen as source gas and argon as collision gas. Regular operating parameters were: capillary voltage = 3.5 kV, cone voltage = 50 V, source temperature = 100°C, desolvation temperature = 500°C, cone gas flow = 50 L h–1, and desolvation gas flow = 500 L h–1. Full-scan mass spectra were acquired over the m/z range 50–1000. Solutions (25 μL, MeOH) were introduced into the mass spectrometer via flow injection, rate = 0.2 mL min–1 with a model 1100 HPLC System (Agilent Technologies, Mississauga, ON, Canada). With one exception, spectra to examine dimer formation were obtained either on the Q-TOF or the MDS Sciex QTRAP spectrometers (flow rate = 20 μL min–1) described above. The spectrum showing dimer formation with ammonium ions was obtained from a solution of OOO (ca 1 μM) in acetonitrile-isopropanol (40:60) containing NH4OAc (ca 1 mM) using ESI-MS on a Thermo Exactive Orbitrap spectrometer (Burlington ON, Canada; spray voltage 3.30 kV, capillary 380°C and 32.5 V, tube lens 85 V,

Our approach to the mass spectrometric analysis of triacylglycerols began with considering what structure(s) are formed during electrospray ionization, followed by how the ions might fragment under conditions of collisional activation. Triacylglycerols obviously have conformationally mobile structures, which have been studied in some detail for triacetylglycerol using DFT methods [38] and molecular mechanics [39], but the two results produced different structures having the minimum energy. We have searched the energy surface for triproprionylglycerol using a smaller basis set {B3LYP/6-31 g(d)} than Limpanuparb et al. [38] and reached the same conclusion, namely that the two OCCO groups have dihedral angles of 176° (anti) and 62° (gauche). Three conformers (A, B, C) are shown in the Supplementary Material Figure 1S. In order to assess binding affinities and fragmentation pathways of cationized TAGs, we have also computed a structure for 1,3-diundecanoyl-2-(9Z-undecenoyl)-glycerol (Supplementary Material Figure 2S). The short-chain species were used to study fragmentation and association into dimers of cationized TAGs while the long-chain species were suitable as a model for studying the binding energies of natural TAGs having a double bond in the Δ-9 position of one of the chains, but principally the sn-2 chain. Structures of Cationized Triacylglycerols Triacylglycerols are very weak bases and form protonated molecules under conditions of chemical ionization, but not using electrospray ionization. However, they are readily complexed by cations, such as ammonium, lithium, silver, or sodium ions [7, 14, 16, 17]. Altough both lithiated and sodiated TAGs can yield useful structural information in MS2 spectra, only the former yield useful MS3 spectra [15, 18]. While ammoniated TAGs produce simpler MS2 spectra than do


the lithiated species [19, 24, 40] and give useful MS3 spectra [24, 40], formation of these ions requires an excess of ammonium acetate relative to the concentration of TAG. Our examination of the literature has not uncovered a comprehensive picture of the structures of cationized TAGs that could be formed from ESI-MS which prompted us to do DFT computations on a range of cationized TAGs [19]. Complexation of tripropionylglycerol to a lithium cation leads to a large set of structures, a selection of structures being shown in Figure 2, ranging in relative energies from the most stable form with tetracoordinate binding, 1A, to complex 1H with dicoordinate binding. The energy range (gas phase electronic plus zero point energies) within this set is greater than 150 kJ mol–1. While some structures have different coordination levels to the lithium cation, others are simply conformers. We have also identified other complexes having structures with similar energies, a selection of which is shown in the Supplementary Material Figure 3S. We have examined a wide range of structures of lithiated 1,3-diundecanoyl-2-(9Z-undecenoyl)-glycerol, some examples of which are shown in Figure 3 and Supplementary Material Figure 4S. The structures 2A – 2C are shown as lithiated TAGs, but essentially identical structures were found for the sodiated and argentiated species, allowing for the longer bonds to these cations. Thus 2A has the cation tetracoordinate to oxygen atoms, 2B and 2C are dicoordinate, in one case to two oxygen atoms and in the other to one oxygen and the pi cloud of the alkene. Eight other computed structures having a lithium cation complexed in both tricoordinate and dicoordinate bonding to various carbonyl and alkene groups are shown in Supplementary Material Figure 4S. These structures again show the wide range of possible structures that can be formed and, as expected, the energy of these depends on the coordination level. At the same level of coordination, energies are slightly different if the coordination involves the sn-1/3 or sn-2 isomeric chains. While the attraction between carbonyl groups and a cation is strong, the attraction between an alkenyl π bond and either a silver or a lithium cation is

substantial and must be considered when attempting to unravel the details of ESI-MS on cationized TAGs. We propose that the ions exiting from an ESI source have a distribution of structures based on some function of their relative energies. However, given the conformational mobility of these complexes, this distribution may not be the same for the ions undergoing CID. We have also examined structures with a single coordinating group, but these were higher in energy than the structures shown and were not considered further. While sodium and silver cations are known to form structures with higher levels of coordination in solution [41], in the gas phase tetracoordination is to be expected for lithium and the other cations [42, 43]. Additionally, we have computed structures for selected protonated and ammoniated species, widely used in analyses of TAGs, and shown in the Supplementary Material Figure 5S. Protons may only become dicoordinated and, indeed, this was found to be the case as seen in the structures 5SA and 5SB, although the binding of the ligands in both cases was quite unequal. Achieving tetracoordination of an ammonium ion given the geometrical constraints of the TAG ligands is impossible, and we only found two tricoordinated structures, 5SC and 5SD, neither of which was strongly bound. Structures similar to 5SA and 5SC were reported by Renaud et al. [19] We have attempted to expand the current understanding of cationized TAGs by experimentally measuring relative binding efficiencies and behavior under conditions of CID of selected cationized TAGs and then comparing these to relative computed values of binding energies. Experimental Relative Binding Efficiencies of TAGs with Li+, Na+, and Ag+ Cations Any experimental binding study involving alkali metal ions will be complicated by the presence of a background of such ions leached from glassware or introduced unintentionally by other means [44]. The level of this contamination is said to be approximately 1×10–6 M [45]. Metal ion content is rarely listed in

Figure 2. Selected computed structures of lithiated tripropionylglycerols


Figure 3. Computed structures of some lithiated C-11 triacylglycerols: 2A, Li+ tetracoordinate to 4 oxygen atoms; 2B, Li+ dicoordinate to sn-1/3 C = O groups; 2C, Li+ dicoordinate to sn-1 C = O and sn-2 C = C groups (figure insets show binding site details)

the specifications for solvents. For example, of the 33 grades of methanol at present available from Sigma-Aldrich, only five grades list either the Na+ or K+ content. This varies typically from ≤ 0.5 ppm to ≤ 0.05 ppm for both ions (1.7×10–5 to 1.7×10–6 M for Na+ in methanol). Even the TraceSELECT grade, which is recommended for metal speciation analysis, lists the Na+ content as ≤ 0.25 ppm (8.6×10–6 M) and the K+ content as ≤ 0.01 ppm (2.0×10–7 M). The only grade of Caledon Laboratories Ltd. methanol listing ionic impurities, Ultra LC/MC, shows the Na+ content as 0.35 ppm (1.2×10–5 M) and the K+ content as 0.20 ppm (4.0×10–6 M). This explains the observation in ESI of sodiated and potassiated TAG ions from methanol solutions in the absence of any added cationization reagents. Complexation of metal cations to various molecules under ESI conditions has been widely studied [46–49]. We were able to measure the Na+ content of Caledon HPLC grade methanol, the solvent used for the competitive binding studies, and the stock 1.5×10–3 M Li+ solution by flame emission spectrometry (lab-built instrumentation) as 1.2×10–6 M and 4.0× 10–6 M, respectively. These values were used in the preparation of solutions which contained 5.0×10–5 M of both Li+ and Na+ and 1.0×10–6 M of the TAGs PSP (saturated), MEM (five double bonds), and LnLnLn (nine double bonds). The peak height ratio in ion counts, i[TAG + Na]+/i[TAG + Li]+, varied from 2.4 to 2.9, indicating that the sodiated TAG was the preferred ion formed by ESI and that unsaturation was not an important factor in the competition between Na+ and Li+. A more thorough study was undertaken with the tags PSP, POP, PPO, and OOO using various ratios of [Li+]/ [Na+] and [Ag+]/[Na+], where the sum of the added ionic concentrations was held constant at 1.0×10–4 M, well above the background concentration. If the following simple chemical equilibrium is assumed to exist in solution MTAGþ þ Naþ →NaTAGþ þ Mþ

ð2Þ

where M = Li or Ag, the equilibrium constant for this reaction is ½Mþ ½NaTAGþ ð3Þ ½Naþ ½MTAGþ If it is assumed that the ESI ion current is proportional to the concentration of cationized TAG in the spray droplet, K¼

where iNa is the ion current due to the sodiated TAG, [TAG + Na]+ and iM is the ion current due to the lithiated or argentiated TAG, [TAG + M]+, then iNa ¼ k Na ½NaTAGþ and

ð4Þ

iM ¼ k M ½MTAGþ

ð5Þ

where kNa and kM are sensitivity coefficients [50], which depend on the ability of the ion to become charged at the drop surface and escape into the gas phase. This leads to K¼

½Mþ k Mi Na : ½Naþ k Nai M

ð6Þ

Finally, if the sensitivity coefficients are approximately equal—a reasonable assumption given the similarity of the ions, K¼

½Mþ iNa : ½Naþ iM

ð7Þ

Equation 7 indicates that the ratio of ion currents when multiplied by the ratio of calculated concentrations should be constant, assuming that the ratio of the actual metal ion concentrations is the same as that calculated from the added solution components. If K is greater than unity, binding between the TAG and Na+ is indicated to be stronger than between the TAG and M+. Experimental results are shown in Table 1. On average, binding to Na+ is indicated to be somewhat stronger than to Li+, regardless of the degree of unsaturation in the acyl side chains or regioisomerism. The binding of Na+ to a TAG with three unsaturated acyl chains (OOO) does appear to be slightly more favorable than with only one unsaturated side chain (POP or PPO). When unsaturation is not involved (PSP), the binding of Na+ is indicated also to be stronger than that of Ag+, with a magnitude comparable to that with Li+. However, with Ag+, the presence of unsaturation has a large effect, as would be expected, shifting the binding efficiency strongly in favor of Ag + over Na + . Regioisomerism does not seem to play a large role in binding efficiency, as indicated by the similarity in results for POP and PPO. However, increasing the amount of


unsaturation increases the binding efficiency of Ag+ as indicated by the results for OOO. The model giving rise to Equation 7, however, is obviously an oversimplification, since the K values in Table 1 are not constant, especially those involving the competition between Na+ and Li+. These latter values decrease monotonically with a decrease in the ratio of [Li+]/[Na+]. This behavior indicates that binding favors the metal ion that is in low concentration, since K91 at low [Na+] and KG1 at low [Li+]. Peaks for sodiated and potassiated TAGs are observed in solutions containing 1× 10–4 M or higher Li+ concentrations with no added Na+ or K+, even though the concentration of lithium ions is about 100 times or more higher than that of the background ions. This is another observation of the same phenomenon. For the [Li+]/[Na+] binding studies, reasonable changes were made to the optimal instrumental tuning parameters, such as ESI spray voltage, source declustering potential and ion energy, to determine if these changed the above K values. No significant effects were observed. The assumption that the ratio of the actual metal ion concentrations is the same as that calculated from the added solution components neglects any influence on these concentrations from the formation of dimeric [2TAG + M]+ species, of mixed [TAG + X + 2 M]+ species formed with the cationization salt (trifluoromethanesulfonate), or of any metal ion clusters with this anion. Such interactions could shift the [TAG + M]+ equilibria significantly. Other factors affecting the observed results could also involve solubility effects, since a considerable evaporation of solvent must occur prior to ion evaporation, perhaps increasing the concentration of either or both the electrolyte or TAG to or beyond its solubility limit. Increasing the concentrations will also result in an increase in the nonideality of solution. Other phenomena at the drop surface may also be involved. Nevertheless, the general observations that sodium ion binding is preferred over that of lithium or silver ions in the absence of unsaturation, that unsaturation Table 1. Values of K for Various Ratios of [Li+]/[Na+] and [Ag+]/[Na+] Conc. Ratio

K for selected TAGs, Na+ versus Li+

[Li+]/[Na+] 9.0 4.0 1.5 0.67 0.25 0.11

PSP 3.26 2.22 1.52 1.13 0.76 0.61

POP 2.74 2.20 1.35 1.11 0.79 0.67

PPO 2.68 2.16 1.61 1.32 0.91 0.68

OOO 5.35 2.45 1.78 1.39 1.05 0.82

K for selected TAGs, Na+ versus Ag+ [Ag+]/[Na+] 9.0 4.0 1.6 0.67 0.25 0.11

PSP 3.82 2.36 1.80 1.47 2.08 2.37

POP 0.38 0.14 0.11 0.098 0.12 0.21

PPO 0.35 0.17 0.099 0.13 0.15 0.41

OOO nd 0.0087 0.010 0.030 0.068 0.12

does not play a large role in alkali metal ion binding and that silver ion binding is strongly affected by unsaturation but not by regioisomerism are still valid. This suggests that Li+ and Na+ bind preferentially to oxygen atoms on the glycerol backbone, as will Ag+ in the absence of unsaturation. The small values of K for Ag+ and TAGs with unsaturation indicate that Ag+ ions bind more readily to carbon–carbon double bonds than either Li+ or Na+. Several authors have reported that separation of positional isomers in silver-ion chromatography (Ag-HPLC) is dependent on the steric availability of double bonds for interaction with silver ions [5, 8]. Using this method, silver ions incorporated in the chromatographic stationary phase interact reversibly with the π electrons of double bonds in fatty acyl residues. An example of these effects is that PPO is retained more strongly than POP, which enables the two isomers to be separated. These effects are in line with HSAB theory [51] which describes how hard bases such as oxygencontaining species bind better to hard acids such as H+, Na+, whereas softer bases such C–C π bonds bind preferentially with softer acids such as Ag+. One might expect to observe a similar effect on the cationization of POP and PPO by silver ions during the ESI process, but in our work both similar absolute ion counts for [TAG + Ag]+ adducts of POP and PPO, as well as similar K values for POP and PPO, were obtained. This indicates that the binding of TAGs with silver cations is not influenced by the position of a fatty acid on glycerol to any large extent. It is apparent that double bonds in unsaturated TAGs should be considered in determining the structures of the cationized TAGs as they can become coordinated to all of the cationic species we studied. We have not studied any polyunsaturated TAGs in detail.

Computed Binding Energies of Cationized TAGs Using B3LYP computations with various basis sets, we have computed the energies, (Eelec + EZPVE), for up to six possible geometries of adduct ions [TAG + M]+ where M = Li or Ag. Results only for the structures having the lowest energies are given in Table 2. While the absolute values of binding energies varied somewhat between a small basis set 631G(d) and the larger 6-311G(2d,p) basis set, the relative values were the same. The DZVP basis set was found to be suitable for M = Ag complexed with amino acids [35] and was, therefore, used for comparison of argentiated, lithiated, and sodiated complexes of 2. The in vacuo binding energies presented in Table 2 provide an understanding of the stability of [TAG + M]+ complexes in the gas phase and show that all cations bind to both carbonyl groups and C–C π bonds, but that for Ag+, binding to a π bond is especially important. Computed bond distances and vibrational frequencies for the structures are also presented in Table 2 since these yield information about the binding between atoms and groups. In all cases, the bond distances and vibrational frequencies show the same trends


Table 2. Computed Propertiesa of Selected Cationized TAGs, Including TAG–Cation Bond Distances, O–cation, N–H, and C=O Vibrational Frequencies, and Gas-Phase Binding Energies. Argentiated and Sodiated TAGs Had Similar Structures to Those Shown in Figure 3 for the Lithiated Species; Ammoniated and Protonated Species Are Shown in Supplementary Figure 5S, with dimers in Supplementary Figure 7S Structure

TAG – cat. distances, Å

O – cat. or N – H vib. cm–1

C = O str. cm–1

Gas-phase ion affinities (GIA) kJ mol–1 b

1SA 1A, M = Li 1A, M = Na 2S 2A, M = Li 2B, M = Li 2C, M = Li 2A, M = Na 2B, M = Na 2C, M = Na 2A, M = Ag 2B, M = Ag 2C, M = Ag 5SA, M = H 5SB, M = H 5SC, M = NH4 5SD, M = NH4 7SA, M = Li 7SB, M = Na 7SC, M = NH4

– 1.92, 1.93, 2.04, 2.17 2.27, 2.28, 2.38, 2.43 – 1.91, 1.93, 2.04, 2.16 1.81, 1.82 1.78(O); 2.35, 2.39(CC) 2.26, 2.28, 2.38, 2.42 2.16, 2.18 2.14(O); 2.68, 2.76(CC) 2.43, 2.49, 2.48, 2.64 2.29, 2.30 2.24(O); 2.46, 2.47(CC) 1.01, 1.63 1.02(O); 2.07, 2.14(CC) 1.82, 1.83, 1.83 1.76, 1.94 (O); 2.39, 2.42(CC) 1.96, 1.97, 1.97, 1.97 2.27, 2.27, 2.27, 2.27 1.81, 1.81, 1.84, 1.84

– 413, 440, 481 164, 204, 211 – 406, 461 620, 641 616, 644 205, 222 – 299 189, 198 – 245 2975 (OH str) 2931 (OH str) 3220, 3242, 3267, 3543 e 3184, 3287, 3514 e 375, 416 257 3264, 3312, 3328, 3546e

1824, 1829, 1832 1753, 1772, 1791 1771, 1786, 1798 1821, 1826, 1830 1750, 1770, 1789 1705, 1733, 1816 1712, 1801, 1834 1768, 1784, 1795 1731, 1751, 1810 1736, 1796, 1833 1739, 1749, 1765 1695, 1717, 1791 1693, 1788, 1809 1595, 1759, 1785 1657 d,1801, 1824 1733, 1751, 1764, 1786, 1746, 1754, 1769, 1790, 1749, 1750, 1754, 1785, 1757, 1758, 1763, 1783, 1768, 1770, 1777, 1793,

– 310 220 – 300 303 244 225 232 179 238 243 251 907 913 194 144 443 347 280

1791 f 1835 f 1815, 1817 1811, 1813 1814, 1815

c c

a

Structures 1, 7 fully optimized with G09/B3LYP/6-31G(d), 2, 5 with G09/B3LYP/DGDZVP. Single-point energies (kJ mol–1) corrected for basis set superposition errors (BSSE) by the counterpoise method, 1, 7 with G09/B3LYP/6-311++G(2d,p), 2, 5 with G09/B3LYP/DGDZVP. c Single-point energy, no BSSE correction needed. d Combined O–C, C–O str, and OCO bend. e NH vibrations. f Includes C=O str and HNH bend. b

as the computed binding energies, with weaker interactions having longer bonds or lower wave numbers. In the case of the carbonyl stretching frequencies, a weaker interaction between the metal ion and the carbonyl group resulted in a larger wave number for the C = O stretch. The binding energies for protonated TAGs, 5SA, 5SB show how tightly protons can be bound to carbonyl oxygen atoms or to C–C π bonds, but as these ions are not formed by ESI, the gas-phase binding of protons to the alcohol solvent used is greater [52]. The binding between TAG atoms and ammonium ions is relatively weak, which can be correlated with the high concentrations of ammonium ions needed to obtain an adequate signal of [TAG + NH4]+ ions. Modeling of the [TAG + M]+ adducts in which the M cation (M = H, Li, Na, or Ag ) is bound to the O atoms of the carbonyl groups of the sn-1 and sn-3 fatty acids (sn-1/3 complex), or the sn-1 and sn-2 fatty acids (sn-1/2 complex), showed that the sn-1/3 complex is more stable than the sn-1/2 by 31 kJ mol−1 (H+), 24 kJ mol−1 (Li+), 20 kJ mol−1 (Na+), and 15 kJ mol−1 (Ag+). Moreover, the sn-1/3 and sn-1/2 complexes of [TAG + M] + adducts of a triacylglycerol containing two of the same saturated acid chains, A, and one unsaturated chain, B, may form the two possible positional isomers, ABA and AAB, which have comparable stability. It is likely that the same result would apply to TAGs from two different saturated acids (A, A′) and one unsaturated acid (B), where three

combinations are possible, AA′B, ABA′, and A′AB (see Supplementary Material Figure 4S). Solvent Effects in the Binding of TAGs with Cations With regard to using ESI-MS in analytical methods for determination of TAGs, we have found that for solutions containing both Li+ and Na+ (added independently or as background) ESI favors production of sodiated TAGs despite the fact that the binding energies of TAGs in the gas phase clearly favor Li+ over Na+. The nature of ions generated in an ESI source, and their relation to ions present in solution, has attracted much attention [45, 50, 53–56]. In cases involving competitive ion formation, the nature of the ions observed in the mass spectrometer can be correlated with the solvent, with analyte concentrations, and with the relative energies of the competing species. TAGs, essentially nonpolar molecules, will have little affinity for methanol, whereas ions will have stronger interactions with this solvent. However, the binding energy of Li+ to methanol is substantially greater than that of Na+ [57, 58]. Thus, Li+ ions with respect to Na+ ions will be preferentially associated with methanol, resulting in more sodiated TAG ions being ejected with respect to lithiated TAG ions. To explore how the difference between the interactions of Li+ and Na+ with TAGs might be described, we repeated some of the computations shown in Table 2 using the polarizable continuum model (PCM) in a self-consistent


reaction field (SCRF) with a universal force field set for the dielectric constant of methanol [27], as shown in Table 3. The net effect of using this force field is to increase the bond distances between the binding atoms and the cation, which results in changes to the infrared stretching frequencies, but more importantly to the energetics between the ion and neutral TAGs. In each case for both the ion affinity and the relative free energy, the value for the sodiated TAG was more positive than the lithiated TAG, which can be interpreted that solvation energy shifts the equilibrium in favor of formation of sodiated TAGs over lithiated TAGs. This is consistent with the observation that for equal concentrations of Na+ and Li+, the ESI signal for [TAG + Na]+ ions is more intense than for [TAG + Li]+ ions in methanol.

Formation of Dimeric [2TAG + M]+ Species Covalently bonded dimeric TAG ions have been reported [10], but these ions appear to be radical-induced oxidation products of the fatty acid chains in TAGs, resulting in crosslinking. There appear to be few reports of dimeric ions in which a cation is complexed to a pair of TAGs, producing ions in the form of [2TAG + M]+, in which the two TAGs may or may not be different [49, 59–61]. The fact that these ions appear not to have been widely studied is undoubtedly due to the fact that most analyses involve [TAG + M]+ species, with little need to scan above m/z 1,300. Lévêque and coworkers, using ESI and an LTQ ion trap, have studied the MS2 spectra of [2TAG + Li]+ ions [59] as a means of determining regioisomer composition. Bird et al. published a spectrum which showed low-intensity peaks corresponding to [2TAG + NH4]+ ions [60], but made no comment on these. Gerbig and Takáts, using DESI as an ionization method and an LTQ Orbitrap, observed dimers of triacylglycerols as ammonium adducts [61], the formation of which

“was associated with van der Waals forces between the long hydrocarbon chains of the fatty acid residues.” Since the presence of TAG dimeric ions has potential analytical significance, we investigated their formation and performed computations to assess the binding energies for selected species. Spectra of complex natural TAG samples would be expected to show mixed TAG dimers (e.g., [TAG1 + TAG2 + M]+). However, if a chromatographic separation precedes MS analysis, the spectra will be much simpler. For this reason and to reduce overall complexity, only dimers of single TAGs were considered in our analysis. Spectra of different cationized TAGs are shown in Figure 4a, b and Supplementary Material Figure 6S in which examples of lithiated, sodiated, potassiated and ammoniated ions were observed showing both structures [TAG + M]+ and [2TAG + M]+. Similar results were obtained from QTOF, QTRAP, and single-stage Orbitrap (Exactive) instruments, which showed that this behavior was not instrument-specific. As shown in the figures, there appears to be no difference in relative intensities of monomer versus dimer ion formation if the TAG has saturated or unsaturated fatty acid residues. There are, however, clear differences in relative intensities of dimer ions with respect to the [TAG + M]+ cation. Thus, ammoniated dimer ions are much lower in intensity relative to the monomer compared with lithiated or sodiated ions, which corresponds to weaker binding of the ammonium cation to the pair of TAGs. We addressed this observation by computing the gasphase binding energies for dimeric species of model TAGs having three-carbon saturated chains plus ammonium, lithium and sodium cations with the results being shown in Table 2. Computed energies, including a universal force field set for the dielectric constant of methanol, are shown in Tables 2 and 3 and structures are shown in Supplementary Material Figure 7S. Geometrically, it is very possible for the two pairs of carbonyl groups in two TAGs having the

Table 3. Computed Properties of Selected Cationized TAGs Using a Universal Force Field with the Dielectric Constant for Methanol Solvent, Showing TAG–Cation Bond Distances, O–Cation and C=O Vibrational Frequencies, and Relative Binding Energies. Structures of Tripropionylglycerols (1) Are in Supplementary Figure S1 and Figure 1, C11 TAGS (2) Are in Supplementary Figure 2S and Figure 3, Where Argentiated and Sodiated Species Had Similar Structures to the Lithiated Ion, and Dimers (7) Are in Supplementary Figure 7S Structure

TAG – cat. distances, Å

O – cat. vib., cm

1SA c 1A, M = Li c 1A, M = Na c 1B, M = Na c 2Sd 2A, M = Lid 2A, M = Nad 2A, M = Agd 2C, M = Agd 7SA, M = Li c 7SB, M = Na c

– 2.00, 2.00, 2.15, 2.63 2.37, 2.37, 2.49, 2.51 2..30, 2.32 – 2.09, 2.09, 2.19, 2.52 2.42, 2.43, 2.52, 2.56 2.54, 2.56, 2.71, 2.71 2.35(O); 2.53, 2.55(CC) 1.98, 1.99, 2.01, 2.01 2.32, 2.32, 2.33, 2.33

– 283, 123, 177 – 307, 141, – – 345, 197

302, 356 140 338, 394 144 373, 405

–1

C = O str., cm 1795, 1749, 1768, 1755, 1760, 1735, 1746, 1728, 1717, 1743, 1756,

1797, 1761, 1777, 1763, 1760, 1745, 1752, 1735, 1758, 1744, 1758,

–1

1800 1800 1779 1795 1767 1765 1756 1752 1762 1746, 1762, 1796, 1797 1762, 1766, 1794, 1795

IA = – E[TAG + cat] + E[TAG] + E[cat], E = (electronic energy + zero-point energy) in kJ mol–1. ΔG = as above, but E = electronic + thermal free energy in kJ mol–1. c Optimized using G09/B3LYP/6-31G(d) computations. d Optimized using G09/B3LYP/DGDZVP computations. a

b

Ion Affinity, IA – -33 -18 5 – -34 -14 -3 20 49 63

a

Relative free energy, ΔG b – -70 -55 -28 – -78 -42 -44 -35 -46 -15


by four carbonyl groups and are thus similar to the dimer ions from TAGs reported on here.

Fragmentation of Cationized TAGs As mentioned earlier, there have been few detailed studies of fragmentation mechanisms when cationized triacylglycerols are subjected to collisional activation. We have explored several approaches to the problem, including a study of breakdown curves, isotopic labeling, MS3 spectra and extensive DFT computations. Breakdown Curves

Figure 4. ESIMS (MeOH) of (a) LaOP (1.0 μM) + LiOAc (100 μM) on a QToFMS, showing [2TAG + M]+ dimers, as well as adduct ions from extrinsic [TAG + Na]+ and [TAG + K]+ ions; (b) OOO (1.0 μM) + NH4OAc (100 μM) on an Exactive Orbitrap MS, showing [TAG + NH4]+, [TAG + Na]+, [2TAG + NH4]+, and [2TAG + Na]+ ions

geometry of 1B or 2B to bind to a cation resulting in tetrahedral coordination. The binding energies in the gas phase mirror those for the monomeric cations, with lithiated ions being the most tightly bound and ammoniated ions being the least. Relatively, the binding energy (in the gas phase) gained by dimer formation is less for ammonium than it is for the other ions, which is in accord with the observed spectra. Computations of the dimers were somewhat challenging, given the size of the floppy species, and it should be noted that the B3LYP functional is not parameterized to compute long-range van der Waals interactions. Application of the solvent force field was successful in the cases of Li+ and Na+ and, as in the case of the monomers, the results suggest that sodiated dimer formation from a methanolic solution should be favored over lithiated dimer formation. We suggest that dimer formation might have implications for analyses of complex lipid mixtures if quantification is based solely on intensities of [TAG + M]+ species. Apart from loss of signal intensity of the monomers, minor changes in LC-MS mobile phase composition could alter the distribution between monomer and dimer, thereby affecting the robustness of the method. The fact that cations could become complexed by two TAG molecules, geometry permitting, should not be surprising since such dimeric species must be inherently of lower energy than a [TAG + M]+ as the charge is more delocalized. Indeed, if the geometry is favorable, higher coordination numbers can be observed [62]. Recently, it was found that two molecules of ethyl pyruvate can become complexed to sodium ions in ESI-MS, leading to dimer ion formation [48]. These ions have sodium cations complexed

Ham and Cole [63] reported on product-ion formation and breakdown curves when lithiated mono- and diacylglycerols were subjected to low-energy CID. They observed product ions from chain degradation and water losses in a QqQ spectrometer, which they were able to correlate with computed energies and propose mechanisms for the fragmentations. We chose to examine the sodiated ions and observed much simpler spectra. The only product ion we observed when both sodiated glycerol and monopalmitin were subjected to CID at modest collision energies (QqQ, 25 eV, laboratory frame) and multiple collisions was m/z 23, the sodium ion. By contrast, under the same conditions, CID on sodiated dipalmitin (m/z 591) yielded product ions at m/z 335, 313, and 279. The first two ions correspond to loss of neutral palmitic acid and its sodium salt, respectively. The ion at m/z 279 is sodiated palmitic acid, [C16H32O2 + Na]+, and had the lowest intensity at all collision energies. Breakdown curves for sodiated mono- and dipalmitin are shown in Supplementary Material Figure 8S. At lower collision energies, the ion at m/z 335 was predominant, but at higher collision energies, the ion at m/z 313, [C19H37O3]+, became predominant. The fact that we did not observe formation of m/z 23 from sodiated dipalmitin indicated that the second ester group significantly increased binding to the cation compared with the single ester group in monopalmitin, or to the hydroxyl groups in glycerol. Furthermore, the second ester group facilitates fragmentation of the diacylglycerol moiety, and we will provide evidence (vide infra) that the interaction of ester groups with the cation controls the fragmentation of cationized TAGs. Given these data, we turned our attention to a sodiated triacylglycerol and determined breakdown curves for [POP + Na]+ under similar conditions. These are shown in Supplementary Material Figure 9S. The results were comparable to those from sodiated dipalmitin, namely, no evidence for the formation of an ion at m/z 23 was found, but as the collision energy was increased, all four possible product ions were detected as the intensity of the precursor ion at m/z 855 decreased. By contrast, m/z 23 was observed under highenergy collision conditions using a MALDI-TOF/TOF


instrument [22]. The ion observed at m/z 599 was due to [POP + Na – C16H32O2]+ (i.e., to loss of neutral palmitic acid), whereas that at m/z 577 was due to [POP + Na – C16H31O2Na]+ (i.e., to loss of neutral sodium palmitate). Similarly, the minor ions observed at m/z 573 and 551 corresponded to the analogous losses for the oleyl group in the sn-2 position of POP. The intensity of the ion at m/z 599 began to decrease at higher collision energies, which implies that it is undergoing further fragmentation [14]. In order to assess whether unsaturation at different positions on the glycerol moiety affects fragmentation, we determined breakdown curves for four lithiated TAGs, namely PPP, POP, PPO, and OOO. By repeating these experiments on the argentiated ions of the same TAGs, we could determine differences, if any, of cationization by a hard and a soft cation. The results are presented in Figure 5. It is apparent that if the TAG is symmetrical, unsaturation has no effect on fragmentation of the lithiated species (e.g. [PPP + Li]+ versus [OOO + Li]+), but the corresponding argentiated ions gave significantly different curves. When the substituents of the TAGs are not symmetrically arranged (e.g., POP versus PPO), the product ion distributions were different for both the lithiated and argentiated species. This is consistent with the known fact that loss of sn-1/3 substituents is more facile than the loss of sn-2 substituents [14]. It was also obvious that the predominant fragmentation of both argentiated POP and PPO was loss of neutral palmitic acid, even as the collision energy was increased. This indicates that the Ag+ ion is more tightly bound to the unsaturation site in the oleyl chain than is the Li+ ion. It the case of [POP + Li]+, the intensity of the ion [PO_ + Li]+ formed from loss of neutral palmitic acid decreased as the collision energy was increased, indicating that it was undergoing further fragmentation, which could be studied by examining MS3 spectra (vide infra). Corresponding work on MS n spectra for argentiated ions has been reported [6]. In a previous study of fragmentation products as a function of collision energy using a tandem quadrupole time-of-flight MS, it was found that the relative ease of generating DAG-like ions from [TAG + M]+ adducts followed the order [TAG + Na]+ G [TAG + Ag]+ G [TAG + Li]+ G [TAG + NH4]+, showing that the ammoniated species are the most labile [16].

Computed Mechanisms for the Loss of an sn-1/3 RCOOH Group from [TAG + Li]+ Ions by an Initial Proton Transfer As a result of the detailed work published by Hsu and Turk [14, 15], certain experimental facts need to be incorporated into any proposed mechanism for the formation of product ions from a [TAG + Li]+ ion. These include that for the loss of a neutral acid moiety RCOOH, the proton comes from a methylene group alpha to a carbonyl group in one of the

Figure 5. Breakdown curves showing fractional abundances plotted against center-of-mass collision energy for selected cationized TAGs, (AA_ or _AB = loss of the neutral acid AH, [AA]+ or [AB]+ = loss of a neutral salt [Acat] or [Bcat])

ester chains, which was demonstrated by labeling of these groups with deuterium. There should also be some energetic factor in a mechanism to account for the preferred loss of a neutral acid from a primary ester (i.e., from an sn-1/3 position), as opposed to the loss of an acid from the secondary ester (i.e., from the sn-2 position). A further factor is that if an acid is lost from an sn-1/3 position, an important fragment in a second stage of fragmentation (MS3) is a molecule with a formula equivalent to an α,ß-unsaturated acid lost from the sn-2 position. The mechanisms proposed by Hsu and Turk [14, 15] offered structures that accommodated, but did not verify, the first and third of these points, but addressed neither the necessary geometric requirements nor the energetic requirements for reactions to occur. We have approached the fragmentation processes for losses of neutral acids and neutral lithium salts from [TAG + Li] + ions using DFT computations on lithiated tripropionylglycerol [3C3 + Li]+ as a model system (see Figure 2). These ions are of adequate size to probe the above points, yet small enough to allow for reaction pathways to be computed with reasonable computational efficiency. In the first instance, we focused on abstraction of a proton from the methylene group alpha to a carbonyl group, which could only occur if the proton was made sufficiently acidic by complexation of a lithium cation on the adjacent carbonyl group. The proton would have to be abstracted by one of the only reasonably basic sites in the [TAG + Li]+ ion, namely


an uncomplexed carbonyl group (i.e., there is a restrictive geometric requirement to reach a saddle point for proton transfer. These constraints limited the possible structures in the series presented in Figure 2, and certainly the most stable structure, 1A, would not lead to product ions as all four oxygen atoms are complexed to the cation. It was apparent that the energetic requirements for transfer of a proton from a methylene group alpha to a carbonyl group complexed to a lithium ion were high, sufficiently high that we initially became concerned about the validity of the experimental data that supported this. Repetition of the experiment in which a cationized d5-TAG (fully deuterated glyceryl backbone) was subjected to CID left no doubt that the proton in the loss of RCOOH must indeed come from a methylene group alpha to a carbonyl group, which indicated that finding a viable saddle point for proton transfer required a search through a range of computed geometries of [3C3 + Li]+ ions (see Figure 2). This search ultimately led to a series of ions for the fragmentation pathway, shown in classic “arrow-pushing” format in Schemes 1a (proton transfer from an sn-2 chain) and 1c (proton transfer from an sn-1/3 chain), while detailed pathways from computations are shown in Scheme 1b and d, respectively. Given the symmetry properties of a TAG molecule, two pathways should be expected and, indeed, the fragmentation pathways not only turned out to be different but also led to different products. As the work progressed, it became obvious that different product ion structures were required in order to understand the subsequent fragmentations observed in MS3 experiments. For the pathway with proton transfer from an sn-2 chain (Scheme 1a, b), only structure 1E appeared to have the required geometry, which appeared to be stereoselective in that only one of the two protons bound to C(3) (marked in green (Scheme 1b), 252 pm from the closest carbonyl oxygen atom) was accessible for abstraction. Simultaneously, there must be a cleavage of the C–O bond involving the black carbon (sn-1) of the glycerol chain and also a rotation of the C–O bond to the sn-3 carbon of the glycerol chain so that the sn-3 carbonyl oxygen atom labeled 20 becomes associated with the black (sn-1) carbon atom. Together with the departing oxygen atom, these two oxygen atoms become aligned in a trigonal bipyramidal geometry of the transition state, analogous to that for a classical SN2 displacement reaction. These significant electronic and geometric realignments to form saddle point SP11 have a substantial energy requirement as indicated in Scheme 1b. However, once this saddle point has been reached, formation of the ion-neutral complex IN11 can occur with the loss of some 150 kJ mol−1. Ion-neutral complexes are known to be important in fragmentation processes [64, 65]. The proposed formation of a 1,3-dioxan-2-yl cation in IN11 differs from the proposals advanced by Hsu and Turk, who proposed the intermediacy of 2-alkylidene-1,3-dioxolanes (Supplementary Material Figure 10S) in their mechanisms [14, 15]. Examination of structure IN11 indicated that C(3) was likely electron-rich with respect to the white (sn-3) carbon atom in

the 1,3-dioxan-2-yl ring and, indeed, we were able to find a saddle point (SP12) for a second SN2-type displacement reaction that was only some 53 kJ mol−1 higher in energy than IN11. This saddle point led to the formation of an ionneutral species (IN12) containing a γ-lactone, which had a lower energy than the precursor ion. Loss of a molecule of neutral acid gave a product ion Lac11 as a five-membered lactone ring, which was close to being isoenergetic with the postulated precursor ion 1E. To our knowledge, lactones have not been proposed previously as possible structures of product ions from the CID of cationized TAGs. Attempts to find an analogous pathway in which initial proton transfer occurred from an sn-1/3 moiety were unsuccessful. After further exploration, it became obvious that keto-enol tautomerism from a lithiated ester was energetically possible also. The pathway ultimately found is shown in Scheme 1c and d. This began from a conformer, 1A′, of the low-energy cationized TAG 1A with proton transfer leading via saddle point SP13 to an ester enol (sn-1) also having 4 oxygen atoms complexed to the lithium cation, 1A″. Proton transfer from the sn-1 chain to the sn-3 chain (SP14) led to a novel cationized 1,5-dioxocane ring 1A′′′, only 140 kJ mol−1 higher in energy than 1A′. Collapse of this ring via saddle point SP15 (also via an SN2-type transition structure) led to the ion-neutral complex IN13 in which charge was on the 1,3-dioxolan-2-yl ring rather than on the lithium. The lithium enolate carbon atom in IN13 can be expected to be nucleophilic as discussed previously, which can lead with a very modest energy input to the saddle point SP16, from which formation of the ion-neutral species IN14 is highly exothermic, as was the case in the formation of IN12. Loss of the sn-3 neutral acid from IN14 leads to a 6-membered cationized lactone ring, Lac12. While the absolute energy of SP15 was computed to be 392 kJ mol−1 higher in energy than 1A′ the step from 1A′′′ only involved an energy input of 250 kJ mol−1, thus making the initial proton transfer (enolization) step at 310 kJ mol−1 the highest barrier in the process. This is lower than the barrier of 342 kJ mol−1 for proton transfer from 1E to SP11, although with these energy requirements, neither process could be considered to be facile. However, we have been unable to find any lower energy pathways for an initial proton transfer process, a process required by the experimental data.

Computed Mechanism for the Loss of an sn-1/3 RCOOLi Group from [TAG+Li]+ Ions Computing a reasonable pathway for the loss of a neutral salt moiety from an sn-1/3 position turned out to be relatively straightforward, as shown in “arrow-pushing” format in Scheme 2a and as outputs from computations in Scheme 2b. In this case also, significant geometric requirements appeared to be essential in order to lead to a viable reaction pathway. Thus, structure 1G, a relatively highenergy isomer of the base structure 1A, had the appropriate


Scheme 1. Mechanisms for the loss of an sn-1/3 RCOOH group from [TAG + Li]+ ions

geometry for the carbonyl group of the ester on the sn-1 carbon atom to attack the electrophilic carbon atom at the sn3 position, which led to saddle point SP21 in which a carbon atom has a trigonal bipyramidal geometry and long bonds to

a pair of oxygen atoms. This saddle point was at a relatively low energy with respect to structure 1G. Saddle point SP21 can collapse to form the ion-neutral complex IN21 containing a neutral lithium carboxylate and the charged moiety as a

Scheme 2. Mechanism for the loss of an sn-1/3 RCOOLi group from [TAG + Li]+ ions


1,3-dioxan-2-yl cation. Species IN21 can dissociate by loss of the neutral lithium carboxylate into a product ion Cat21. However, compared with the case of loss of the neutral acid, this formation of a product ion is substantially endothermic. It should be noted that as with all conformationally mobile species, other conformers of IN21 having similar energies were also found. Thus, loss of a lithium carboxylate from an sn-1/3 position begins with a lithiated TAG that must be a minor component of the mixture of such ions being ejected from the ESI source and while the energy needed to reach the saddle point SP21 is low, formation of the product ion is significantly endothermic. Computed Mechanism for the Loss of an sn-2 RCOOLi Group from [TAG + Li]+ Ions The loss of a neutral lithium salt from an sn-2 position appeared to be quite straightforward as outlined in Scheme 3a and b. In this case, a viable geometry for the fragmentation was found in the relatively low-energy structure 1E in which the carbonyl group of the sn-1 ester could displace a leaving group on the sn2 carbon atom activated by complexation with a lithium cation. This led to the saddle point SP31 only 107 kJ mol−1 above structure 1E. The saddle point can collapse to the ion-neutral IN31 from which the product ion Cat31 containing the cationic site in a 1,3-dioxolan-2-yl ring can be formed by loss of the neutral lithium carboxylate and injection of some 112 kJ mol−1. The energy requirements for loss of a lithium carboxylate from the sn-2 position are lower than those for the same loss from the sn-1/3 positions, but losses from these positions are favored statistically. Computed Mechanism for the loss of an sn-2 RCOOH Group from [TAG + Li]+ Ions Finding a viable mechanistic pathway for this minor process turned out to be challenging; the results are outlined in Scheme 4a and b. The “arrow-pushing” scheme in Scheme 4a represents only key ion-neutral complexes, with the actual pathway that is proposed from computations being much more complex. The same experimental constraint applied for loss of a neutral acid from the sn-1/3 positions, namely that the proton abstracted came from the methylene group alpha to the carbonyl group on an sn-1/3 chain. Generating the requisite geometry led us to structure 1H,

which, at 153 kJ mol−1 above 1A, must be a minor component of the mix of structures emitted from the ESI source. The lithium cation is complexed to three oxygen atoms at bond distances of 198, 198, and 208 pm with only one bond being to a carbonyl oxygen atom; these bond distances are the longest Li–O bonds in the structures in Figure 2. Abstraction of a proton (colored green) from a C10–H bond requires adequate polarization of the bond, and this was apparently achieved by bond rotations leading to the lithium cation retaining coordination to three oxygen atoms, but being bound more tightly to one atom; the computed bond distances were 187, 204, and 216 pm. Reaching saddle point SP41 (Scheme 4b) required the input of some 233 kJ mol−1 to enable the sn-2 carbonyl oxygen atom (O31) to abstract the green-colored proton from C10. The bond distance between C8 and C10 at 136 pm is now that of a typical bond between sp2-hybridized carbon atoms. The imaginary frequency from the computed infrared spectrum of SP41 (–220 cm–1) does not correspond to that of a typical proton bound between two basic sites, but rather represents skeletal oscillations in which the green-colored proton and C10 can become close or distant. The latter motion leads to an ion-neutral complex IN41 in which the lithium cation is tricoordinate, bound to three oxygen atoms in a sevenmembered ring. If the sn-3 ester group is now allowed to undergo rotation about the C–O bond, the carbonyl oxygen atom, O15, can displace a neutral acid (formerly the sn-2 group) leading to a new saddle point SP42 in which a central carbon atom is partially bound to two oxygen atoms in the trigonal bipyramidal geometry of a classic SN2 transition state. The product of this reaction has structure IN42. However, as proposed previously for the loss of a neutral acid from the sn-1/3 position, IN42 is not the terminal structure in the fragmentation process since C10 is nucleophilic, being part of an enolate anion, and both atoms C4 and C5 may be considered to be electrophilic. Ion-neutral complex IN42 can collapse in three different ways leading to product ions Lac41–Lac43 having structures of cationized δ- and γ-lactones, as detailed in Scheme 4b. The complexity leading to the formation of these ions arises from the fact that whereas IN11 contains a symmetrically substituted ring, IN42 does not. In this case, ring opening at the primary position proceeds via SP43 and IN43 to give the lithiated δ-lactone Lac41, whereas opening at the secondary position leads to the lithiated γ-lactones Lac42

Scheme 3. Mechanism for the loss of an sn-2 RCOOLi group from [TAG + Li]+ ions


Scheme 4. Mechanisms for the loss of an sn-2 RCOOH group from [TAG + Li]+ ions

and Lac43, which are geometric isomers. The energetics of all three pathways are comparable, which suggests that the loss of an sn-2 acid from a lithiated TAG leads to product ions having a mixture of structures.

MS3 Spectra of [TAG + Li – sn-1/3 RCOOH]+ Ions The product ion from the loss of a neutral acid from the sn1/3 position has been shown to have analytical utility because when it is subjected to further fragmentation in an MS3 experiment, the nature of the sn-2 and sn-3/1

substituents is revealed [15, 18]. Results showing the major product ions from a series of such experiments using a linear ion trap are given in Table 4; typical spectra, using lithiated PSP, are shown in Figure 6. Similar results were obtained on generating the initial product ions in-source in a QTOF MS and dissociating these ions by CID in the collision cell. The results are thus not dependent on instrument type. The power of the technique is illustrated using the example of lithiated LaOP, [C49H92O67Li]+, where MS3 on the product ion at m/z 527 resulting from the loss of neutral palmitic acid gave three major product ions, namely m/z 289 (lithiated oleic acid, [C18H34O27Li]+), m/z 207 (lithiated


Table 4. MS3 Data from CID Experiments on Selected Lithiated TAGs After Loss of a Neutral Acid from the sn-1/3 Positions (MS2) TAG

m/z [ABC + Li]+

m/z [AB_ + Li]+

m/z MS3 (most intense ions)

LaOP POLa MEM PLP POP PSP PEP OPO SOP a LnLnLn PDP LOL DPD

783 783 803 837 839 841 859 865 867 879 885 887 957

527 583 575 581 583 585 603 583 611 601 629 607 629

289, 289, 309, 287, 289, 291, 309, 329, 291, 285, 335, 327, 375,

[O + Li]+; 247, [La__ + 2H + Li]+; 207, [La + Li]+ [O + Li]+; 303, [P__ + 2H + Li]+; 263, [P + Li]+ [E + Li]+; 275, [M__+2H+Li]+; 235, [M+Li]+ [L + Li]+; 303, [P__ + 2H + Li]+; 263, [P + Li]+ [O + Li]+; 303, [P__ + 2H + Li]+; 263, [P + Li]+ [S + Li]+; 303, [P__ + 2H + Li]+; 263, [P + Li]+ [E + Li]+; 303, [P__ + 2H + Li]+; 263, [P + Li]+ [O__ + 2H + Li]+; 289, [O + Li]+; 263, [P + Li]+ [S + Li]+; 331, [S__ + 2H + Li]+; 289, [O + Li]+ [Ln + Li]+; 325, [Ln__ + 2H + Li]+ [D + Li]+; 303, [P__ + H + Li]+; 373, [_D_ + Li]+; 263, [P + Li]+ [L__ + 2H + Li]+; 287, [L + Li]+; 289, [O + Li]+ [D__ + 2H + Li]+; 335, [D + Li]+; 287, [C18H32O2Li ]+ b

a

from ref [15], F.-F. Hsu and J. Turk, J. Am. Soc. Mass Spectrom. 21, 657-669 (2010) loss of C23H34O2, i.e. D plus one methylene group from the glycerol moiety, likely as the methyl ester of D. Abbreviations for the TAGs, in ABC format, are La: = 12:0, M=14:0, P=16:0, S=18:0, O=18:1, L=18:2, Ln=18:3, E=20:5, D=22:6, AB_ = loss of the neutral acid corresponding to C and (A__+2H)=the loss of the neutral acid B minus 2 hydrogen atoms (B – 2H), Li = 7Li isotope. b

lauric acid, [C12H24O27Li]+), and m/z 247, [C15H28O27Li]+. By contrast, MS3 on the product ion at m/z 583 resulting from the loss of neutral lauric acid gave three complementary product ions, namely m/z 289 (lithiated oleic acid, [C 18 H 34 O 2 7 Li] + ), m/z 263 (lithiated palmitic acid, [C16H32O27Li]+), and m/z 303 [C19H36O27Li]+. It is noteworthy that in almost every case, two of the ions were the lithiated acids remaining after the loss of an sn-1/3 neutral acid at the MS2 stage. The third ion was invariably one containing glycerol atoms and the remaining sn-1/3 acid, but with a transfer of two hydrogen atoms. In the literature, this has been described as the loss of an α,ß-unsaturated acid with the lost hydrogen atoms being proposed to come from carbon atoms alpha and beta to a carbonyl group [14, 15].

Figure 6. Product ion spectra of [PSP + 7Li]+. (a) MS/MS spectrum of m/z 841. (b) MS/MS/MS of m/z 841 → m/z 585

To us, it seemed problematic to explain the structural rearrangements required for formation of the MS3 product ions shown in Table 4 by using the literature structure shown in Supplementary Material Figure 10S. By contrast, the structures of the cationized lactones Lac11 and Lac12 appeared to offer direct, simple routes to the three major product ions. Our proposals are shown in Scheme 5a (“arrow pushing”) and 5b (detailed paths from DFT computations). We draw attention to the simplicity of the three pathways outlined in Scheme 5a. Path a requires the rearrangement of two bonds and leads not to the loss of an α,ß-unsaturated acid as proposed in the literature, but rather to the loss of a structurally isomeric α-lactone. The computational process proceeds via saddle point SP51, which can collapse to ion-neutral IN51 and the formation of product ion Cat51. Attempts using computations to find a pathway leading to an α,ß-unsaturated acid were unsuccessful. The fact that after loss of an sn-3 neutral acid the major MS3 losses lead to both the lithiated sn-1 and sn-2 acids makes it highly improbable that this could happen from an intermediate ion having a single structure. In other words, it is highly probable that the ion from loss of an sn-3 (or sn-1) neutral acid has more than one structure, as proposed in Scheme 1a–d. Thus, in Scheme 5a paths b and c are complementary and both proceed via well-precedented 1,2elimination processes, namely the cleavage of a bond, followed by abstraction of a proton alpha to the cleavage position [66, 67]. Computations revealed that this was indeed feasible, although the fragmentation appears to be more complex than the simple approach shown in Scheme 5a. As laid out in Scheme 5b, path b proceeds from Lac11 with an input of some 320 kJ mol−1 to saddle point SP52, from which the protonated lithium salt of what was the sn-3 acid, sn3HLi+, can be eliminated, giving an ion which can be observed in the MS3 spectrum. Plausible parallel paths, c1 and c2, are laid out in Scheme 5b proceeding from Lac12 since in this case, two different


Scheme 5. Fragmentation mechanisms (MS3) of the product ion from loss of an sn-1/3 RCOOH group from a [TAG + Li]+ ion

protons may be abstracted by the departing lithium salt of what was the sn-2 acid. Formation of either saddle point SP53 or SP54 requires similar energy inputs, but both less than for the formation of SP52. The protonated lithium salt of what was the sn-2 acid, sn2HLi+, can be formed from either saddle point but the ion-neutral complexes IN53 and IN54 are very different yet easier to form than IN52. Separation of the ion-neutrals into free ions was somewhat more favorable for sn2HLi+. Given the various energy differences shown in Scheme 5b, it would appear reasonable that all three processes are likely to be operating in the formation of product ions during an MS3 experiment. Finally, we observed a ubiquitous minor product ion at [M – 44]+ ([TAG + Li – RCOOH – 44]+) in the MS3 spectra reported in Table 4. This ion is also present in a spectrum from the literature [15]. Given the atoms present in lithiated TAGs, this loss must be a molecule of carbon dioxide. The literature proposal for the structure of the MS2 product ion after loss of a neutral acid from the sn-1/3 position of a lithiated TAG (Supplementary Material Figure 10S) was made using the techniques available at that time [14, 15]. It is difficult to explain loss of CO2 based on this structure, as multiple bond cleavages and bonding rearrangements would be required. However, loss of CO2 from a lactone, although somewhat energy-demanding since at least two bonds must be broken, is more probable since no atom rearrangements are required. We suggest that even though this is a minor ion, its ubiquity is important and is further evidence that

structures of the [TAG + Li – R1/3COOH]+ ions should possess a lactone functionality.

Conclusions Most of the many approaches to the analysis of the complex mixtures found in naturally occurring TAG samples involve LC in combination with various forms of mass spectrometry. Despite the fact that many details of these procedures have been worked out, fundamental understanding of what occurs is limited. In the present work, we have presented evidence that when TAGs are cationized by ESI-MS, multiple structures may be formed. When the cations are larger than a proton, dimeric complexes between a pair of TAG molecules and the cation are readily formed, which has analytical implications as the ESI ion current is now divided among several species. Sensitivity of ESI-MS for lithiated or argentiated TAGs using protic solvents is further complicated by the ubiquitous presence of sodium and potassium ions in such solvents. We have also shown that although cation binding to TAGs is primarily to oxygen atoms from the glycerol moiety, binding to π bonds in unsaturated chains can also be important, especially for a soft cation such as Ag+. The effects of having multiple π bonds in a chain await further work. Our studies on the low-energy CID of sodiated mono-, di-, and triacylglycerols have shown that the cation–oxygen binding in a monoacylglycerol is sufficiently weak that it may be broken to produce sodium cations, observed at m/z 23. By contrast, the


presence of two or three ester groups tightens the binding of the cation to the glyceryl oxygen atoms sufficiently that fragmentation now occurs by loss of part of the molecular skeleton. The experimental and computational results from the fragmentation of cationized triacylglycerols under collisional activation indicate the complexity of the processes and suggest that fragmentations only occur from tightly defined geometries, even though the ions are conformationally mobile and the chains are undoubtedly rotating rapidly. Fragmentation pathways for losses of neutral acids or neutral metal salts from either the sn-1/3 or sn-2 positions are presented. These pathways were computed using the B3LYP functional [29, 30] and a modest basis set for model TAGs having only saturated three-carbon chains. Natural TAGs contain many more carbon atoms, which generally have some level of unsaturation. The B3LYP functional is not parameterized for long-range dispersion interactions (van der Waals forces) such as must occur especially in tightly defined geometries of the saddle points shown in Schemes 1, 2, 3 and 4, and the computed energies will thus represent trends, but cannot be expected to correlate closely with observed product-ion spectra. The mechanistic pathways could be modeled better using a more modern functional such as wB97XD/6 (from G09) [68], but such computations including longer chains with at least one double bond would be significantly more difficult than the present work and must constitute a separate project. Detailed structural information can be obtained from the MS2 spectra of lithiated TAGs followed by MS3 spectra on the product ion from loss of a neutral acid [15]. As is evident in Figure 5 and Supplementary Material 9S, the intensity of these product ions decreases as the collision energy is increased, which indicates that these ions are then undergoing further fragmentation. We believe that our proposal that these product ions have a lactone core in their structure provides a sound basis from which the MS3 spectra can be interpreted, which should facilitate the use of these techniques in the analysis of complex mixtures of TAGs from natural sources.

Acknowledgments Felix Kannemann provided invaluable advice on the computations. The authors thank Karen MacDougall, Sue Penny, and Robert L. White for assistance and gratefully acknowledge access to computational facilities at the ACEnet Regional High Performance Computing Consortium for universities in Atlantic Canada, funded by the Canada Foundation for Innovation, the Atlantic Canada Opportunities Agency, and the provinces of Newfoundland and Labrador, Nova Scotia and New Brunswick.

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