7.2.1 Double and Triple Bonds
Lipids can form double- or triple-bond positional isomers. The position and stereoi- somerism of unsaturated bonds play critical roles in the biophysical and biochemi- cal properties of lipids and can significantly influence the development of many pathologies [15].
The localization of double bonds in unsaturated aliphatic chains of lipids is a clas- sical analytical problem that can be addressed by mass spectrometry. The first meth- ods developed for localizing double bonds were based on EI. Fragments indicating the double-bond position can be obtained from lipids derivatized at either the double- bond site [16] or a distant functional group [17]. In the first case, the newly introduced groups weaken the carbon–carbon bond at the original double-bond site, while the second approach relies on gas-phase decompositions that occur physically remote from the charge site. The derivatization strategies utilizing charge-proximal and charge-remote fragmentations were later developed for soft ionizations; some of these methods will be discussed in the following sections. Distinguishing double-bond ste- reoisomers (cis/trans isomers) is comparatively easy using chromatography, but it might be difficult from mass spectra because it usually relies on relatively small differ- ences in fragment ion abundances. Evidence for double-bond geometry can be found in high-energy CID [18], EID [19], or ozone-induced dissociation (OzID) [20] spectra.
Only a few methods for determining the position of triple bonds in lipids have been developed to date. EI spectra of 4,4-dimethyloxazoline (DMOX) derivatives of fatty acids with triple bonds show CRF ions separated by 10 Da [21]. Fatty acids with triple bonds were also investigated by acetonitrile covalent adduct chemical ioniza- tion (CI) [22]. Probably the only atmospheric-pressure ionization method for pin- pointing triple-bond positions utilizes gas-phase chemistry of acetonitrile in APCI [23].
7 Structural Characterization of Lipids Using Advanced Mass Spectrometry Approaches 186
7.2.1.1 Charge-Switch Derivatization of Fatty Acids
Fatty acid identification and structural analysis have traditionally been performed by (GC)/EI-MS [24]. The most informative spectra are obtained for derivatives with a readily chargeable functional group where a positive charge is fixed after ioniza- tion, e.g. pyrrolidides [25, 26], picolinyl (3-hydroxymethylpyridinyl) esters [17], or DMOXs [17, 27]. CRF pathways in straight, unsubstituted alkyl chains yield groups of ions separated by 14 mass units. The presence of a functional group causes changes in the peak pattern, which is helpful for the localization of double bonds [28], alkyl (most frequently methyl) branching [28], hydroxyl [29], epoxy [30], cyclopropane [31], and other groups within the acyl chain. In addition to positively charged ions, deprotonated fatty acids can be created by fast atom bombardment (FAB) and fragmented by high-energy CID to obtain informative CRF ions [32].
The advent of atmospheric-pressure ionization techniques, especially electro- spray, has made it possible to analyze fatty acids by HPLC/MS or direct infusion of a liquid sample into a mass spectrometer. Analysis of underivatized fatty acids by electrospray ionization (ESI-MS) is not very useful because the detection sensitivity is low, and low-energy CID spectra are uninformative regarding the structure of the aliphatic chains. A dramatic increase in the signal is achieved after derivatizations that introduce a permanent positive charge [33–36]. Since the derivatives with a permanent positive charge originate from molecules that are easily deprotonated to negatively charged ions, the reaction is referred to as charge-switch derivatization.
Among the derivatives, N-(4-aminomethylphenyl)pyridinium (AMPP+) amides proved particularly helpful in the structural analysis of fatty acids [37] (Figure 7.1).
The reagent was designed to minimize fragmentations of the derivatives close to the permanent charge site, thus providing CRF ions at appreciable intensities.
Importantly, AMPP+ is commercially available.
The CID spectra of AMPP+-derivatized saturated fatty acids [37] show a periodic pattern of fragments separated by 14 mass units. These ions are products of carbon–
carbon bond cleavages, likely formed by the 1,4-H2 elimination mechanism. The presence of double bond(s) disrupts the pattern by lowering some ions’ intensities and creating new fragments that unambiguously identify the positions of double bonds. All AMPP+-derivatized fatty acids provide the same fragments by the amide and benzyl bond cleavages, which can be utilized for precursor ion scan (PIS) profil- ing of a wide range of fatty acids (Figure 7.2).
Besides double bonds, AMPP+ derivatives help localize other features on acyl chains like methyl branching sites [38, 39] or hydroxy groups [40, 41]. To increase the abundance of CRF ions providing information on structural features in the ali- phatic chains, new derivatives capable of generating odd-electron ions were
N+ N+
R R
OH NH
O O
H2N +
Figure 7.1 Reaction scheme for derivatization of fatty acid with N-(4-aminomethylphenyl) pyridinium (AMPP+).
developed [42]. The radical ions are produced by photodissociation of electrospray- generated, even electron precursors containing photocleavable iodine. The derivatives, 1-(3-(aminomethyl)-4-iodophenyl)pyridin-1-ium (4-I-AMPP+) or 1-(4-(aminomethyl)- 3-iodophenyl)pyridin-1-ium (3-I-AMPP+), are conjugated to fatty acids using a similar protocol developed for AMPP+ [36].
Irradiation of the mass-selected precursor ions with a UV (266 nm) laser yields [M−I]•+ radical cation and secondary fragment ions that correspond to acyl chain cleavages and to the elimination of the AMPP+ moiety (Figure 7.3).
7.2.1.2 Ozone-Induced Dissociation
The reaction of the double bond with ozone was described in 1847 by Christian F. Schửnbein [43], and its mechanism was proposed a 100 years later by Rudolf Criegee [44]. The reactants first form an unstable molozonide (1,2,3-trioxolane), which reverts to its corresponding carbonyl oxide (Criegee intermediate) and
307.2
#
349.2
# 211.2
183.2
169.2
Relative intensity (%)
100
80
60
40
20
0
226.2239.1
281.1
323.1
449.3
#
#
*
377.2 337.1
295.1 *
150 200 250 300 350 400
m/z
450 500
477.1 449.3 449.3
a. 18 : 1–Δ6
b. 18 : 1–Δ9
c. 18 : 1–Δ11
d. 20 : 1–Δ11 253.1
267.2
*
309.1
*
Figure 7.2 Tandem mass spectra of AMPP+-derivatized monounsaturated FAs having different double-bond positions. Source: Reprinted with permission from Yang et al. [37].
Copyright 2013 American Chemical Society.