B American Society for Mass Spectrometry, 2017 J. Am. Soc. Mass Spectrom.

(2017)
DOI: 10.1007/s13361-017-1675-2

RESEARCH ARTICLE

Broad Separation of Isomeric Lipids by High-Resolution

Differential Ion Mobility Spectrometry with Tandem Mass
Spectrometry
Andrew P. Bowman,1 Rinat R. Abzalimov,1,2 Alexandre A. Shvartsburg1
1
Department of Chemistry, Wichita State University, 1845 Fairmount, Wichita, KS 67260, USA
2
Present Address: City University of New York, 85 Saint Nicholas Terrace, New York, NY 10031, USA

Resolved 3/4 of isomers Abstract. Maturation of metabolomics has brought a deeper appreciation for the
across all four types: importance of isomeric identity of lipids to their biological role, mirroring that for
proteoforms in proteomics. However, full characterization of the lipid isomerism has
been thwarted by paucity of rapid and effective analytical tools. A novel approach is
ion mobility spectrometry (IMS) and particularly differential or field asymmetric wave-
form IMS (FAIMS) at high electric fields, which is more orthogonal to mass spectrom-
etry. Here we broadly explore the power of FAIMS to separate lipid isomers, and find
a ~75% success rate across the four major types of glycero- and phospho- lipids (sn,
chain length, double bond position, and cis/trans). The resolved isomers were iden-
tified using standards, and (for the first two types) tandem mass spectrometry. These
results demonstrate the general merit of incorporating high-resolution FAIMS into lipidomic analyses.
Keywords: Ion mobility spectrometry, FAIMS, CID, Lipids

Received: 20 January 2017/Revised: 13 March 2017/Accepted: 27 March 2017

Introduction constituent FAs differs across sn- isomers [4, 14]. Such isomers
with distinct FAs exchanged between sn1 and sn3 positions are

P roteomics and metabolomics measure the complement of

proteins and metabolites in living tissues presumed to
reflect and predict the health and disease states [1, 2]. Expres-
enantiomers [15, 16], and proper chirality also matters. Ac-
cordingly, characterizing the lipidome to individual isomer
level should be inherent to biomedical analyses [17].
sion of metabolites (unlike proteins) is not coded in the ge- The prevailing “shotgun” lipidomics approach [18] based
nome, creating an open-ended number of options that incorpo- on tandem mass spectrometry (MS) via collision-induced dis-
rates massive isomeric complexity. For instance, a common sociation (CID) effectively profiles lipid stoichiometries, but
lipid subclass is glycerolipids made by esterifying fatty acid the CID spectra of isomers typically differ only in the fragment
(FA) chains to one or more of the three glycerol hydroxyls. ratios that are sensitive to the excitation energy and collision
These feature four major isomer types (Figure 1): (i) acyl chain cross-section and thus depend on the ionization source, gas
substitution or stereonumbering (sn-) that results from ex- pressure, and details of the instrument design and operation that
change of FA chains (transacylation), (ii) chain length from
control the ion temperatures and product mass discrimination
swapping pieces between FAs, and, for unsaturated lipids, (iii)
[12, 13, 19, 20]. Thus relying on those ratios requires thorough
double bond (DB) positional isomers and (iv) cis/trans (Z/E)
calibration using pure standards for each conceivable isomer
geometry at each DB [3–12]. Permuting these variations can
and is difficult for mixtures. In a new ozone-induced dissocia-
yield hundreds of isomers with often distinct biological activ-
tion (OzID) mode, ozone mixed into the CID collision gas
ities, many present in vivo [3–13]. As is widely known,
trans-fats differ in uptake rates from the cis- analogs and are selectively severs the DBs in unsaturated lipids, revealing their
implicated in health issues, including cardiovascular and psy- position [21–23]. One can also subject CID products to OzID
chiatric diseases and slow infant growth [8–11]. In another [15, 24]. Unfortunately, OzID is blind to other isomer types.
example, the nutritional absorption and bioavailability of This situation calls for isomer separations before the MS/MS
step. This has been traditionally pursued with liquid chromatog-
raphy (LC) [16, 25–31], using both the usual reversed phase and
Correspondence to: Alexandre Shvartsburg; tailored non-aqueous reversed phase [27], silver-ion normal phase
e-mail: [email protected] that exploits Ag ions binding to DBs [28–30], and their
 A. P. Bowman et al.: Broad Separation of Isomeric Lipids by High-Resolution FAIMS

some subclasses (e.g., sphingolipids and phospholipids [36] or

(i) ceramides and lysophospholipids [41]), isomer resolution has
been rare: most isomers resolved by LC were not separated by
IMS in LC/IMS experiments [43]. Poor orthogonality between
IMS and MS dimensions can be mitigated by high IMS resolv-
(ii) ing power (R), and the latest DT IMS systems [44] operated at
>1 Atm to attain R > 100 distinguish some isomers such as
phosphocholines (PC) 18:1 with DB in positions 9 and 6.
However, some separations claimed in that work were elicited
by sophisticated peak fitting.
(iii) A newer FAIMS approach exploits nonlinear ion motion in
electric fields of high intensity (E) to sort ions by the average
derivative of K(E) function over a certain range [45, 46]. This
quantity is extracted by a periodic asymmetric field applied
across a gap between two electrodes, through which ions are
(iv) carried by the gas flow. Ions injected into the gap are pushed
toward either electrode, but a given species can be balanced
Figure 1. Scheme of four major lipid isomer types: (i) sn-, (ii) (and thus passed and detected) by superposing a particular
chain length, (iii) DB position, (iv) cis/trans compensation voltage (CV) on the waveform. As the K(E)
derivative is correlated to m substantially weaker than the
mobility itself, FAIMS is far more orthogonal to MS than linear
IMS [46–51]. The specific advantage depends on the analyte
combination [31]. Despite dramatic advances in HPLC of lipids, nature and is about 4-fold for lipids [48]. Hence, FAIMS tends
many isomers could be separated only partly (if at all) despite to resolve isomers much better than linear IMS with equal R
lengthy gradients [28]. Other protocols involve complicated and metric.
laborious prior derivatization [28, 29]. Hence, the assessment that The resolving power maximizes in planar gaps where ho-
“the inability to distinguish isomeric lipids is the major limitation mogeneous electric field equilibrates only one species at any
of existing lipidomics weaponry that seriously impedes the under- CV, although finite resolution may allow similar species to
standing of lipid biochemistry” [22]. pass [46, 52]. The resolution is sensitive to the carrier gas, and
An alternative or complement to condensed-phase methods is generally augmented by addition of He or H2 to N2 because
is rapid post-ionization ion mobility spectrometry (IMS) in of higher ion mobilities in lighter gases and pronounced non-
gases that has unique selectivity [32]. Linear IMS devices Blanc effect in mixtures of molecules with disparate masses
based on absolute mobility (K) comprise the original drift- [46, 53–56]. The latter is theoretically maximized at ~80% He,
tube (DT) IMS with constant electric field and dynamic-field in line with the findings in FAIMS chips (where microscopic
systems such as traveling-wave (TW) IMS implemented in the gaps permit extreme fields by the Paschen law) [56, 57].
Synapt platforms (Waters, Milford, MA). These can be However, the resolving power of these chips is low, and the
“nested” between LC and MS stages, with the peaks eluting electrical breakdown in macroscopic devices employed here
from LC dispersed by IMS and analyzed by (time-of-flight) [53, 54] constrains the He fraction to ~50%–75%.
MS without slowing the LC step [33]. The resulting domains in High-resolution FAIMS has been deployed to separate var-
the IMS/MS palette differ for major biomolecular classes (e.g., ious biomolecular isomers, including the peptide sequence
peptides, lipids, carbohydrates, oligonucleotides), for some inversions and variants differing in the localization of post-
enough to classify by the peak position [34, 35]. However, translational modifications [49], protein conformers [50], and
finer categorization is obstructed by tight correlation between isotopomers [51]. There were three such studies for lipids: a
the ion mass (m) and K for homologous compounds that have survey mapping subclasses in the FAIMS/MS space and dem-
analogous elemental composition and global morphology [34, onstrating first resolution of isomers (sn- for diacylglycerols)
35]. This especially holds for lipids that normally form +1 (−1) [48], targeted separation of two phospholipid isomers (requir-
ions and therefore one trend line in the IMS/MS space (unlike ing Ag+ cationization, as the usual protonated species could not
peptides where multiply-charged ions distribute over several be pulled apart) [58], and resolution of sn-isomers for triacyl-
lines with unequal slopes) [33]. Further, the mean and top glycerols using alcohol vapors doped into the gas [59]. Frac-
deviations of those +1 ions from the central trend are just tionation of complex samples into subclasses (e.g., based on
2.6% and ~5% (versus 7.3% and ~30% for peptides and the head group) without isomer separation was also document-
8.8% and ~35% for carbohydrates) [36], which stringently ed [60, 61].
constrains the power of linear IMS in isomer separations. Hence, FAIMS has been shown to separate sn- but
Hence, while linear IMS can clean up lipid mixtures from not the other three isomer types. Here, we explore them
isobaric and matrix interferences for superior component elu- all using the system with highest resolving power pro-
cidation [36–42] and tissue imaging [41] and largely separate vided by He/N2 buffers and maximum feasible voltages.
A. P. Bowman et al.: Broad Separation of Isomeric Lipids by High-Resolution FAIMS

The results prove broad utility of FAIMS for lipid iso- 16:0), S (stearic, 18:0), A (arachidic, 20:0), and B (behenic,
mer separations. 22:0) for saturated FAs and Pe (palmitelaidic, 16:1[9E]), Po
(palmitoleic, 16:1[9Z]), E (elaidic, 18:1[9E]), O (oleic,
18:1[9Z]), Ps (petroselenic, 18:1[6Z]), Pd (petroselaidic,
18:1[6E]), Lo (linoleic, 18:2[9Z,12Z]), and Le (linoelaidic,
Experimental 18:2[9E,12E]) for unsaturated FAs. For example, the DG
We employed a planar (p-) FAIMS unit with the gap width (g) 16:0/0/18:0 is P0S and PC 18:0/18:1(9Z) is SO.
of 1.88 mm [51]. The high-definition bisinusoidal waveform Comparisons of FAIMS spectra acquired not concurrently
[62] with adjustable profile and frequency is put out by a are complicated by shifts due to variations of DV, ambient
supply protected from electrical shorts that enables operation pressure, and temperature. Like with MS, the increasing re-
near the arc breakdown threshold without risking equipment solving power of IMS and FAIMS renders such shifts more
damage [51]. Here we adopted the 2:1 harmonic ratio, 1 MHz prominent. As with “lock mass” in MS [64], this problem in
frequency, and zero-to-peak amplitude (dispersion voltage, FAIMS of peptides was addressed using internal calibrants
DV) of 4 kV, leading to the dispersion field (ED) of 21.3 kV/ exhibiting consistent CV shifts as a function of experimental
cm. The compensation voltage (CV) was scanned at 0.3–1 parameters [48]. Good calibrants are homologous to the
V/min. Ions are pulled along the gap by flow formulated by analyte(s) with close (but not overlapping) mass and CV to
digital meters (MKS Instruments, Andover, MA, USA) from (1) maximize the correlation between the CV dependences on
UHP He and N2 with up to ~70% (v/v) He, the breakdown instrumental parameters for the two species, and (2) minimize
limit. The total flow rate Q was varied from 2 to 0.7 L/min to the time between the appearances of those species and, thus, the
control the resolution/sensitivity trade-off by adjusting the parameter drift during it. A calibrant must be easily ionized to
filtering time (roughly proportional to 1/Q) from t = 0.2 to yield strong peaks from traces in the sample, yet not materially
0.5 s [55, 62]. The inflow is split, with one part desolvating suppress the analyte ionization. It should also produce one or
incoming ions in a curtain plate/orifice inlet and other carrying few well-defined CV peaks and not complex with the analyte,
ions through the device [52]. The optimum rate depends on the which could cause artifacts for the analyte and/or calibrant
ion nature and gas composition, increasing to offset faster upon the fragmentation of complexes during or after the
diffusion for smaller ions or lighter gases. FAIMS step. By these criteria, we selected lipids similar but
The FAIMS stage is coupled to an LTQ XL mass spectrom- not isomeric to targeted analytes: LeLeLe for EEE and its
eter (Thermo Fisher Scientific, Waltham, MA) by an electro- isomers, PdPdPd for other TGs and MS/SM pair, P0A for
dynamic ion funnel interface [51, 52, 62]. The inlet features a M0E, and M0O, M0E for other DGs, and MS for other PCs
multi-hole slit of ~6 mm length that fits the rectangular shape of (with the EC scale anchored by PdPdPd and others being
ion beams leaving the FAIMS gap while matching the conduc- secondary calibrants). Spectra were aligned by linear CV scal-
tance of regular cylindrical capillary [51]. The funnel assem- ing to match the calibrant peaks. In some cases, two calibrants
bled from metalized plastic electrodes has the length of 80 mm, (straddling the analyte peak) were employed for utmost accu-
a 25-mm entrance orifice, and 2-mm exit aperture, and operates racy. For transferability of findings across FAIMS devices with
with axial DC voltage of ~100 V, confining rf voltage of unequal gaps, CV is converted to the compensation field (EC)
~1 MHz frequency, and ~110 V amplitude. This funnel effec- deemed positive for type C ions (where ∂K/dE < 0).
tively captures a supersonic jet expanding from the inlet capil- We first inspected the positive-mode mass spectra for all
lary and focuses it into the first quadrupole of the LTQ. lipids to clarify the stoichiometry of ions from ESI. As expect-
Ions are generated by electrospray ionization (ESI) source ed, those were singly ammoniated and protonated species for
biased at ~2.5 kV versus the FAIMS curtain plate (with subse- glycerides and PC, respectively [48]. We then obtained the EC
quent ~1 kV drop to the orifice) from samples infused at ~0.5 spectra for individual isomers over a range of gas compositions
μL/min. We focused on ubiquitous glycerolipids and phospho- and flow rates, and applied CID at the apexes of peaks of
lipids in the 500–900 Da range, represented by 32 species interest or during EC scans. The calibrated spectra for isomers
(Table 1): 10 diacylglycerols (DG), 11 triacylglycerols (TG), were superposed to optimize the resolution (EC spread divided
and 11 phosphocholines (PC) that form 19 isomer pairs (5 for by the mean peak width at half maximum, w) and signal at
DGs, 7 for PCs, and 7 for TGs). These were procured from sufficient resolution. Those results were validated by FAIMS/
Larodan (Solna, Sweden) or Avanti (Alabaster, AL, USA) and MS/(MS) analyses for isomeric mixtures, in some instances
dissolved to 10 μM in 30:70 chloroform/methanol (Fisher with several component ratios.
HPLC grade) salinized by ammonium acetate (Fisher ACS
grade) at 2% by weight. We adopt the lipid nomenclature
outlined by Liebisch [63], Holcapek [27, 59], and Blanksby
[15] with the subclass (DG, TG, or PC) followed by giving FAs
Results
(or entering 0 if none) at sn1, sn2, and (for DG and TG) sn3 As expected, all lipid ions in all He/N2 compositions are type C
sites. The FAs are identified by the number of carbons, number [46, 48]. Typical behaviors are seen in the separation of type
of DBs, and (if any) location and symmetry of DBs. They are (iii) isomers trielaidin and tripetroselaidin (TGs comprising
abbreviated as L (lauric, 12:0), M (myristic, 14:0), P (palmitic, FAs with single trans-DB in the w9 or w12 position). The EC
 A. P. Bowman et al.: Broad Separation of Isomeric Lipids by High-Resolution FAIMS

Table 1. Presently explored lipid isomer pairs with monoisotopic molecular masses (Da) and separation outcomes, ordered by class (DG, PC, TG) and mass within
class

Class Isomer 1 Isomer 2 Isomer type Mass Resolution

DG 1-Palmitin-2-Laurin 16:0/12:0/0 PL0 1-Palmitin-3-Laurin 16:0/0/12:0 P0L sn- 512.4 Full*

1-Laurin-2-Stearin 12:0/18:0/0 LS0 1-Stearin-2-Laurin 18:0/12:0/0 SL0 sn- 540.5 Full
1-Myristin-3-Elaidin 14:0/0/18:1(9E) M0E 1-Myristin-3-Olein 14:0/0/18:1(9Z) M0O cis/trans 566.5 Full
1-Palmitin-3-Stearin 16:0/0/18:0 P0S 1-Stearin-2-Palmitin 18:0/16:0/0 SP0 sn- 596.5 None
1,2-Dioelin 18:1(9Z)/18:1(9Z)/0 OO0 1,3-Diolein 18:1(9Z)/0/18:1(9Z) O0O sn- 620.5 None
1-Myristin-3-Behenin 14:0/0/22:0 M0B 1-Palmitin-3-Arachidin 16:0/0/20:0 P0A chain length 624.6 Full
PC 1,2-Dipalmitelaidoyl 16:1(9E)/16:1(9E) PePe 1,2-Dipalmiteleoyl 16:1(9Z)/16:1(9Z) PoPo cis/trans 729.5 Full
1-Myristoyl-2-Stearoyl 18:0/14:0 MS 1-Stearoyl-2-Myristoyl 14:0/18:0 SM sn- 733.6 Full
1-Oleoyl-2-Palmitoyl 18:1(9Z)/16:0 OP 1-Palmitoyl-2-Oleoyl 16:0/18:1(9Z) PO sn- 759.6 Full
1,2-Dielaidoyl 18:1(9E)/18:1(9E) EE 1,2-Dipetroselenoyl 18:1(6Z)/18:1(6Z) PsPs DB pos.+cis/trans 785.6 Full
1,2-Dioleoyl 18:1(9Z)/18:1(9Z) OO 1,2-Dipetroselenoyl 18:1(6Z)/18:1(6Z) PsPs DB pos. 785.6 None
1,2-Dielaidoyl 18:1(9E)/18:1(9E) EE 1,2-Dioleoyl 18:1(9Z)/18:1(9Z) OO cis/trans 785.6 Full
1-Stearoyl-2-Oleoyl 18:0/18:1(9Z) SO 1-Oleoyl- 2-Stearoyl 18:1(9Z)/18:0 OS sn- 787.6 Full
TG 1,2-Dipalmitin-3-Elaidin PPE 1,2-Dipalmitin-3-Olein PPO cis/trans 832.8 Full
16:0/16:0/18:1(9E) 16:0/16:0/18:1(9Z)
1-Palmitin-2-Olein-3-Linolein POLo 1-Palmitin-2-Elaidin-3-Linolein PELo cis/trans 856.8 None
16:0/18:1(9Z)/18:2(9Z,12Z) 16:0/18:1(9E)/18:2(9Z,12Z)
Trilinolein 18:2(9Z,12Z)/ LoLoLo Trilinoelaidin 18:2(9E,12E)/18:2(9E,12E)/ LeLeLe cis/trans 878.7 Full
18:2(9Z,12Z)/18:2(9Z,12Z) 18:2(9E,12E)/
Triolein 18:1(9Z)/18:1(9Z)/18:1(9Z)/ OOO Trielaidin 18:1(9E)/18:1(9E)/18:1(9E) EEE cis/trans 884.8 None
Triolein 18:1(9Z)/18:1(9Z)/18:1(9Z) OOO Tripetroselaidin 18:1(6E)/18:1(6E)/18:1(6E) PdPdPd DB pos.+cis/trans 884.8 Full
Trielaidin 18:1(9E)/18:1(9E)/18:1(9E) EEE Tripetroselaidin 18:1(6E)/18:1(6E)/18:1(6E) PdPdPd DB pos. 884.8 Full
1-Palmitin-2-Arachidin-3-Olein PAO 1-Arachidin-2-Olein-3-Palmitin AOP sn- 888.8 Partial
16:0/20:0/18:1(9Z) 20:0/18:1(9Z)/16:0

For each isomer, we give the common name, systematic nomenclature and abbreviation. “Full” separation means each isomer cleanly filtered from the other at FAIMS
peak apex, “partial” means a dip between the two peaks in 1:1 mixture
* Separation shown in an earlier publication [48]

values go up upon He addition (Figure 2a), while peaks narrow The peak widths in p-FAIMS devices scale as t1/2 fun-
(Figure 2 b–d) as the ion mobility increases [48, 65]. These damentally and actually faster in view of fringe effects [65,
observations track those for peptides, with EC and peak widths 68]; hence, halving Q to ~1 L/min should decrease w by
somewhat exceeding those for 1+ ions of similar mass under >1.4 times. The actual narrowing of ~1.7 times delivers
identical conditions [53, 54]. For example, at 65% He we find peak resolution at half-maximum and perfect filtering of
w ~2–2.5 V/cm versus 1.6 V/cm on average for 1+ peptides either isomer at its apex (Figure 3 a, b), at a sensitivity
[53] around 900 Da (at Q = 2 L/min). Broader peaks may penalty of ~5× (a) and ~30× (b). Yet higher resolution
reflect larger cross-sections and, thus, lower K for lipids com- (near the detection limit) is achievable at Q = 0.7 L/min.
pared with isobaric peptides [66] and/or more diverse confor- Spectral annotations were confirmed by varying the isomer
mations. Hence, the resolving power for lipids approximates ratios in a mixture, e.g., 2:1 and 1:2 (Figure 3c). This
that for 1+ peptides [53], increasing from ~10 in N2 to ~40 at illustrates the method for realistic samples, where those
the maximum He content. ratios are generally unequal.
The EC difference between EEE and PdPdPd likewise Given these trends, we settled on 65% He, throttling the
expands at higher He content (Figure 2b–d). Combined gas as needed for isomer resolution. With peptides, one
with narrower peaks, this elevates the resolution from none commonly has to lower the He fraction (often to ~30%–
in N2 and marginal at 40% He to fair at 65% He. The 40%) to maintain reasonable ion counts even at the “stan-
improved resolving power also pulls away two minor fea- dard” Q = 2 L/min [49]. This is not the case for lipids
tures a1 and a2 on the low-EC sides of dominant peaks b. because of (1) lower K values compared with multiply
These species with intensities sensitive to ESI conditions charged peptides generated by ESI [43], and perhaps (2)
may be protomer-like [67] isomers with distinct ammoni- lack of “self-cleaning”, which eliminates ions that
ation sites. (Probable attachment of NH4+ to DB may place underwent significant E C change upon isomerization
it on any FA, although that would not engender three peaks (unfolding) induced by field heating inside the FAIMS
as FA1 and FA3 are equivalent). Although these features gap [69, 70].
confound the sought separation in principle, at the highest Another isomer of EEE is OOO, comprising the oleic
He fractions they lie too far from the base peaks to inter- FA with cis-DB in the same place. These formed essen-
fere (Figure 2d). Such secondary features potentially en- tially coincident peaks at any Q down to 0.7 L/min (Fig-
hance the isomer resolution, although here their difference ure 3d), but PsPsPs was thus resolved from OOO as well
(if any) is less than that for the base peaks. as from EEE. To gauge the prevalence of two outcomes,
A. P. Bowman et al.: Broad Separation of Isomeric Lipids by High-Resolution FAIMS

(a) lipids are polyunsaturated, allowing isomers with multiple

80
DB shifts and/or cis/trans transitions. Such plural varia-
60
tions should intuitively augment the structural difference
EC, V/cm

between isomers and, thus, their separation. Indeed,

40 trilinoelaidin and trilinolein that feature two DBs with
trans- or cis-configuration (in the w6 and w9 positions)
20 in each FA are separated by >4 V/cm, which is more than
baseline (Figure 3g) and better than the three cis/trans
0 isomers with one transition. Here, conversely to Figure 3e,
0 20 40 60 EC is higher for the trans- than cis-geometry. Some sn- TG
He, % isomers with P, S, or O chains were resolved using vapor
dopants [59]. Here, the arachidin-containing PAO and
EEE PdPdPd AOP are partly resolved at lower flow rates (Figure 3h).
N2 b (b) The DGs have lower mass than TGs (here ~600 versus
~800–900 Da) and higher EC values [48], but exhibit
w 3.1 similar isomer separations overall (Figure 4). Some sn-
w 3.8 isomers were resolved well: e.g., SL0 and LS0 (Figure 4a)
or PL0 and P0L [48]. Others were not: e.g., SP0 and P0S
(Figure 4b), where only the lack of customary peak com-
a pression for the mixture upon Q decreasing from 2 to 0.9
L/min suggests slight separation. This is not readily
interpreted as a consequence of larger length difference
He 40 % b (c) between S and L (6 carbons) or P and L (4 carbons)
w 2.9
Relative Intensity

compared with S and P (2 carbons): transacylation changes

w 3.0 the chain length in sn1/sn3 and sn2 positions by 6 carbons
in the SL0/LS0 pair but 16 carbons in SP0/P0S as P and
hydroxyl switch places. The O0O and OO0 isomers with
a2 same transposition of O are merged too, although here both
exhibit two substantial peaks (Figure 4c). However, the
a1 PL0/P0L pair [48] shows that such chain-hydroxyl switch
isomers are not necessarily problematic.
He 65 % Mixture b (d) The type (iv) M0E/M0O pair is separated well, with
w 2.4 higher EC for the cis-geometry (Figure 4d). The isomers
M0B and P0A are resolved almost baseline, despite B and
w 2.1 A differing by the minimal two carbons or <10% of the FA
length (Figure 4e). How representative is this single exam-
a2 ple of type (ii) isomers remains to be determined. As
a1 diglyceride ions are smaller and more mobile than triglyc-
erides, their peaks are slightly narrower under identical
conditions (e.g., w = 1.8–1.9 versus 2.1–2.4 V/cm at Q =
20 40 60 80 100
2 L/min). However, the associated sensitivity drop requires
EC, V/cm somewhat higher Q for diglycerides and the final peak
Figure 2. Separations of ammoniated trielaidin and widths compare. The enantiomers with exchanged FAs
tripetroselaidin (m/z = 902.8) at Q = 2 L/min: (a) EC for EEE as are not resolvable by IMS methods unless converted to
a function of He content (circles - measurements, line - qua- diastereomers or with a chiral vapor doped into the gas.
dratic regression), and (b)–(d) spectra for both with peak widths In this respect, the LC separations are currently
listed. The data measured for 1:1 mixture are added in (d) indispensable.
Our phospholipids lie between DGs and TGs in mass
(~700–800 Da) and EC range. Their FAIMS spectra tend to
we probed other type (iv) TG isomer pairs with one elaidic be cleaner with stronger signal and fewer minor peaks,
or oleic FA. The PPE and PPO are fully resolved at likely reflecting a more efficient and specific protonation
reduced flow rates, but PELo and POLo are not (Fig- at the unique PC group versus ammoniation of glycero-
ure 3e, f). It is tempting to rationalize the trans-to-cis lipids possible on multiple similarly favorable sites. All
transition (that bends FA chains) as less impactful in an three sn-isomer pairs tried are well resolved despite modest
FA in the middle sn2 location than sn1 or sn3, but this fails differences between the FAs involved: four carbons for
to explain why EEE and OOO co-elute. Many important MS/SM, two carbons and one DB for OP/PO, and a DB
 A. P. Bowman et al.: Broad Separation of Isomeric Lipids by High-Resolution FAIMS

1:1 Mixtures (except c)

Q 1.2 all (a) Q 1.2 all

65 1.2 L/min PPE (e)
PPO
PdPdPd
EEE

w 1.4 w 1.3
w 1.3 w 1.4
Q 0.9 (b) Q 0.9 (f)
POLo
Q 0.9 PELo
w 1.3 w 1.2
w 1.3 Q 0.7 w 1.2
Relative Intensity

Q 1.2 all (c) Q 2.0 Q 0.9 (g)

Q 0.9 LeLeLe
w 1.0
1:2
2:1
Mix LoLoLo w 1.1
Mix w 1.6

w 1.2
Q 0.7 all (d) Q 1.2 PAO (h)
Q 1.2 w 1.7
EEE
w 0.9 Q 0.9
OOO
w 0.9 AOP
w 1.3

84 86 88 90 86 88 90 92 94 96
EC, V/cm
Figure 3. (a), (b) FAIMS spectra for triglyceride isomer pairs (flow rates Q marked for each trace) with widths w (fwhm, V/cm) shown
for all peaks. Panel (c) presents the spectra for 2:1 and 1:2 EEE/PdPdPd mixtures. Resolution was improved using Q = 0.9 L/min (e)
and 0.7 L/min (h)

for OS/SO (Figure 5a–c). However, each species features a both (Figure 5e), again in contrast to the co-eluting OOO
minor peak coincident with the major one for its isomer at and EEE (Figure 3d). The other type (iv) pair PoPo/PePe
all He fractions, with the height ratios conserved regardless was well-separated too (Figure 5f), also with the trans-
of the ESI conditions. This pattern already encountered for geometry at higher EC.
DGs [48] was ascribed to isomeric contaminants resulting The summary FAIMS/MS palette for lipids studied
from imperfect synthesis and/or transacylation during the here (Figure 6) reproduces the main trends found at
storage or ESI process [71]. Their abundance is commonly 50% He [48]: the negative correlation between EC and
[38, 71] ~10%–15%, in line with the present range of mass within each subclass, higher EC for phospholipids
~2%–30%. The OO and PsPs (with single cis-DB on each than glycerolipids of similar mass, and lower EC for
FA in the w9 and w12 position) coincided even at the unsaturated lipids. Consistency of these trends despite
maximum resolving power (Figure 5d), in contrast to the much higher EC here (~80–140 V/cm versus ~40–80
parallel move involving the trans-DB in TGs (Figure 3a– V/cm in [48]) confirms their utility for classifying lipids
c). Their isomer EE with trans-DBs was resolved from based on the domains in FAIMS/MS space.
A. P. Bowman et al.: Broad Separation of Isomeric Lipids by High-Resolution FAIMS

1:1 Mixtures 1:1 Mixtures

Q 1.2
(a)
MS SM (a)
Q2 SL0 Q 0.9 w 1.6
w 1.3
Q 0.9
Q2 w 1.8 w 1.2
w 1.3
Q 1.2 w 1.2
LS0
a w 1.4
w 1.2 OP Q 1.2 all (b)
a b/b w 1.4
PO
w 1.4 w 1.5

(b)
SP0 w 1.2
w 1.8 Q2
112 114 116 118 120 122 124
Q2
Q 0.9 Q 1.2 OS (c)
P0S Q 1.2 w 1.5 SO
Q 0.9 w 1.7
w 1.8
w 1.8
w 0.9 w 1.2
126 128 130 132 134 136 138
PsPs (d)

Relative Intensity
w 1.5
Q 0.9 all (c) OO Q 1.2
Relative intensity

w 1.5
Q 1.2
Q 0.7
O0O OO0
w 1.0 w 1.2 w 0.9
PsPs (e)
w 1.0
EE
w 1.2 w 1.3
Q 1.2
Q 0.9
Q 0.7
106 108 110 112
108 110 112 114 116
Q2 M0E (d)
Q 1.2 PoPo (f)
Q2 w 1.9 M0O Q 0.9 w 1.6 PePe
Q 0.7 w 2.1
Q 0.9 w 1.8

w 1.2 w 1.7
114 116 118 120 122 124
w 1.0 w 0.9 EC, V/cm

Q 2.0 P0A (e) Figure 5. FAIMS spectra for phosphocholine isomer pairs (no-
Q 2.0 w 1.8 tation as in Figure 3)
Q 1.2

M0B Lipids are commonly identified by CID. For example,

w 1.9 both sn-isomers PO and OP can eliminate O or P as
ketenes, yielding fragments at m/z = 522.6 and 496.6,
w 1.4 w 1.4 respectively [38, 72]. However, an FA is easier to sever
at sn2 than at sn1 (because the tertiary α-hydrogen
forming an H-bond with the carbonyl oxygen hinders
118 120 122 124 126 the latter process), and the ion counts at 522.6 exceed
EC, V/cm those at 496.6 by ~3–4 times for OP and vice versa for
Figure 4. FAIMS spectra for diglyceride isomer pairs (notation PO [20, 38, 72]. This was also observed here (Figure 7),
as in Figure 3) confirming the isomer separation found with standards
 A. P. Bowman et al.: Broad Separation of Isomeric Lipids by High-Resolution FAIMS

140
OP -P SM -M
DG0
130 -O -S
PC0
120
EC, V/cm

DG1-2
110 PC1-2
-P -M

Relative Intensity
-O -S
100

90 TG1-6 PO -O MS
-P -S
80
-M
500 600 700 800 900
Molecular mass, Da
Figure 6. The FAIMS/MS palette for all lipids studied. Domain -O
labels identify the class followed by the number of double -S
bonds. Filled and empty symbols are for saturated and unsat- -P -M
urated lipids, respectively. The lines are 1st-order regressions
for glycerides and phosphocholines
460 480 500 520 460 480 500 520
(Figure 5b). The features at 18 units below these frag-
-E EE
ments (m/z of 504.6 and 478.6) arise from the elimina- -E
tion of same FAs as free acids [72]. These two frag-
ments were previously reported as similarly intense [38,
72], but we see greater losses at the sn1 position. Al- -PC head group
though this preference appears weaker than that for ke-
Relative Intensity

tene loss, it can help to confirm the assignments. These

trends extend to PCs with saturated FAs, e.g., MS and -O OO
SM (Figure 7). The chain-length isomers (where two -O
chains differ in length) are trivially disentangled by two
FA elimination products: e.g., at m/z of 284 and 398 for
-PC head group
M0B versus 314 and 370 for P0A. However, CID cannot
identify the DB position and cis/trans isomers, as this
information resides in the lost chain. For instance, EE, -Ps PsPs
-Ps
OO, and PsPs eliminate E, O, and Ps as ketenes and free
acids, leaving identical products (m/z = 522.6 and 504.6),
and the PC head group (through two 1,2-hydride shifts)
[38] to yield the fragments at m/z = 604 (Figure 7). -PC head group
Concluding, high-resolution FAIMS has separated ~3/4 of
lipid isomer pairs across major types (including one pair from 400 500 600 700 800
[48]): 7 out of 9 sn- and chain length (i, ii), 3 out of 4 DB m/z
position (iii), and 7 out of 9 cis/trans (iv). (The only type (ii) Figure 7. CID spectral windows for exemplary isomers select-
pair was considered together with (i), and the numbers do not ed by FAIMS from binary mixtures (at peak apexes): sn-isomers
total to 15 out of 20 because the two pairs belonging to two (top panels) and DB position and cis/trans isomers (bottom
types were counted in each. For isomers differing in either the panels). The FAs lost are marked and underlined for ketenes
DB position or cis/trans arrangement, 1 out of 2 and 5 out of 7
were separated, respectively.) To address the remaining pairs,
we are raising the FAIMS resolving power and investigating
cationization by Ag+ and other metal ions that benefited some
lipid separations [59]. All species resolved were assigned using Acknowledgments
standards, with confirmation by CID for most sn- and chain The authors thank Gordon A. Anderson (GAACE) and Dr.
length-isomers. Integration of OzID (in progress) will extend Keqi Tang (PNNL) for broad aid with instrument development,
the a priori identification to DB position isomers. Overall, this Matt Baird and Julia Kaszycki for experimental help, and
work showcases FAIMS as a powerful tool for rapid lipid Professor Stephen Blanksby for numerous insightful discus-
isomer elucidation. sions. This research was funded by NIH K-INBRE (P20
A. P. Bowman et al.: Broad Separation of Isomeric Lipids by High-Resolution FAIMS

GM103418), NSF First (EPS-0903806), and NSF CAREER atmospheric pressure chemical ionization mass spectrometry. Rapid
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