UV Lamp as a Facile Ozone Source for Structural Analysis of Unsaturated Lipids Via Electrospray Ionization-Mass Spectrometry

Article abstract of DOI:10.1007/s13361-017-1861-2

Ozonolysis of alkene functional groups is a type of highly specific and effective chemical reaction, which has found increasing applications in structural analysis of unsaturated lipids via coupling with mass spectrometry (MS). In this work, we utilized a low-pressure mercury lamp (6?W) to initiate ozonolysis inside electrospray ionization (ESI) sources. By placing the lamp near a nanoESI emitter that partially transmits 185?nm ultraviolet (UV) emission from the lamp, dissolved dioxygen in the spray solution was converted into ozone, which subsequently cleaved the double bonds within fatty acyls of lipids. Solvent conditions, such as presence of water and acid solution pH, were found to be critical in optimizing ozonolysis yields. Fast (on seconds time scale) and efficient (50%–100% yield) ozonolysis was achieved for model unsaturated phospholipids and fatty acids with UV lamp-induced ozonolysis incorporated on a static and an infusion nanoESI source. The method was able to differentiate double bond location isomers and identify the geometry of the double bond based on yield. The analytical utility of UV lamp-induced ozonolysis was further demonstrated by implementation on a liquid chromatography (LC)-MS platform. Ozonolysis was effected in a flow microreactor that was made from ozone permeable tubing, so that ambient ozone produced by the lamp irradiation could diffuse into the reactor and induce online ozonolysis post-LC separation and before ESI-MS. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s13361-017-1861-2

B American Society for Mass Spectrometry, 2017
J. Am. Soc. Mass Spectrom. (2017)
DOI: 10.1007/s13361-017-1861-2
RESEARCH ARTICLE
UV Lamp as a Facile Ozone Source for Structural Analysis
of Unsaturated Lipids Via Electrospray Ionization-Mass
Spectrometry
Craig A. Stinson,1,2 Wenpeng Zhang,1,3 Yu Xia1,3
1Department of Chemistry, Purdue University, West Lafayette, IN 47907-2084, USA
2Present Address: Intel Corporation, Hillsboro, OR 97214, USA
3Department of Chemistry, Tsinghua University, Beijing, 100084, China
UV Induced Ozonolysis
Abstract. Ozonolysis of alkene functional groups is a type of highly specific and
effective chemical reaction, which has found increasing applications in structural
analysis of unsaturated lipids via coupling with mass spectrometry (MS). In this work,
we utilized a low-pressure mercury lamp (6 W) to initiate ozonolysis inside
electrospray ionization (ESI) sources. By placing the lamp near a nanoESI emitter
that partially transmits 185 nm ultraviolet (UV) emission from the lamp, dissolved
dioxygen in the spray solution was converted into ozone, which subsequently
cleaved the double bonds within fatty acyls of lipids. Solvent conditions, such as
presence of water and acid solution pH, were found to be critical in optimizing
ozonolysis yields. Fast (on seconds time scale) and efficient (50%–100% yield)
+/-
O
3
+/-
h
185 nm
O2
from LC
h
2O(3P)
to ESI
O2
O
3
AC
ozonolysis was achieved for model unsaturated phospholipids and fatty acids with UV lamp-induced ozonolysis
incorporated on a static and an infusion nanoESI source. The method was able to differentiate double bond
location isomers and identify the geometry of the double bond based on yield. The analytical utility of UV lamp-
induced ozonolysis was further demonstrated by implementation on a liquid chromatography (LC)-MS platform.
Ozonolysis was effected in a flow microreactor that was made from ozone permeable tubing, so that ambient
ozone produced by the lamp irradiation could diffuse into the reactor and induce online ozonolysis post-LC
separation and before ESI-MS.
Keywords: Ozonolysis, Unsaturated lipid, Electrospray ionization, Lipidomics, LC-ms
Received: 1 November 2017/Revised: 27 November 2017/Accepted: 27 November 2017
properties and due to its compatibility with liquid chromatog-
raphy (LC) separations [7, 8].
Introduction
ipids have gained increased interest in biomedical fields in
recent years as mounting evidence indicates their critical
A long-standing issue with mass spectrometry-based anal-
ysis is the resolution of molecular isomers, which is exacerbat-
ed in lipid analysis due to large structural diversity [9]. A well-
encountered situation is determination of alkene positional and
geometric isomers in lipid fatty acyls, which often requires a
battery of analytical methods. For instance, conjugated linoleic
acids (CLAs), which are found to have health benefits, includ-
ing weight reduction and cancer prevention among others,
typically exist in several isomeric forms [6]. The most abundant
naturally occurring CLA is FA18:2 (9Z, 11E), with many other
isomers existing in low abundance [10]. Although gas chroma-
tography (GC) and silver ion liquid chromatography can sep-
arate most isomers of CLA fatty acid methyl esters, complete
structural characterization of individual isomers requires col-
lective efforts from MS, nuclear magnetic resonance, and Fou-
rier transform infrared spectroscopy [11].
L
function in cell signaling [1, 2] and as biomarkers of health and
disease [3]. There are also wide industrial and consumer appli-
cations for using lipids as chemical feedstocks [4], surfactants
[5], and nutraceuticals [6]. Accompanying these advances, the
demand is surging for accurate and rapid identification of lipids
of interest from complex mixtures. Mass spectrometry (MS)-
based techniques have emerged as powerful methods for lipid
analysis, with electrospray ionization (ESI) being commonly
employed as the ionization method due to its ability to ionize a
large spectrum of lipids with different physical/chemical
Correspondence to: Yu Xia; e-mail: xiayu@mail.tsinghua.edu.cn

C. A. Stinson et al.: UV-lamp Induced Ozonolysis for Unsaturated Lipid Analysis
Recently, there have been promising developments in MS,
concentrations are reported to reach 0.001% in ambient air
[43]. Although producing much lower concentrations of ozone
compared with the ozone generators, LP Hg lamps enjoy
advantages of low power consumption, small physical size,
and wide accessibility (commercially available at relatively
low cost). Considering the traits of LP Hg lamps, we attempted
to develop an ozonolysis reactor that could be coupled online
with ESI-MS by employing a LP Hg lamp for ozone produc-
tion. Using unsaturated lipid standards (FAs and GPs), includ-
ing their C=C location isomers and configuration isomers as
model compounds, we demonstrated that UV-induced
ozonolysis from an LP Hg lamp in ambient air was highly
efficient and could be online coupled with ESI from nanoflow
to microflow regimes. The compatibility of the online
ozonolysis reactor with LC-ESI-MS was further demonstrated
using a mock mixture of unsaturated FA isomers.
aiming to address the analytical challenge of carbon–carbon
double bond (C=C) characterization with simplified proce-
dures, increased sensitivity, and throughput. These include
solution derivatization (ozonolysis [12], the Paternò-Büchi re-
action [13, 14], charge-switch derivatization [15], etc.), new
gas-phase ion activation/dissociation methods (charge remote
fragmentation [16, 17], ion/molecule reactions [18], radical-
based dissociation [19, 20], electron-based excitation [21],
ultraviolet photo-dissociation [22, 23], etc.), and coupling ion
mobility separation with MS [24–26]. Among these,
ozonolysis of unsaturated fatty acyls has historically been a
reliable method for locating C=C by detecting the aldehyde
products [27, 28]. During ozonolysis, ozone reacts with the
alkene functional group to form a primary ozonide, which
rapidly dissociates to a site-specific aldehyde and a carbonyl
oxide (Criegee intermediate) [29]. The products can then re-
combine to form secondary ozonides or the Criegee intermedi-
ate subsequently reacting with adjacent solvent molecules. In
the past decade, ozonolysis has been adapted to ESI-MS and
applied to glycerophospholipids (GPs), glycerolipids (GLs),
and fatty acid methyl esters (FAMEs) [12, 30]. Online
ozonolysis ESI has been implemented via corona discharge at
the ESI tip region, adding ozone to the ESI desolvation gas
(ozone ESI), and ionization via low temperature plasma ioni-
zation at ambient air [31–33]. Direct information can be ob-
tained on the lengths of chains being cleaved off at each C=C
from the unsaturated fatty acyls. Because it is necessary to link
the ozonolysis products to their intact precursors to achieve
C=C location determination, ozone ESI is not well suited for
complex lipid mixture analysis. To solve this problem, the
Blanksby group subsequently implemented ion/molecule
(ozone) reactions to achieve ozonolysis of mass-selected ions
inside a mass spectrometer, termed OzID, which has been
demonstrated for complex mixture analysis on different instru-
ment platforms [34–38]. Alternatively, the Curtis group dem-
onstrated in-line ozonolysis after one- or two-dimensional LC
separations prior to MS analysis. Owing to excellent lipid
separation with LC, identification of geometric and positional
lipid isomers of conjugated CLA FAMEs and GPs in complex
mixtures have been achieved [39–41].
Experimental
Lipid Nomenclature
Shorthand notation from the Lipid Maps project was used for
lipid structural identification [44, 45]. For GP standards, the
head group, fatty acyl stereo position, fatty acyl carbon num-
ber, degree of unsaturation, and alkene stereo orientation were
specified. For example, PE 16:0/18:1(9Z) signifies the
glycerophosphoethanolamine head group with 16 and 18 car-
bon fatty acyl chains on the sn1 and sn2 glycerol positions,
respectively. The B0^ and B1^ after the carbon number refers to
the degree of unsaturation of the respective fatty acyl. Alkene
bond location was defined by counting sequential carbons
starting from alpha carbon of the fatty acyl ester and proceeding
towards the terminal carbon, i.e., C=C bond at the Δ9 position
is located between carbon 9 and 10 of the fatty acyl. Z and E
nomenclature for alkene bond stereo configuration follows the
alkene location identifier.
Chemicals
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC 16:0/
18:1(9Z)) and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphate
(PA 18:0/18:1(9Z)) dissolved in 10 mg/mL chloroform were
purchased from Avanti Polar Lipids, Inc. (Alabaster, AL,
USA). Stock solutions in chloroform were diluted with
isopropanol (LC grade; Macron Fine Chemicals; Center Val-
ley, PA, USA) before diluting to the final working solution.
Primary organic solvents used in ESI working solutions were
all LC grade and consisted of methanol (Macron Fine
Chemicals), acetonitrile (Sigma Aldrich; St. Louis, MO,
USA), and isopropanol. Ultrapure H2O was obtained from a
purification system at 0.03 μS∙cm (model: Micropure UV;
Thermo Scientific; San Jose, CA, USA). Ammonium hydrox-
ide (28%–30% as NH3; Macron Fine Chemicals), glacial acetic
acid (Mallinckrodt Chemicals; Hazelwood, MO, USA), formic
acid and ammonium formate (Sigma Aldrich, St. Louis, MO,
USA) were used as solution modifiers to enhance lipid
In the above-mentioned ozonolysis applications, dedicated
ozone generators have been used as sources for ozone produc-
tion. These ozone generators utilize dielectric barrier discharge
or corona discharge of dioxygen gas to achieve high ozone
concentrations (up to 10%) [42]. Photodissociation of
dioxygen by vacuum UV (VUV) photons is an alternative
method to generate ozone. For instance, low pressure mercury
(LP Hg) lamps are known as Bozone producing^ lamps when
the emission wavelength at 185 nm is not filtered. Photodisso-
ciation of dioxygen at 185 nm results in the production of two
O (3P) atoms. The O (3P) atom then combines with molecular
oxygen to form ozone in the presence of a non-reacting species
(Figure 1a). Resulting from irradiation by the 254 nm wave-
length emission of the LP Hg lamp, a significant portion of
ozone decomposes into O (1D) and O2, and ozone

C. A. Stinson et al.: UV-lamp Induced Ozonolysis for Unsaturated Lipid Analysis
[M+H]+
PC 16:0/18:1(9Z)
c
e
a
UV Off
O2 + h
2 O(3P)
O3 + M
O2 + O(1D)
185 nm
760.5
m/z 760.5
O(3P) + O2 + M
[M+Na]+
782.4
O3
Aqueous solvent
O3 + h
254 nm
400 500 600 700 800 900 1000
m/z
m/z 650.4
1 cm
b
d
UV On
650.4
f
borosilicate glass
UV off
UV off
m/z 650.4
MS
inlet
background
m/z 760.6
tip o.d.
[M+H]+
763.4
UV on
1.0
UV on
HV
760.6
h
185 nm
~ 20 nL/min
electroosmotic flow
2.0
3.0
4.0
5.0
6.0
400
500
600
700
m/z
800
900
1000
Time, min
AC
Figure 1. (a) Ozone formation and decomposition in ambient air near an LP-hg lamp. (b) Experimental setup for implementing UV
lamp-induced ozonolysis on a static nanoESI source. Positive ion mode MS spectra of 5 μM PC 16:0/18:1(9Z) dissolved in 7:3
ACN:H2O, 1% acetic acid (c) before UV irradiation, and (d) during UV irradiation. (e) Structures of protonated PC 16:0/18:1(9Z) and
the aldehyde product resulting from ozonolysis. (f) XIC of on/off ozonolysis depicting the baseline peak area intensities of precursor
ion (m/z 760.6) and reaction product (m/z 650.4) as a function of time
ionization via ESI. 1-Stearoyl-2-arachidonoyl (PC 17:0/20:4),
cis-vaccenic acid (FA 18:1 (11Z)), and oleic acid (FA 18:1
(9Z)) were purchased as purified oils and dissolved in chloro-
form for final concentrations of 1 mg/mL and subsequently
diluted into working solutions.
positioned vertically and 0.5–1.5 cm orthogonal from the emit-
ter tip depending on the experiment with more details described
in the Results and Discussion. Ozone concentration under
ambient conditions was measured using an ozone analyzer
(model 49i; Thermo Scientific).
NanoESI
Mass Spectrometry
Ozonolysis was effected with two different nanoESI sources
made from fused silica capillary or borosilicate glass. Fused
silica tips were purchased prefabricated from New Objective,
Inc. (Woburn, MA, USA) and were connected to a stainless-
steel union (Valco; Houston, TX, USA), which served to join
the solution propelled by syringe pump (Harvard Apparatus;
Holliston, MA, USA model: 11 plus) to the nanoESI emitter
and was also the point of high voltage application to effect
electrospray (~ 1.5 kV). Borosilicate glass tips were made
from capillary (1.5 mm o.d. and 0.86 mm i.d.) pulled to a
~10 μm i.d. tip using a micropipette puller (P-1000 Flaming/
Brown; Sutter Instrument, Novato, CA, USA). A stainless-steel
wire (~4 cm in length) was inserted through the back of the tip
and was in contact with the sample fluid. High voltage DC
potential of ~ 1.5 kV was applied between the source and MS
inlet, which enabled a stable ion current via electroosmotic
flow of ~20 nL/min.
All data were collected on a 4000 QTRAP mass spectrometer
(Sciex, Toronto, Canada) equipped with a home-built nanoESI
source. The characteristic parameters of the mass spectrometer
used in this study were set as follows: spray voltage, 1200 to
1500 V; curtain gas, 10 psi; and declustering potential, 40 V.
Data acquisition, processing, and instrument control were per-
formed using Analyst 1.5 software.
Online Ozonolysis-LC-MS for Fatty Acid Mixture
Analysis
The LC system consisted of a binary pump (Agilent 1200;
Waldbronn, Germany) and a manual injector with a 10 μL-sample
loop. A C18 column (150 mm × 4.6 mm i.d., 5 μm particle size,
Agilent) was used for chromatographic separation. An ozone
reactor was made by ozone permeable Teflon AF-2400 tubing
(0.02-in. o.d., 0.01-in. i.d., 80-cm in length; Biogeneral Inc., San
Diego, CA, USA). The AF tubing was coiled concentrically at
3 cm radius with the UV lamp placed in the center. The reactor
connected the LC and a homemade splitter (1:50 v/v), which
decreased the flow rate to 10 μL/min for ESI-MS. Mobile phase
consisted of acetonitrile with 0.5% formic acid (v/v, A) and
ammonium formate buffer (10 mM, B). Gradient elution was
used as follows: 80% B (5 min), 80%–85% B (5–10 min),
85%–90% B (10–15 min), and 95%–100% B (22 min). At the
end of elution, the gradient was set back to 80% B and the system
was allowed to equilibrate.
Ozone Reactions
A 20 mA, 2.54 cm lamp length, 0.64 cm diameter, double bore
tubing low pressure mercury (LP Hg) lamp (model number:
81–1057-51; BHK, Inc.; Ontario, CA, USA) was used to create
ozone. Lamp specifications from the manufacturer state the
emission intensities at 20 cm distance for 254 and 185 nm is
73 and 42 μW/cm2, respectively (185 nm intensity measured in
argon atmosphere). For most experiments, the lamp was

C. A. Stinson et al.: UV-lamp Induced Ozonolysis for Unsaturated Lipid Analysis
a
PA 18:0/18:1(9Z)
Results and Discussions
The formation of ozone from an LP Hg lamp used in this study
was monitored by an ozone analyzer via photometric detection,
sampling at 1 cm distance from the lamp. Ozone concentration
was found around 10 ppm (data not shown). We then placed
the lamp 0.5 cm away from a static-nanoESI emitter made from
a pulled borosilicate glass tip to test if efficient ozonolysis can
be implemented at the nanoESI-MS interface. Figure 1c and d
compare the nanoESI MS spectra of an unsaturated model
lipid, PC 16:0/18:1(9Z) (5 μM in 7:3 ACN: H2O, 1% acetic
acid) before and during UV irradiation in positive ion mode.
Before the lamp was turned on, protonated PC ([M + H]+, m/z
760.6) was detected as the most abundant species accompanied
by a small sodium adduct peak (m/z 782.4). Upon UV irradi-
ation, the intact PC signal dropped significantly, while a peak at
m/z 650.4 appeared, corresponding to cleavage of the Δ9 C=C
and formation of an aldehyde at this position (structure shown
in Fig. 1e). Combining the information from ozonolysis with
that obtained from conventional MS/MS experiments, relative-
ly complete structural information of this lipid molecule, in-
cluding the head group (obtained from CID in the positive ion
mode), fatty acyl chain composition (obtained from CID in the
negative ion mode), and C=C location, can thus be determined.
Figure 1f shows the extracted ion chromatogram (XIC) of
intact PC (m/z 760.6) and the ozonolysis product (m/z 650.4)
for two consecutive events when the lamp was turned on for
12 s and subsequently turned off. With the lamp on, the
precursor ion was converted approximately 100% to m/z
650.4 and the intact PC was completely consumed. After the
lamp was turned off, ozonolysis product was observed for
approximately 2 min, suggesting that most of the reaction
occurred in solution near the spray tip. Ozone might be formed
from two sources: (1) formation in the tip due to trace oxygen
dissolved in the solution, or (2) diffusion of ozone from the
spray plume region into the solution through the tip. Borosili-
cate glass does not transmit UV photons of wavelengths shorter
than 250 nm due to the presence of additives in glass; however,
the glass thickness near the tip approaches micrometer dimen-
sions, which increases the likelihood of photon transmission to
the solution. On the other hand, it has been reported that
dioxygen can diffuse into the solution from the ESI tip that is
exposed to atmospheric pressure [46]. Given the low flow rate
at which the static nanoESI operates (around 20 nL/min), it is
possible that ozone formed at the vicinity of the tip can effec-
tively diffuse into the nanoESI solution and react with the
unsaturated lipid alkene.
591.5, 639.5
[M-H]-
701.7
UV on
591.5
639.5
background
732.6
550
600
650
m/z
700
750
PC 17:0/20:4(5Z,8Z,11Z,14Z)
728.5 688.5 648.4 608.4
b
[M+H]+
UV on
796.6
background
648.4
688.5
608.4
728.5
763.8
500
600
700
m/z
800
900
Figure 2. UV lamp-induced ozonolysis of unsaturated phos-
pholipids: (a) negative ion mode nanoESI of 5 μM PA 18:0/
18:1(9Z) in 1:1 ACN:MeOH with 1% acetic acid added, and (b)
positive ion mode nanoESI of 10 μM PC 17:0/20:4
(5Z,8Z,11Z,14Z) in 69:30:1 ACN:H2O:MeOH with 1% acetic
acid added. The structure motifs of ozonolysis products are
indicated in figure insets
methoxyhydroperoxide product (structure motif shown in the
inset of Fig. 2a). This product results from reaction between
methanol and the Criegee intermediate and has been previously
demonstrated when methanol is used as a co-solvent for in-
source ozonolysis-MS experiments [32]. Detection of α-
methoxyhydroperoxide product provided strong evidence that
ozone was indeed involved in unsaturated lipid alkene cleavage
and site-specific aldehyde formation for the reactions reported
in this study.
Figure 2b demonstrates the application of UV lamp
ozonolysis for the analysis of lipid molecules consisting of
polyunsaturated fatty acyls. The nanoESI spray solution of
PC 17:0/20:4 (5Z,8Z,11Z,14Z) was prepared at 10 μM in
69:30:1 ACN:H2O:MeOH with 1% acetic acid added. As
shown in the reaction spectrum (Figure 2b), the aldehyde
products are formed abundantly, viz. ions at m/z 608.4, 648.4,
688.5, and 728.5, resulting from ozonolysis at the Δ5, 8, 11,
and 14 double bonds. Minor α-methoxyhydroperoxide prod-
ucts were also detected (not labeled) due to the presence of 1%
MeOH in the solution. This experiment suggests UV lamp-
induced ozonolysis is applicable for alkene localization in
polyunsaturated lipids.
The UV lamp-induced ozonolysis setup was further tested
for unsaturated phospholipid analysis in negative ion mode.
Figure 2a shows the reaction spectrum of PA 18:0/18:1(9Z)
prepared at 5 μM in 1:1 ACN:MeOH with 1% acetic acid
added upon UV irradiation. Two major reaction products were
observed at m/z 591.5 and 639.5, respectively. The product at
m/z 591.5 is the aldehyde formed from cleavage of the Δ9 C=C.
The product at m/z 639.5 is 48 Da higher in mass than the
aldehyde product, characteristic for the formation of the α-

C. A. Stinson et al.: UV-lamp Induced Ozonolysis for Unsaturated Lipid Analysis
Being successful in coupling UV lamp-induced ozonolysis
on a static nanoESI source, we further tested its compatibility
with infusion nanoESI operating in a higher flow rate regime
(300 nL/min to 1 μL/min). Figure 3a shows schematics of the
experimental setup. The nanoESI emitter (15 μm tip i.d.) was
constructed from fused silica capillary (75 μm tip i.d.) with
polyamide coating removed from the tip (~7 mm) during
manufacturing. The LP Hg lamp was placed at 1.5 cm away
from the tip. Oleic acid (FA 18:1(9Z)), consisting of a single
C=C in the fatty acyl chain, was used a model compound.
Figure 3b shows the MS spectrum of 5 μM oleic acid prepared
in 7:3 ACN:H2O (0.5% formic acid added) infused at 500 nL/
min in negative ion mode. The deprotonated oleic acid peak
([M–H]−) was clearly detected at m/z 281.4. When the lamp was
turned on, the ozonolysis product at m/z 171.3, which would be
Note that there was approximately 0.5 s delay for the lamp to
warm up, resulting in approximately 2.5 s effective illumina-
tion time. With the lamp on for 2.5 s the oleic acid ion intensity
decreased to 1.35e6 counts per second (cps) from 3.8e6 cps,
accounting for 65% decrease in intensity (Fig. 3b and c are the
corresponding MS1 spectra). Simultaneously, the 9-oxo-
nonanoic acid ion increased to 50% the original oleic acid ion
intensity before UV exposure. When the lamp was turned off
the ion signal at m/z 171.3 began to decrease while m/z 281.4
increased, clearly demonstrating that the peaks were affected
by irradiating the fused silica nanoESI tip with UV light from
the lamp. When the experiment was completed after 32 s, the
precursor signal at m/z 281.4 returned to its original intensity.
At 500 nL/min solution flow rate and 7 mm exposure length,
the solution traverses this length in 3.7 s. Upon close observa-
tion of the XIC in Fig. 3d, the ozonolysis product (m/z 171.3)
lasted for ~4.2 s after the lamp was turned off. This timing
indicates that the majority of the ozonolysis occurs in solution,
which likely results from ozone formation from oxygen dis-
solved in the solution. When N2 purged solution was subjected
to the same experiments, the yield of 9-oxo-nonanoic acid was
significantly reduced. The remaining small amount of
ozonolysis product was caused by ambient oxygen diffused
into the ESI solution through the tip opening. Similarly, exper-
iments at the same flow rate but using a stainless-steel nanoESI
source also resulted in drastically reduced yield of 9-oxo-
nonanoic acid ion, further corroborating the hypothesis of
solution ozonolysis. Using the fused silica nanoESI setup,
UV lamp-induced ozonolysis was achieved in reasonable
yields (>50%) for flow rate from 300 to 1000 nL/min.
−
consistent for C9H15O3 , immediately appeared, corresponding
to deprotonated 9-oxo-nonanoic acid (Fig. 3c). This product
allowed identification of the alkene functional group between
C9 and C10, counting from the carboxylic acid side [47].
Turning the lamp on and off the process of C=C cleavage
and 9-oxo-nonanoic acid ion formation can be activated or
deactivated, respectively, on the seconds timescale. Figure 3d
represents an XIC of intact oleic acid ([M–H]−, m/z 281.4) and
the ozonolysis product (m/z 171.3) from a sequence of 3 s lamp
on/2 s lamp off (manually repeated six times) in a span of 30 s.
direct infusion
a
a
300 – 1000 nL/min
7 mm
MS
inlet
tip i.d.
fused silica capillary
h
185 nm
Effect of Water in the Solvent for Ozonolysis
HV
FA 18:1(9Z)
The Beauchamp group studied the interfacial ozone reactions of
oleic acid (FA 18:1(9Z)) with field-induced droplet ionization
and reported detection of abundant azeleic acid (m/z 187) besides
9-oxo-nonanoic acid ion (m/z 171) and oleic acid dimers [47]. It
was hypothesized that azeleic acid (HOOC(CH2)7COOH) was a
product resulting from the Criegee intermediate. Interestingly,
this product was not detected in this work. Instead, the aldehyde
was observed as the only ozonolysis product for lipid samples
dissolved in ACN/H2O solvent system (Fig. 1d and Figure 3c).
We attributed this phenomenon to the presence of water in the
solution. It has been reported that nucleophilic addition of water
to the alpha carbon of the Criegee intermediate releases H2O2 and
forms an aldehyde (Scheme 1) [48]. Because only aldehydes are
formed from ozonolysis, the reaction spectra are simple, leading
to higher sensitivity for detection and straightforward elucidation
of C=C location.
b
b
c
UV on
171.3
[M-H]-
UV off
[M-H]-
171.3
281.4
281.4
175.1
150 200 250 300 350 400
150 200 250 300 350 400
m/z
m/z
3 s UV on / 2 s UV off
m/z 281.4
d
m/z 171.3
0
6
12
18
24
30
36
Time, s
Figure 3. (a) Schematics of the reaction setup for UV lamp-
induced ozonolysis coupled with infusing nanoESI. Negative
ion mode MS spectra of oleic acid (FA 18:1(9Z), 5 μM, dissolved
in 7:3 ACN: H2O, 0.5% formic acid added) at 500 nL/min flow
rate: (b) before UV irradiation, and (c) after 2.5 s UV irradiation.
(d) XIC of intact oleic ion ([M-H]−, m/z 281.4) and deprotonated
9-oxo-nonanoic acid (m/z 171.3) with the lamp manually turned
on for 3 s and off for 2 s sequentially
Impact of Solution pH
In the course of exploring optimal conditions for ozonolysis,
we found it was critical to keep solution pH low. For instance,
when FA or PA solutions were added with 1% ammonium
hydroxide, a common additive for negative ion mode ESI, no

C. A. Stinson et al.: UV-lamp Induced Ozonolysis for Unsaturated Lipid Analysis
Aldehyde Product
alkene functional groups absorb UV radiation where conjugat-
ed double bonds are sensitive to ~233 nm wavelength, whereas
mono- and polyunsaturated species absorb below 200 nm
wavelength. Bond geometry isomerization and subsequent
photochemical reactions are common events following photon
absorption [52]. Irradiation of ESI solution containing unsatu-
rated lipids with UV light from a LP Hg lamp could potentially
result in direct photon absorption by the alkene functional
group. These photochemical reactions appear, though, to not
have a significant impact during the UV-induced ozonolysis as
evidenced by the minimal observation of side reaction prod-
ucts. The geometry of cis/trans isomers also appears to be
conserved through observation of different ozonolysis reaction
rates (to be discussed in subsequent sections).
H2O2
+/-
+/-
O3
+/-
+/-
+/-
Primary
Ozonide
Criegee
Intermediate
Scheme 1. Formation of aldehyde as the main product for
ozonolysis in the presence of water as solvent. For simplicity,
only the reaction product consisting of charge moiety is
depicted in the scheme
obvious ozonolysis products were observed. It has been shown
that hydroxide anion (OH−) reacts with ozone at rate constant
in the range of 48–70 M−1 s−1 (Reaction, 1) [49]. At basic
solution pH, the half-life of ozone in solution decreases signif-
icantly, resulting in negligible alkene cleavage products. The
replacement of ammonium hydroxide by 1% acetic acid indeed
improved ozonolysis to ~15% yield for oleic acid solution upon
UV irradiation. Interestingly, addition of 0.5% formic acid in
the solution (in place of acetic acid) produced higher yield
ozonolysis (~60%, Figure 3c). The pKa of formic acid is 3.75
relative to 4.75 of acetic acid, corresponding to theoretical pH
values of 2.3 and 2.7, respectively, for compositions of 0.5%
formic acid and 1% acetic acid in 1 mL aqueous solution. For
GPs, it was experimentally determined that addition of 1%
acetic acid to the ESI solution produced abundant aldehyde
reaction products, therefore not requiring the use of formic
acid. It is not clear at this time exactly why ozonolysis of GPs
is more efficient at higher pH relative to FAs. However, it is
evident that conditions of low pH are critical for efficient
ozonolysis and this could be due to the relatively low concen-
tration of ozone produced with the LP Hg lamp used in this
study.
Analysis of Mixtures of C=C Location Isomers
FAs having the same number of carbons but with different
alkene locations are termed positional isomers and during
mixture analysis cannot be resolved with MS or MS/MS at
low CID energy conditions. Ozonolysis is a powerful method
for determining C=C location and can be utilized for isomer
differentiation. To demonstrate this ability, a mixture of oleic
acid (FA 18:1(9Z)) and cis-vaccenic acid (FA 18:1(11Z))
(5 μM each in 7:3 ACN:H2O, 0.5% formic acid added) was
subjected to UV-induced ozonolysis using the static nanoESI
setup. Figure 4a shows the reaction spectrum, in which the
aldehyde products of each isomer are observed abundantly at
m/z 171.2 and 199.2 besides the remaining intact FA ions (m/z
281.4). The aldehyde ions provide direct evidence for locating
C=C at Δ9 and Δ11, respectively, which otherwise would not
be obtainable on standard ESI MS/MS platforms. In addition to
identification, there is the potential for relative isomer quanti-
tation based on intensity ratios of ozonolysis products.
Figure 4b shows such a plot as a function of cis-vaccenic/oleic
acid molar ratios, while keeping the total concentration of cis-
vaccenic and oleic acid at a constant 10 μM. A linear relation-
ship (R2 = 0.9925) was obtained for the FA 18:1(11Z)/FA
18:1(9Z) molar ratios ranging from 0.075 to 13.3 and the limit
of detection for each isomer in the mixture was 0.2 μmol.
Conjugated linoleic acids (CLA) are a widely studied class
of natural and synthetically derived FAs that have potential
human health benefits, including anti-cancer properties [6].
Online ESI MS ozonolysis of CLA FAMEs has been demon-
strated in both the solution and gas phase with ozone generators
[41, 53]. Figure 4c shows UV lamp-induced ozonolysis of a
mixture of two CLA isomers, FA 18:2(9Z, 11E) and FA
18:2(10E, 12Z) ([M–H]−, m/z 279.3). The aldehyde products
at m/z 171.2 and 197.2 derived from ozonolysis of FA 18:2(9Z,
11E), whereas ions at m/z 185.2 and 211.2 resulted from FA
18:2 (10E, 12Z). Peaks at m/z 295.4 and 313.5 are sequential
oxidation reaction products from the CLA precursor, whereas
all other ions were present before application of UV and are
considered as background. In the literature it is reported that
trans-double bonds have faster ozonolysis reaction kinetics
relative to the cis-geometry [35, 54]. This phenomenon was
ˉ
•
O3 þ OHˉ→O2 þ O2H k ¼ 48−70 M−1s−1
ð1Þ
Other Possible Reactions
In addition to ozonolysis, we acknowledge the complex
chemistry/photochemistry that occurs in solution from UV
radiation and therefore do not rule out the possibility of other
mechanisms for the observed alkene cleavages. The 185 nm
emission from an LP Hg lamp can also be absorbed by H2O in
the solution phase, resulting in photodissociation producing
hydroxyl radical, a process that is used extensively in water
purification [50]. The concentration of dioxygen in solution is
approximately 2 mM and a solution containing 30% H2O has
an [H2O] of 17 M. Lipid peroxidation with hydroxyl radical
typically involves rearrangement of the alkene upon hydrogen
abstraction and oxygen addition [51]. The related reaction
products were not observed when performing the reaction with
standard lipids, which provides evidence that hydroxyl radical
was not ultimately involved in alkene cleavage. In addition,

C. A. Stinson et al.: UV-lamp Induced Ozonolysis for Unsaturated Lipid Analysis
bond positions in lipid mixtures (FAMEs and PCs) [39–41].
FA 18: (11Z)
199.3
In their studies, the reactor is produced from of thin-walled
(0.02-in. o.d., 0.01-in. i.d.) Teflon AF-2400 tubing, which
passes through a bottle filled with gaseous ozone at 35.6 g m
a
b
UV on
FA 18:1(9Z)
171.2
[M-H]-
281.4
−3
(~2.5%). Ozone can diffuse into the tubing due to the
283.4
background
semipermeable nature of the material and react with unsaturat-
ed lipids efficiently in solution at even relatively high flow rate
(~0.5 mL/min) prior to ionization and MS analysis. Inspired by
their work, we also tested coupling UV lamp-nduced
ozonolysis with online LC-MS. Figure 5a shows the schematic
representation of the flow reactor placed in after the LC column
and before a home-built ESI source. The flow reactor was made
from the same Teflon AF-2400 tubing, which was concentri-
cally coiled (80 cm in length) at 3-cm radius, while the UV
lamp (185 nm) was placed in the center. The distance between
the coil and the lamp was about 2.5 cm. At a flow rate of
0.5 mL/min, the effective reaction time was 15 s, equivalent to
the residence time of the solution traveled in the UV exposed
region of the tubing. A mock mixture of four unsaturated fatty
acids, including FA 18:2 (9Z, 12Z), FA 18:2 (10E, 12Z), FA
17:1 (10Z), and FA 18:1 (9Z), was subjected to reversed-phase
LC separation, post-column ozonolysis, and ESI-MS analysis.
As shown in the ion chromatogram (Figure 5b), all five species
were well separated, even for the C=C location isomers, FA
18:2 (9Z, 12 Z) and FA 18:2 (10E, 12Z). Figure 5c–f present
the ozonolysis spectra (negative ion mode) of each fatty acid
eluent. The aldehyde products corresponding to cleavage of
each individual C=C in the fatty acid were clearly detected,
150
200
250
m/z
300
350
10.0
y = 0.5977x + 0.4117
R² = 0.9925
5.0
0.0
0
5
10
15
molar ratio, FA 18:1 (11Z) /(9Z), 10 µM total
FA 18:2(9Z,11E)
c
FA 18:2(10E,12Z)
171.2
197.2
11E
UV on
211.2 185.2
10E
185.2
197.2
313.5
12 Z
211.2
[M-H]-
279.5
9Z
171.2
311.4
295.4
150
200
250
m/z
300
350
b
a
FA 17:1 (10Z)
h
185 nm
Figure 4. Application of UV-induced ozonolysis for FA C=C
isomer analysis. (a) Negative ion mode MS spectrum of
ozonolysis of 1:1 mixture of oleic acid [FA 18:1(9Z)] and cis-
vaccenic acid (FA 18:1 (11Z)). (b) a plot of ratios of aldehyde
products of vaccenic/oleic acid versus their molar concentra-
tion ratios (total of 10 μM). (c) Negative ion mode ozonolysis MS
spectrum of a mixture of two CLA isomers, FA 18:2(9Z, 11E)
and FA 18:2(10E, 12Z) ([M–H]−, m/z 279.5)
100
from LC
FA 18:2 (10E, 12Z)
FA 18:2 (9Z, 12Z)
FA 18:1 (9Z)
to ESI
Teflon AF tubing
(0.02” o.d., 0.01” i.d.)
0
10
12
14 16
Time, min
18
20
22
c
e
d
100
279.2
279.2
100
FA 18:2 (9Z, 12Z)
FA 18:2 (10E, 12Z)
indeed observed during UV lamp-induced ozonolysis. For
instance, the intensity of the 11E (m/z 197.2) and 10E (m/z
185.2) products are higher than the 9Z (m/z 171.2) and 12Z
(m/z 211.2), respectively. Based on the ozonolysis reaction rate
difference, the intensity differences between cis/trans geome-
tries may be used to identify alkene geometries during analysis
of unknown samples. Furthermore, observation of the different
reaction rates between cis/trans geometries gives further indi-
cation that, at least during initial stages of UV lamp-induced
ozonolysis, geometry is conserved during UV induced
ozonolysis.
background
183.0
background
183.0
185.1
211.1
171.1
211.1
200
0
0
200
250
250
m/z
m/z
f
267.3
281.3
FA 17:1 (10Z)
100
FA 18:1 (9Z)
100
background
183.0
background
183.0
185.1
283.3
171.1
229.0
0
0
200
250
200
250
m/z
m/z
Figure 5. UV lamp-induced ozonolysis coupled online with
LC-MS for fatty acid mixture analysis. (a) Cartoon depiction of
the experimental setup. (b) Reversed-phase liquid chromato-
gram of four unsaturated fatty acids. ESI-MS spectra in nega-
tive ion mode of (c) FA 18:2 (9Z, 12Z), (d) FA 18:2 (10E, 12Z), (e)
FA 17:1 (10Z), and (f) FA 18:1 (9Z) after online ozonolysis
Coupling UV-Lamp Induced Ozonolysis with Online
LC-MS
The Curtis group has successfully demonstrated coupling an
ozonolysis reactor in-line with LC-MS for locating double

C. A. Stinson et al.: UV-lamp Induced Ozonolysis for Unsaturated Lipid Analysis
allowing confident assignment of C=C location. Compared
species since ozonolysis occurs in the ESI solution. UV lamp
allows fast Bon/off^ ozonolysis on the seconds time scale (data
in Figure 3). This feature, although not demonstrated herein,
could be useful for LC-MS platform, where the Boff^ cycle can
be used to for identifying lipid structural information, such as
head group and fatty acyl composition via linked scans (MS/
MS), while the Bon^ cycle could lead to the C=C location
information, thus allowing high level structural characteriza-
tion. Due to the scope of this study, only fatty acids and
phospholipids were explored for ozonolysis. These two classes
of lipids have relatively high solubility in ACN/H2O solvent
system for ozonolysis and the response in ESI is comparable to
the commonly used CHCl3/MeOH system. In future studies,
UV lamp-induced ozonolysis conditions will be tailored for
analyzing neutral lipids, which might require use of a more
nonpolar solvent system to enhance both solubility and re-
sponse in ESI.
with the in-line ozonolysis reactor developed by the Curtis
group, UV lamp-induced ozonolysis from our work showed
lower reaction efficiency. We attributed this to high solution
flow rate (0.5 mL/min), which resulted in inefficient interaction
between ozone and lipids and low ozone concentration
(10 ppm) produced in the current setup. Reaction efficiency
could be improved by using a higher power UV lamp or
multiple UV lamps. It is also worth mentioning that the low
power UV lamp is well suited for nanoflow and microflow LC-
MS systems as demonstrated by data in Figs. 2 and 3.
Conclusions
In this study, a low power (6 W) LP Hg lamp was employed to
facilitate ozonolysis in solution, which allows its direct cou-
pling with static or infusion nanoESI and implementation on an
LC-ESI-MS platform. Ozone formation is initiated by 185 nm
UV emission from the lamp via the well-studied dioxygen
photolysis pathway. To effect ozonolysis on the static (20 nL/
min) and infusion (300–1000 nL/min) nanoESI sources, the
lamp just needs to be placed near the spray tip region. In those
two setups, experimental data prove that majority of ozonolysis
happens in solution at the tip region of a nanoESI emitter. The
presence of trace oxygen in spray solution and partial trans-
mission of 185 nm UV photon by the material made for those
nanoESI emitters (thin borosilicate glass and fused silica cap-
illary without polyamide coating) are responsible for the reac-
tions. Using unsaturated fatty acids and phospholipids as model
compounds, efficient ozonolysis (yield: 50%–100%) has been
achieved regardless of the geometry of the C=C, degree of
unsaturation, and polarity of ionization using above two
nanoESI sources. Given relatively low concentrations of ozone
generated by the lamp, optimal solution condition is key to
obtaining high reaction yields. It is important to include water
in the solvent system, e.g., ACN/H2O (7/3, v/v). This is be-
cause solvent water converts Criegee intermediate to aldehyde,
consolidating other possible ozonolysis products into alde-
hydes. This phenomenon not only simplifies spectral interpre-
tation for locating C=C positions but also increases detection
sensitivity. The role of low pH condition is to keep [OH−]
concentration low, so as to reduce side reactions between ozone
and OH−. Besides nanoESI, a flow microreactor made from
Teflon tubing, which is permeable to ozone formed in ambient
air, has been developed for coupling UV lamp-induced
ozonolysis directly with LC-MS. A mock mixture of unsatu-
rated fatty acids was used as a proof-of-principle demonstration
on the capability of assigning C=C location in each FA (in-
cluding isomers) via baseline separation of FAs from the mix-
ture, online ozonolysis, and subsequent ESI-MS detection.
Overall, UV lamp-induced ozonolysis has attractive analytical
features, such as easy implementation, not instrument vendor-
specific, no modifications on MS needed, and low cost. How-
ever, it also inherits the same limitations as other ozoneESI
techniques, including the need of a prior separation of lipid
Acknowledgments
Financial support from NSF CHE-1308114 and NIH R01-
GM118184 is greatly appreciated. C.A.S. acknowledges the
Purdue Department of Chemistry for the Emerson Kampen
Fellowship Award.
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