A study of the ozonolysis of model lipids by electrospray ionization mass spectrometry

Article abstract of DOI:10.1002/rcm.6183

RATIONALE The ozonolysis of vegetable oils has been proposed as step to functionalise unsaturated lipids for the production of bio-based chemicals and materials. The observation of ozonolysis products by mass spectrometry will facilitate mechanistic studies that are essential for the further development of industrial processes based on lipid ozonolysis. METHODS Ozonolysis of the model lipids methyl oleate and triolein was performed and samples were taken over a range of reaction times. Ozonlysis products were separated by normal and non-aqueous reversed-phase (NARP) liquid chromatography (LC) coupled to electrospray ionization (ESI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) using a hybrid quadrupole-time of flight instrument. Post-column addition of ammonium acetate solutions aided ionization. Volatile reaction products were observed using gas chromatography/electron impact mass spectrometry (GC/MS). RESULTS Secondary ozonides, reaction intermediates and previously unreported high molecular weight product dimers were observed as intact molecular ammonium ion adducts under positive ESI. The main fragment ions obtained by MS/MS were from cleavage of the trioxolane group, loss of fatty acyl chain and fragmentation between n-8 and n-9 on the fatty acyl chain. The MS/MS spectra and exact mass measurements explain most of the ozonolysis reaction products. CONCLUSIONS LC/MS, LC/MS/MS and GC/MS results demonstrate that the products from the ozonolysis of the model lipids methyl oleate and triolein are consistent with known ozonolysis reaction pathways. LC/MS techniques using non-aqueous chromatography have permitted the direct observation of mono-, di- and tri-1,2,4-triloxanes which are the secondary ozonides formed by the ozonolysis of triolein. It was also shown that intermediates formed during the ozonolysis of triolein can combine to form high molecular weight ozonide dimers. Copyright

Full text of DOI:10.1002/rcm.6183

Research Article
Received: 30 November 2011
Revised: 31 January 2012
Accepted: 1 February 2012
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2012, 26, 921–930
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6183
A study of the ozonolysis of model lipids by electrospray
ionization mass spectrometry
*
Chenxing Sun, Yuan-Yuan Zhao and Jonathan M. Curtis
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada
RATIONALE: The ozonolysis of vegetable oils has been proposed as step to functionalise unsaturated lipids for the produc-
tion of bio-based chemicals and materials. The observation of ozonolysis products by mass spectrometry will facilitate
mechanistic studies that are essential for the further development of industrial processes based on lipid ozonolysis.
METHODS: Ozonolysis of the model lipids methyl oleate and triolein was performed and samples were taken over a range
of reaction times. Ozonlysis products were separated by normal and non-aqueous reversed-phase (NARP) liquid chromato-
graphy (LC) coupled to electrospray ionization (ESI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS)
using a hybrid quadrupole-time of flight instrument. Post-column addition of ammonium acetate solutions aided ionization.
Volatile reaction products were observed using gas chromatography/electron impact mass spectrometry (GC/MS).
RESULTS: Secondary ozonides, reaction intermediates and previously unreported high molecular weight product
dimers were observed as intact molecular ammonium ion adducts under positive ESI. The main fragment ions obtained
by MS/MS were from cleavage of the trioxolane group, loss of fatty acyl chain and fragmentation between n-8 and n-9 on
the fatty acyl chain. The MS/MS spectra and exact mass measurements explain most of the ozonolysis reaction products.
CONCLUSIONS: LC/MS, LC/MS/MS and GC/MS results demonstrate that the products from the ozonolysis of the model
lipids methyl oleate and triolein are consistent with known ozonolysis reaction pathways. LC/MS techniques using non-
aqueous chromatography have permitted the direct observation of mono-, di- and tri-1,2,4-triloxanes which are the
secondary ozonides formed by the ozonolysis of triolein. It was also shown that intermediates formed during the ozonolysis
of triolein can combine to form high molecular weight ozonide dimers. Copyright © 2012 John Wiley & Sons, Ltd.
Recently, global interest in using renewable resources in place
of fossil fuels for the production of energy, fine chemicals and
polymers has greatly increased. Much research has focused
on transforming vegetable oils, which are abundant and
renewable in nature, into forms that are amenable to these
applications.[1] Ozonolysis is one such transformation of
vegetable oils that has shown potential in the manufacture of
bio-based polymers and chemicals. Ozone, a very reactive
oxidizing agent, can cleave the double bonds of the unsaturated
triacylglycerides (TAGs) found in vegetable oils. Ozonolysis
has been used as one of the steps to make vegetable oil based
polyols;[2,3] these are polyhydroxyl compounds that are
precursors used in polyurethane manufacture. However,
formation of bio-based polyols by this pathway includes
multiple steps, catalysts, and there are many challenges to
producing high yields of polyols.[4] It is hoped that further
elucidation of the products from the ozonolysis of vegetable
oils will aid in the development of ozonolysis processes for
the commercial production of bio-based polyols and other
chemicals.
expected products for the ozonolysis of methyl oleate. The
initial reaction occurs when an ozone molecule attacks the
double bond and the primary ozonide forms. This high-energy
primary ozonide can readily decompose into the Criegee
intermediates 1 and 2, nonanal and 9-oxononanoic acid methyl
ester. In the absence of any participating solvent, Criegee inter-
mediates can recombine with nonanal and 9-oxononanoic acid
methyl ester to form the secondary ozonide 1,2,4-trioxolanes,
which include compounds 1, 2 and 3. The Criegee intermedi-
ates are very important intermediates of ozonolysis because
they can readily undergo further reactions. In the liquid
phase, 1,3-dipolar cycloaddition results in the formation
of 1,2,4-trioxolanes. Step 3 in Scheme 1 is an example of
1, 3-dipolar cycloaddition between the Criegee intermediate
2 and nonanal.
Several methods have been used for observation of both
the intermediates and the products of the ozonolysis of free
fatty acids and vegetable oils, including Fourier transform
infrared (FTIR) spectroscopy and nuclear magnetic reso-
nance (NMR) spectroscopy. Soriano et al. used FTIR to follow
the ozonolysis of sunflower oil; they observed the peaks in
the IR spectrum that are characteristic of the formation of
The mechanism proposed by Criegee, describing the reaction
of ozone with olefins, is the widely accepted one.[5] Scheme 1 is
a simplified version showing the important intermediates and
both aldehydes and ozonides.[6] Sega et al. used 1H NMR
13
and
C NMR for analysis of sesame oil before and after
ozonolysis to identify characteristic peaks that show the
trend of ozonide formation and simultaneous decrease in
double bonds. The relationship between the NMR signal
and physicochemical measurements, including peroxide
value and viscosity, was also established.[7] Even though
* Correspondence to: J. M. Curtis, Department of Agricultural,
Food and Nutritional Science, University of Alberta,
Edmonton, Alberta T6G 2P5, Canada.
E-mail: jonathan.curtis@ales.ualberta.ca
Rapid Commun. Mass Spectrom. 2012, 26, 921–930
Copyright © 2012 John Wiley & Sons, Ltd.

C. Sun, Y.-Y. Zhao and J. M. Curtis
OCH3
OCH3
report of a comprehensive liquid chromatography/tandem
mass spectrometry (LC/MS/MS) method for the identification
of ozonolysis products from unsaturated lipids.
Methyl Oleate (296)
O
O
O3
Step 1
Step 2
In this work, methyl oleate was selected as a simple model
for unsaturated fatty acids, because it is an important compo-
nent of many vegetable oils for which ozonolysis can only
occur on one double bond. Similarly, triolein is selected as the
simple model for unsaturated triacylglycerides (TAGs), which
is an abundant TAG in vegetable oils containing high amounts
of oleic acid such as canola or sunflower oils. The overall
objective of this study is to develop methods for the study of
both the ozonolysis intermediates and products from the model
lipids, methyl oleate and triolein. This was achieved using LC
separation combined with quadrupole time-of-flight (Q-TOF)
mass spectrometer, allowing both exact mass determination
and MS/MS scans for structure identifications.
O
O
O
Primary Ozonide (344)
OCH3
O
O
O
O
Nonanal (142)
Criegee Intermediate 1 (202)
O
OCH3
O
O
O
9-oxononanoic acid methyl ester (186)
Criegee Intermediate 2 (158)
O
Step 3
OCH3
O
O
O
EXPERIMENTAL
O
OCH3
Materials
O
O
O
Methyl oleate and triolein standards were purchased from Nu
Chek Prep Inc. (Elysian, MN, USA). Hexanes, acetonitrile
(ACN), isopropanol (IPA) and methanol (MeOH) were pur-
chased from Fisher Scientific Company (Ottawa, ON, Canada)
and were of analytical grade. HPLC-grade ammonium acetate
(≥99%) was supplied by Sigma (St. Louis, MO, USA). A
solution of porcine renin substrate tetetradecapeptide at 10
pmol/mL in acetonitrile/water (1:1, v/v) in a chemical
standards kit was obtained from Applied Biosystems (Foster
City, CA, USA). ESI-L low concentration tuning mix was pur-
chased from Agilent Technologies Canada Inc. (Mississauga,
ON, Canada).
Compound 1 (344)
Chemical Formula: C19H36O5
Secondary
Ozonide
O
OCH3
H3CO
O
O
O
O
Compound 2 (388)
Chemical Formula: C20H36O7
O
O
O
Compound 3 (300)
Chemical Formula: C18H36O3
Scheme 1. Important intermediates and expected products
for the ozonolysis of methyl oleate.
NMR can reveal lots of structural information, some of the
spectral details cannot be entirely assigned due to the
complexity of spectrum, and a relatively large sample
size is required for NMR analysis. The application of size
exclusion chromatography (SEC) additionally revealed the
formation of high molecular weight ozonolysis products,
but very little structural information can be revealed.[8] Gas
chromatography (GC) has also been widely used to analyze
ozonolysis products, but its application is limited to the light
fraction of the ozonolysis products that are suitable for GC
analysis.[9] Methylation is commonly used to make the
sample amenable to GC analysis, if the starting material is
a triglyceride or vegetable oil.[10] Ozonolysis of simple
lipids has also been studied by mass spectrometry (MS), for
example, the ozonolysis of methyl oleate was analyzed
without any chromatographic separation using direct probe
introduction into chemical ionization mass spectrometry
(CI-MS). This revealed the existence of secondary ozonides
and NMR spectroscopy was used to further assign their
structures.[11] Thornberry and Abbatt designed a coated-wall
flow tube coupled to CI-MS to selectively monitor both
ozone loss and the production of volatile compounds when
ozone reacted on a surface of oleic acid, linoleic acid and
linolenic acid.[12] However, that work focused on gas-phase
products and their experimental design more closely modeled
ozone reactions on a particle surface than ozone reactions in a
bulk solution. To our knowledge there has not to date been a
Ozonolysis of methyl oleate and triolein
A mixture of 0.87 g lipid standards (methyl oleate or triolein)
and 50 mL hexane was placed in a 50 mL flask and the
temperature of the flask was kept at 10 ꢀC a using Julabo F25
circulating chiller (Julabo USA Inc., Allentown, PA, USA). An
ozone/oxygen mixture was introduced into the flask at a rate
of 50 mL/min through a long stainless steel needle with an
ozone concentration of approximately 0.4 g/m3. Ozone was
generated by passing dry oxygen as the feed gas through an
Azcozon ozone generator coupled to a controller unit (RMU
16–16 and RMDC-32D, Azco Industries Ltd., Langley, BC,
Canada). During ozonolysis, 25 mL aliquots were taken
from the reaction at 10, 15, 20, 30, 40, 50, 75, 90, 105, 120, 140,
160, 180, 210 and 240 min. The 25 mL aliquot was mixed with
75 mL hexane and immediately stored at À20 ꢀC. The samples
were diluted 100 times in hexane before any further analysis.
Liquid chromatography/mass spectrometry (LC/MS)
LC/MS analysis was conducted on an Agilent 1200 series
HPLC system (Agilent Technologies Inc, Palo Alto, CA, USA)
coupled to a QSTAR Elite mass spectrometer (Applied
Biosystems/MDS Sciex, Concord, ON, Canada) using electro-
spray ionization (ESI) in positive ion mode. Analyst QS 2.0
software was employed for data acquisition and analysis.
Volumes of 2 mL of sample were injected for LC/MS analysis.
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Rapid Commun. Mass Spectrom. 2012, 26, 921–930

Study of the ozonolysis of model lipids by ESI-MS
For the ozonolysis of methyl oleate, chromatographic
analysis was performed on an Ascentis Si silica column
(150 mm  2 mm i.d.) with a particle size of 3 mm (Sigma,
St. Louis, MO, USA). Mobile phase A was hexane and mobile
phase B was IPA. The gradient was as follows: 0–4 min, 2% B;
4–10 min, 100% B; 10–14 min, 100% B; 14–20 min, 2% B.
40 mmol/L ammonium acetate in MeOH and IPA (3:1, v/v)
was used as post-column flow at a rate of 25 mL/min. The
mass spectra were acquired over the mass range of m/z
100–1000. The ion source temperature was kept at 300 ꢀC.
The other instrumental conditions were as follows: curtain
gas 25; auxiliary gas 20; nebulizing gas 60. All the gas
numbers are in arbitrary units, and nitrogen was used for
all these gases. The ionspray voltage, declustering potential
(DP), focus potential (FP), and DP2 were 5200 V, 35 V, 150 V
and 10 V, respectively. For MS/MS analysis collision-induced
dissociation (CID) at a collision energy of 28 eV and at a
collision gas setting of 8 units was used.
Gas chromatography/mass spectrometry (GC/MS)
GC/MS analysis was only performed on the sample from the
ozonolysis of methyl oleate. A 1 mL aliquot was analyzed
using an Agilent 7890A gas chromatograph equipped with
an Agilent 5975 C mass-selective detector (MSD) (Agilent
Technologies Inc., Palo Alto, CA, USA). An Rxi-5 ms capillary
column (30 m  0.25 mm i.d, 0.25 mm film thickness; Restek
Corp., Bellefonte, PA, USA) was used. The helium flow was
kept constant at 1 mL/min. ꢀThe oven program was 40 ꢀC
ꢀ
for 3 min, then rising to 280 C at a rate of 30 C/min with
ꢀ
a final hold at 280 C for 2 min. Electron impact ionization
was used at 70 eV electron energy and a mass scan range of
m/z 35–500. An Agilent MSD Chem Station E.02.02.1431 was
used for data analysis.
RESULTS AND DISCUSSION
For ozonolysis of triolein, chromatographic analysis was
performed on an Ascentis C18 column (150 mm  2 mm i.d.)
with a particle size of 3 mm (Sigma, St. Louis, MO, USA).
Mobile phase A consisted of ACN and mobile phase B
of IPA. The gradient was as follows: 0–0.1 min, 20% B;
0.1–25 min, 90% B; 25–27 min, 90% B; 27–27.1 min, 90% B;
27.1-36 min, 20% B. The same ammonium acetate solution
was used as post -column flow at a flow rate of 20 mL/min.
The mass spectra were acquired over the mass range of m/z
100–2000. The ion source temperature was kept at 400 ꢀC.
Other mass spectrometer conditions were the same as
the conditions used for the ozonolysis of methyl oleate.
MS/MS analysis used CID at a collision energy of 32 eV
and a collision gas setting of 8 units.
All mass spectra were recorded using the high-resolution
TOF mass analyzer providing mass accuracies typically
below 5 ppm over the range of m/z reported after calibration.
The mass spectrometer was tuned by infusing porcine renin
substrate tetetradecapeptide (m/z 879.9723, doubly charged
ion) at a resolution of above 10 000 full width at half
maximum (FWHM) in positive ion mode. This solution was
also used for calibration of m/z 100–1000; the Agilent
ESI low concentration tuning mix was used for calibration
of m/z 100–2000.
Identification of the products and the intermediates from
the ozonolysis of methyl oleate
Lipids with double bonds on the fatty acyl chain can be
oxidised with ozone. Due to the complexity of products and
intermediates that could be formed by the ozonolysis of
natural lipid mixtures, methyl oleate, which has only one
double bond at the n-9 position, was instead studied as a model
lipid compound. A sample of methyl oleate after 40 min of
ozonolysis was introduced into the mass spectrometer by
flow injection in positive ESI mode using a mobile phase of
hexane and isopropanol (98:2, v/v) and with a post-column
addition of 40 mM ammonia acetate to aid ionization. The
resulting mass spectrum is shown in Fig. 1. The ions seen at
m/z 362 and 406 are ammonium ion adducts of compounds 1
and 2, respectively (see Scheme 1), while ions at m/z 367 and
411 correspond to their sodium ion adducts and ions at m/z
383 and 427 are their potassium ion adducts. Furthermore, the
elemental composition of the ion at m/z 362.2898 was deter-
mined to be [C19H36O5 + NH4]+ (Δ = À0.83 ppm), and that
of m/z 406.2797 was determined to be [C20H36O7 + NH4]+
(Δ = À0.49 ppm), consistent with the elemental compositions
of compounds 1 and 2 (Scheme 1).
Figure 1. Flow injection ESI (+) mass spectrum of the products of the ozonoly-
sis of methyl oleate for 40 min.
Rapid Commun. Mass Spectrom. 2012, 26, 921–930
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C. Sun, Y.-Y. Zhao and J. M. Curtis
In order to confirm this identification, MS/MS spectra of
the [M + NH4]+ ions of compounds 1 and 2 at m/z 362 and
406 were obtained. As can be seen in Figs. 2(a1) and 2(b),
the MS/MS spectra of ions at m/z 362 and 406 are very
similar, showing that ions at m/z 186 and 155 are the main
product ions in both cases. The fragment ion at m/z 142 is
only observed in the MS/MS spectrum of m/z 362; this
fragmentation pathway is illustrated in the inset of Fig. 2(a1).
The cleavage initially occurs at the peroxide bridge of trioxo-
lane, and there can be either ab or ac cleavage. The result of
ac cleavage is an aldehyde radical, which is the product ion at
m/z 186. The other part of this cleavage is the Criegee intermedi-
ate 2, the ion of which is not seen in the MS/MS spectrum.
However, this Criegee intermediate was observed in work of
Thomas where methanol was present in the mobile phase,
which reacts quickly with the Criegee intermediate to form a
stable ion.[13] Apparently, the ion at m/z 155 cannot directly
form by this route but rather is attributed to the cleavage of
the ion m/z 186. Similarly, ab cleavage results in a product
ion at m/z 142 of low intensity. Compound 2 is a symmetric
trioxolane (Scheme 1), so its ab and ac cleavage of ion at m/z
406 produces the same product ion at m/z 186. The pattern of
these product ions also agrees with results of Wu et al., even
though in that work the trioxolane compounds were analyzed
under chemical ionization conditions.[11] Hence, the accurate
mass measurements and MS/MS spectra of the ions at m/z
362 and 406 support the conclusion that they are ammonium
adducts of the secondary ozonides, compounds 1 and 2 in
Scheme 1.
It is interesting to note that the fragment ions arising from
the [M + NH4]+ ion of compound 1 at m/z 362 do not retain
the ammonium adduct but instead are odd-electron species
Figure 2. ESI (+) MS/MS spectra of the ions at (a1) m/z 362, (a2) m/z 367 and
(b) m/z 406 of the products from the ozonolysis of methyl oleate for 40 min.
The inset of (a1) is the fragmentation pathway of secondary ozonide compound
1 (m/z 362). The inset of (a2) is the fragmentation pathway of secondary ozonide
compound 1 (m/z 367).
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Rapid Commun. Mass Spectrom. 2012, 26, 921–930

Study of the ozonolysis of model lipids by ESI-MS
(Fig. 2(a1)). For comparison, the MS/MS spectrum of the
corresponding sodiated ion at m/z 367 [M + Na]+ of
compound 1 shows that product ions are at m/z 225, 209,
180 and 165 (Fig. 2(a2), all of which retain the sodium ion.
These product ions can be explained based on the trioxolane
structure proposed for compound 1 in Fig. 2. For example,
product ions at m/z 225 and 209 are formed by cleavage at
the trioxolane group. This generates either the aldehyde ion
at m/z 209 or the Criegee intermediate that can rearrange into
the carboxylic acid observed at m/z 225.[14] Other fragmenta-
tion due to cleavage of the hydrocarbon chain adjacent to
the trioxolane group results in ions at m/z 180 and 165
(Fig. 2(a2)).[14] Hence, in contrast to the fragmentation observed
for the [M + NH4]+ ion of compound 1, the [M + Na]+ ion of
compound 1 fragments in a manner that is closely analogous
to that previously reported for trioxolane derivatives of other
unsaturated lipids.[14]
In order to confirm the existence of the secondary ozonides,
compounds 1 and 2, as distinct species, normal-phase LC/MS
was performed. The chromatogram shown in Fig. 3(a) is the
total ion current (TIC) trace for the separation of the products
obtained following 40 min of ozonolysis of methyl oleate, using
a silica column. The ions at m/z 362 and 406 are the base
peaks of mass spectra eluting at 2.5 and 2.9 min, respectively.
Since normal-phase chromatography was used, the compound
(m/z 406) eluting at 2.9 min is likely more polar than the
compound (m/z 362) eluting at 2.5 min. This is consistent with
the structures of compounds 1 and 2 in Scheme 1, which show
that compound 2 has two ester groups compared to only one on
compound 1. Another dominant ion seen by flow injection
(Fig. 1) is the ion at m/z 186 which could either be the ammo-
nium ion adduct of an intact compound or else an in-source
fragment ion. Extracted ion chromatograms (XICs) of ions at
m/z 362, 406 and 186 are shown in Fig. 3(b). These indicate that
the ion at m/z 186 has the same retention time as the ion at m/z
362. Furthermore, the MS/MS spectrum of m/z 362 (Fig. 2(a1))
has a major fragment ion at m/z 186. Based on this discussion,
it appears to us that the ion at m/z 186 in the positive ion
ESI spectrum shown in Fig. 1 is indeed a product ion of the
secondary ozonide compound at m/z 362 rather than another
unknown product from the ozonolysis of methyl oleate.
It should be noted that the ion corresponding to intact
methyl oleate is not observed under this experimental condi-
tion, even though methyl oleate was found to still exist in the
sample based on GC/MS analysis. This is because methyl
oleate is a neutral lipid, which does not ionize well by ESI
as we have previously observed. Chemical derivatization,
such as phosphonium labelling of these neutral lipids, has
been shown to improve ionization under ESI.[15] On the other
hand, we have obtained [M + H]+ ions from methyl oleate
using positive ion atmospheric pressure photo ionization
(APPI) and atmospheric pressure chemical ionization (APCI)
from the same ozonolysis sample (data not shown). Methyl
oleate has very low polarity, hence is more easily ionized by
APPI and APCI than by ESI. However, only fragment ions
of secondary ozonides are observed using positive ion APCI
and APPI. Overall, compared to APPI and APCI, ESI with
post-column addition of ammonium ions was found to be a
better choice of ionization method for the observation of
ozonolysis products.
GC/MS results (see Supplementary Fig. 1, Supporting
Information) indicate that nonanal, nonanoic acid and 9-
oxononanoic acid methyl ester are the main volatile ozonolysis
products. Nonanal and 9-oxononanoic acid methyl ester were
seen to form at the beginning of ozonolysis whereas nonanoic
acid is only observed by GC/MS in samples taken after
30 min of ozonolysis. Nonanoic acid can be formed either as a
result of further oxidation of nonanal, which is abundant
after ozonolysis starts, or via an internal rearrangement of
the Criegee intermediate to form a more thermodynamically
stable structure.[16] A mass balance between the reactants
and volatile and involatile products formed during ozonoly-
sis could not be achieved in this study due to a constant loss
of volatiles. Nevertheless, these qualitative observations by
GC/MS are in agreement with the expected products indi-
cated in Scheme 1.
Ions of high m/z value (>500) were not observed in any of
the mass spectra obtained following ozonolysis of methyl
oleate for periods of up to 4 h. Several researchers have
shown that Criegee intermediates are able to react with
other Criegee intermediates or acids to form oligomeric or
polymeric compounds.[17–19] However, there are also reports
Figure 3. (a) Total ion current (TIC) chromatogram of products from the ozono-
lysis of methyl oleate for 40 min by LC/ESI(+)-MS. (b) Extracted ion chromato-
grams (XICs) of the ions at m/z 362, 406 and 186.
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C. Sun, Y.-Y. Zhao and J. M. Curtis
showing that no oligomers are observed either in aerosol parti-
cles or in a bulk solution of oleic acid.[20] In the present study,
both the presence of a large amount of an inert solvent (hexane),
and the absence of the free carboxylic acid group in the methyl
oleate as starting material, likely minimize the probability oli-
gomer formation through the Criegee intermediates and acids.
Ozonolysis has been used for determination of double-bond
position in unsaturated fatty acids and esters.[13,14] For
example, a low-temperature plasma (LTP) probe was recently
used for in situ ozonolysis.[21] The plasma containing ozone
was directed at the sample and the resulting ions were
sampled into a linear ion trap mass spectrometer. For a fatty
acid ester, only aldehyde oxidation products retaining the ester
chain were observed in positive ion mode. In the case of a free
fatty acid, only the aldehyde products from the oxidation of a
double bond with retention of the carboxylic acid head group
were observed in both positive and negative ion modes. The
m/z values of these products can be used to assign the dou-
ble-bond location. However, in general the ozonolysis inter-
mediates, such as ozonides and diperoxides, were not
observed. In contrast, in our study the ozonolysis products are
present at low concentration in the solvent and the observation
of ozonides besides 9-oxononanoic acid methyl ester is facile.
ozonolysis in general. These can be extended in the interpre-
tation of the ozonolyis products of a triolein standard. Inves-
tigation of TAGs is more relevant to the vast majority of lipids
in nature and triolein in particular is the most abundant TAG
found in canola (edible rapeseed) oil. The triolein standard
before and after ozonolysis for 60 min was analyzed using
non-aqueous reversed-phase (NARP) LC/MS, as shown in
Figs. 4(a) and 4(b). The positive ion NARP-LC/MS chromato-
gram of triolein (Fig. 4(a)) shows a single peak for a compound
of m/z 902, consistent with the [M + NH4]+ ion of triolein.
Following ozonolysis (Fig. 4(b)), additional peaks appeared in
the chromatogram and their molecular weight information is
summarized in Table 1. It is evident from Fig. 4(b) that three
prominent groups of peaks appear at retention times of around
12, 15 and 19 min following ozonolysis. It was also observed
that each of these groups is comprised of multiplets of isomeric
compounds, mostly resolved by NARP chromatography.
An ion of m/z 950 is the base peak in the mass spectra of
compounds eluting at 19.3 and 19.6 min. This ion indicates
the formation of mono-ozonide since the exact mass indicates
that this ion contains an additional three oxygen atoms
compared to the [M + NH4]+ ion of triolein (m/z 902). Since
each fatty acyl chain of triolein contains one double bond at
the n-9 position and ozone can attack the double bond on
either the sn-1,3 or sn-2 fatty acyl chain, there can be two
constitutional mono-ozonide isomers that differ in the loca-
tion of trioxolane group. From a statistical consideration, the
probability of forming an sn-1 or 3 isomer is twice that of
forming an sn-2 isomer. Figure 4(b) clearly shows the peak
at 19.3 min is smaller, preceding a larger peak at 19.6 min; this
Identification of the products and intermediates from the
ozonolysis of triolein
Identification of the major intermediates and products arising
from the ozonolysis of methyl oleate should elucidate at least
some of the basic principles and mechanisms important for
Figure 4. LC/ESI(+)-MS TIC chromatograms of (a) the triolein standard and (b) after
60 min of ozonolysis of triolein. Inset (a) is the mass spectrum of the triolein standard.
Inset (b) is the mass spectrum after 60 min of ozonolysis of triolein averaged at a reten-
tion time of 23.1 min. Inset (c) is the mass spectrum after 60 min of ozonlysis of triolein
averaged at a retention time of 13.6 min. The mass spectrum averaged at the retention
time of 13.9 min is the same as that at the retention time of 13.6 min.
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Study of the ozonolysis of model lipids by ESI-MS
Table 1. Retention times, mass accuracy measurements and elemental compositions of molecular ions of ozonolysis inter-
mediates and products
Retention time
(min)
Experimental
m/z (Da)
Error
(ppm)
Elemental
composition
Compounds
Triolein
Mono-ozonide
Di-ozonide
Tri-ozonide
Ozonolysis Intermediates
23.1
19.3, 19.6
15.6, 15.9, 16.2
11.9, 12.3, 12.6, 12.9
13.6, 13.9
9.9, 10.0
902.8211
950.8016
998.7850
1046.7717
792.6704
840.6541
888.6378
1776.2390
1728.2534
1680.2731
1632.2839
1584.3033
4.4
À1.6
À1.6
0.4
À1.0
À2.1
À3.3
À3.8
À4.5
À1.9
À4.7
À2.2
[C57H104O6NH4]+
[C57H104O9NH4]+
[C57H104O12NH4]+
[C57H104O15NH4]+
[C48H86O7NH4]+
[C48H86O10NH4]+
[C48H86O13NH4]+
[C96H172O27NH4]+
[C96H172O24NH4]+
[C96H172O21NH4]+
[C96H172O18NH4]+
[C96H172O15NH4]+
4.9, 6.3
Dimers
15.6, 15.9, 16.2
18.2, 18.4, 18.7
20.9
23.1
25.6
Figure 5. The MS/MS spectra of (a1) the mono-ozonide eluting at 19.6 min, (a2)
the mono-ozonide eluting at 19.3 min, (b) the di-ozonides, and (c) the tri-ozonides.
The insets are examples of the proposed structures with letters indicating fragment
positions. The structures given are examples of the possible isomeric structures
and are not meant to imply unique structural assignments.
Rapid Commun. Mass Spectrom. 2012, 26, 921–930
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C. Sun, Y.-Y. Zhao and J. M. Curtis
indicates that the peak at 19.3 min probably contains an sn-2
isomer. In order to confirm this and obtain more structural
information, tandem mass spectrometry was performed on
the ion at m/z 950 eluting at 19.6 and 19.3 min (Figs. 5(a1) and
5(a2)). It can be seen that the relative intensity of the product
ion at m/z 603 is considerably greater in the former case. The
ion at m/z 603 is the result of neutral loss of ammonia along with
loss of a fatty acyl chain that contains a trioxolane group
[(C57H104O9 + NH4) – C18H34O5 – NH3]+ (cleavage at position
a or equivalent). An ESI study of the ammonium ion adducts
of TAG positional isomers described by Byrdwell and Neff[22]
demonstrates that the loss of a fatty acyl chain from either the
sn-1 or the sn-3 position is energetically favored over the loss
from the sn-2 position. Thus, it is expected that loss of the fatty
acyl chain containing the trioxolane group would be more facile
at the sn-1 or sn-3 positions compared to that at the sn-2
position. Hence, the higher intensity of the fragment ion at
m/z 603 of the compound eluting at 19.6 min compared to that
eluting at 19.3 min suggests that the former has the trioxolane
group at the sn-1 or sn-3 position whereas the latter is substi-
tuted at sn-2. This interpretation is also consistent with the
above discussion concerning the abundance of these isomers.
Other product ions at m/z 493, 509 and 465 are also observed
(Figs. 5(a1) and 5(a2)). The loss of a fatty acyl chain and the
cleavage of the trioxolane group along with neutral loss
of ammonia lead to the formation of the ion at m/z 493
[(C57H104O9 + NH4) – C18H34O2 – C9H18O2 – NH3]+. This is
shown as cleavage at position b and c in the inset of Fig. 5(a1)
for the illustrative example of an sn-1 mono-ozonide with loss
of the sn-3 fatty acyl chain. Similarly, the fragment ion at m/z
509 [(C57H104O9 + NH4) – C18H34O2 – C9H18O - NH3] + results
from cleavage at positions equivalent to b and d. Previously, it
has been proposed that fragmentation at the trioxolane group
can cause the cleavage of the hydrocarbon chain adjacent to the
trioxolane group.[14] For example, cleavage of the bond between
the n-8 and n-9 positions, which results in the formation of a
cleavage at positions a, c and f) and 415 (ÀNH3, cleavage at posi-
tions a, d and g) (Fig. 5(b)) are also observed. These come from the
loss of one fatty acyl chain and the cleavage of both trioxolane
groups, which results in aldehyde/aldehyde, aldehyde/
carboxylic acid and carboxylic acid/carboxylic acid at the former
two n-9 positions. The ion at m/z 635 is the product ion from neu-
tral loss of ammonia along with fragmentation at positions d and
f. The MS/MS spectra of the [M + NH4]+ ions (m/z 998) of the
compounds eluting at 15.6, 15.9 and 16.2 min all contain the
same product ions, but with differences in their intensity ratios.
This probably arises due to the presence of positional and geo-
metrical isomers but at this point there is no further data avail-
able for us to draw any conclusion on which isomers are present.
The mass spectra from the peaks at 11.9, 12.3, 12.6 and
12.9 min all have the ion at m/z 1046 as their base peak. Clearly,
the observation of the [M + NH4]+ ion at m/z 1046 demonstrates
tri-ozonide formation. Since all of the double bonds have been
ozonized, tri-ozonide can only exist as cis/trans geometric
isomers, which is also the reason that there are four peaks
instead of one peak shown between 11.0 and 13.0 min. Figure 5
(c) is the MS/MS spectrum of a tri-ozonide [M + NH4]+ ion,
which has product ions at m/z 399 (ÀNH3, cleavage at posi-
tions a, c and g), 383 (ÀNH3, cleavage at positions a, c and f)
and 415 (ÀNH3, cleavage at positions a, d and g) similar to
the di-ozonide. Product ions at m/z 369 (ÀNH3, cleavage at
positions a, e and g) and 353 (ÀNH3, cleavage at positions
a, e and f) are also observed. The other isomeric tri-ozonide
MS/MS spectra differ only also in the relative intensities of
the product ions.
Besides the formation of ozonides, the peaks at 13.6 and
13.9 min in Fig. 4(b) show the presence of ozonolysis
intermediates. Thus, the ion at m/z 792 is the base peak of the
mass spectra of peaks at 13.6 and 13.9 min (Fig. 4, inset (c)).
From the elemental composition (Table 1) and knowledge
of the ozonolysis reaction pathway, this ion is likely the
decomposition product of a mono-ozonide with an alde-
hyde group at an n-9 position. Since this aldehyde can
be present on the sn-2 or sn-1 position, two isomeric peaks
are observed. Other intermediate ions such as m/z 888 and
840 are also observed, which are the decomposition pro-
ducts of di-ozonides and tri-ozonides with an aldehyde
group at n-9, respectively.
In addition to the intermediates and products listed in
Table 1, high molecular weight products were also formed.
Figure 6 shows the XIC over the range of m/z 1000 to 2000
from the reaction of triolein with ozone for 60, 105 and
120 min. At 60 min of ozonolysis, an ion at m/z 1776 appears
with low intensity in the mass spectrum compared to the base
peak at m/z 998, a di-ozonide (Fig. 6, inset (a)). However, after
120 min of ozonolysis, peaks at similar retention times have
mass spectra in which the ion at m/z 998 is absent whereas
the ion at m/z 1776 is the base peak (Fig. 6, inset (b)). Hence,
longer reaction times are resulting in the formation of higher
amounts of high molecular weight products. The structure of
the ion at m/z 1776 is proposed in inset (c) of Fig. 6, which
might be formed by the cycloaddition of the ozonolysis inter-
mediate having an aldehyde group at n-9 (m/z 888) with the
corresponding Criegee intermediate. It should be noted that
the ozonolysis intermediate with an [M + NH4]+ ion at m/z
888 was also observed (Table 1). Although the dimer
[M + NH4]+ ion at m/z 1776 has the same retention time as
the di-ozonide [M + NH4]+ ion at m/z 998, it can be seen from
product ion at m/z 465 [(C57H104O9 + NH4) – C18H34O2
–
C10H19O3 – NH3]+ (cleavage at position b and e or equivalent).
The peaks in the chromatogram at retention times of 15.6,
15.9 and 16.2 min (Fig. 4(b)) were found to have the same
[M + NH4]+ ion at m/z 998 as the base peak in each case. This
indicates the formation of at least three isomeric forms of the
di-ozonide. Since the di-ozonide has an additional oxygen-
containing functional group with three more oxygens com-
pared to the mono-ozonide, it has higher polarity and would
be expected to elute earlier in NARP chromatography. This is
consistent with the peak elution order seen in Fig. 4(b). The
di-ozonide from triolein can exist as sn-1,3 or sn-1,2 positional
isomers. In addition, each positional isomer can also have
cis/trans geometric isomers,[23] some of which may be separated
under the chromatographic conditions used here. Theoretically,
there can be three cis/trans isomers of an sn-1,3 di-ozonide plus
three cis/trans isomers of an sn-1,2 di-ozonide. However, in the
present experiment, a total of only three peaks are separated, so
assignment of the exact isomeric structure of each peak is not
yet possible.
The MS/MS spectrum of the di-ozonide [M + NH4]+ ion
shows the same product ions at m/z 493 (ÀNH3, cleavage at
positions b and c), m/z 509 (ÀNH3, cleavage at positions b
and d) and m/z 465 (ÀNH3, cleavage at positions b and e) as
was seen for the mono-ozonide. In addition, product ions at
m/z 399 (ÀNH3, cleavage at positions a, c and g), 383 (ÀNH3,
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Rapid Commun. Mass Spectrom. 2012, 26, 921–930

Study of the ozonolysis of model lipids by ESI-MS
Figure 6. Extracted ion chromatograms of m/z 1000–2000 from LC/ESI(+)-MS runs of
samples taken after the ozonolysis of triolein for 60, 105 and 120 min. The insets (a) and
(b) are mass spectra averaged over retention times of 15.0–17.0 min for 60 and 120 min
ozonolysis period samples, respectively. The inset (c) is the proposed structure of the
dimer ion at m/z 1776.
Fig. 6 that the abundance of the former ion is a function of
ozonolysis time and is therefore unambiguously a reaction
product and not formed in the ion source.
acids; secondary ozonides including trioxolanes; and high mole-
cular weight product dimers formed by reaction between ozono-
lysis intermediates.
The mass spectrum of the triolein standard (Fig. 4, inset (a))
and the mass spectrum of the peak at the same retention time
(RT 23.1 min) (Fig. 4, inset (b)) are very similar, except that the
ion at m/z 1632 is only observed after ozonolysis. Also, another
three distinct groups of peaks are present at 18.4 (m/z 1728), 20.9
(m/z 1680) and 25.6 min (m/z 1584) when the reaction time was
60 min (Fig. 6). These four groups of peaks differ in molecular
weight by 48 Th, i.e. three oxygen atoms (Table 1). This is because
these high molecular weight products are formed by reactions
between two ozonided triolein molecules. This occurs through
the linkage between an aldehyde group from one ozonided
triolein molecule and a Criegee intermediate group from another
ozonided triolein molecule. The ions at m/z 1728, 1680, 1632 and
1584 (RT 18.4, 20.9, 23.1 and 25.6 min) have one, two, three, and
four less trioxolane group(s), respectively, compared to the ion
at m/z 1776. The elution order of these oxygen-containing
dimer products is consistent with their polarity so that the
compounds containing more oxygen atoms are more polar and
have shorter retention times. Also, by comparing the XIC of the
range m/z 1000–2000 for the triolein ozonolysis products after
60, 105 and 120 min, it is clear that all of these dimers eventually
convert into the compound of ion at m/z 1776, which is fully
ozonided with no remaining double bonds. It is the first time that
these dimers formed during ozonolysis of triolein have been
separated and observed by NARPLC/(ESI)-MS.
CONCLUSIONS
LC/MS/MS has been used to observe ozonolysis intermediates
and products of the model lipids methyl oleate and triolein.
Secondary ozonide 1,2,4-trioxolanes from both methyl oleate
and triolein can be observed as [M + NH4]+ ions in positive
ESI mode when using post-column addition of ammonia
acetate. Dimeric ozonolysis products from triolein were
detected and separated by LC/MS. Accurate mass measure-
ment and MS/MS provide information on the structure of
secondary ozonides. LC/MS/MS gives a clear picture of the
ozonolysis pathway for model lipids, providing the basis for
understanding the ozonolysis of more complicated lipid
mixtures such as vegetable oils. Future work will focus on
applying this LC/MS/MS method to monitor the ozonolysis
of plant oils, which will be useful in the synthesis of bio-based
chemicals and polymers.
SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article.
Based on the above study of the ozonolysis of methyl oelate
and triolein, we can see that a variety of products are formed
by ozonolysis of unsaturated lipids. These fall into three general
categories: small volatile compounds such as aldehydes and
Acknowledgement
This work was supported by NSERC Discovery Grant No.
197273 awarded to J. M. Curtis.
Rapid Commun. Mass Spectrom. 2012, 26, 921–930
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

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Rapid Commun. Mass Spectrom. 2012, 26, 921–930