Triacylglycerol estolides, a new class of mammalian lipids, in the paracloacal gland of
the brushtail possum (Trichosurus vulpecula)
Electronic Supplementary Material
GC-MS Analyses of Lipids and Fatty Acids
Stuart McLean, Noel W. Davies, David Nichols, Bernie J. Mcleod
Introduction
This electronic supplementary material gives a full account of the GC-MS analyses of
possum proximal paracloacal gland secretions. Some of the findings are referred to in the
main paper.
Methods
Extraction of Gland Contents
The contents of proximal paracloacal glands were extracted into dichloromethane as
described in the main paper.
Saponification and Derivatization
Some samples of gland secretions were saponified by mixing about 100 mg with 400 µl 2 M
KOH in 50% ethanol and heating at 80C for 1 h then standing at room temperature
overnight. After acidification (to pH 5-6) with 200 µl 2 M HCl (which produced a white
precipitate) the solution was extracted twice with ethyl acetate (1.0 + 0.5 ml), which was
separated, evaporated under reduced pressure and the residue dissolved in 1 ml
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dichloromethane. An aliquot (100 µ) was placed in an autosampler insert tube then, just
before GC analysis, trimethylsilyl (TMS) derivatives were formed by adding 50 µl of BSTFA
reagent (bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane, Sigma-Aldrich,
USA) and the tube capped and heated at 65C for 30 min.
In a search for hydroxy fatty acids (hFA), samples were transesterified to form fatty acid
methyl esters (FAME) and treated with TMS reagent to derivatize alcohol groups. To each of
five samples (18-51 mg) of gland contents, 3 ml methanol/chloroform/hydrochloric acid
(10:1:1, by vol) were added, together with 100 µl of a C19 free fatty acid surrogate standard
solution (500 µg/ml). Sample tubes were sealed and heated at 80C for 1 h. Tubes were
allowed to cool, and 1 ml water added followed by 1.5 ml of hexane/chloroform (4:1, by vol).
Samples were mixed and the upper organic layer transferred to a fresh GC vial and
evaporated under a stream of nitrogen. This extraction was repeated. The combined extract
was blown to dryness and 50 µl of BSTFA with 50 µl of chloroform added to vials. Vials
were sealed and heated at 60C overnight. Samples were analysed on a Brüker 300MS GCMS/MS system in full scan mode (m/z 40 to 500) utilising the GC conditions detailed in
Nichols and McMeekin [1] except that the column was an Agilent DB-5MS 30m x 0.25 mm,
0.25 µm phase thickness.
GC-MS Analyses
Lipid extracts were analysed by GC-MS as previously described [2], except that the mass
spectrometer was a Brüker 300-MS Triple Quadrupole instrument using electron ionisation
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(EI). A mix of alkane standards (C7 – C40 in hexane; Supelco, USA) was added to some
samples before GC-MS analysis for determination of Kovats Indices (KI). The glycerides
were investigated by selected ion chromatograms using the masses calculated for the
diacylglycerol (DAG) product ions formed after the loss of an alkanoate group ([M −
RCOO]+), as well as acyl-related ions (RCO+, [RCO − H2O]+, [RCO + 74]+ [3].
Results and Discussion
GC-MS Analyses: TAG
GC-MS analysis of total lipid extracts of proximal gland contents produced a large number of
TAG molecular species, many unresolved, which eluted towards the end of the temperature
program (Fig. 1a, upper panel). Saponification showed that the TAG lipids were formed from
at least 69 short- and long-chain fatty acids, from C6-C24, branched- and straight-chain,
some unsaturated, but dominated by iso- and anteiso-isomers of heptanoic and nonanoic acid,
which have been described previously [2, 4].
For each lipid peak in Fig. 1a, the parent TAG was reconstructed from the mass spectrum
using the diagnostic EI fragment ions. The most abundant high-mass ion in the GC-MS-EI
mass spectrum of TAG corresponds to the loss of one fatty acid carboxyl radical from the
molecular ion to form a diglyceride-type “lyso” ion ([M − RCOO]+) [3]. Chromatograms
were produced for the expected masses of lyso ions formed from the abundant low molecular
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weight acids (Fig. 1a lower panels). These lyso ions have two acyl groups attached to the
remaining glycerol backbone, giving m/z 299 for 7:0/7:0 (14 total acyl carbons) through to
m/z 369 for lyso 9:0/10:0 (C19:0).
A common pattern was for isobaric TAG to elute as a set of three peaks, although each could
include more than one molecular species. In the selected ion chromatogram of m/z 299, the
mass spectrum of the first set of three peaks only showed ions corresponding to C7:0 acyl
groups (principally m/z 95 [RCO − H2O]+ and 113 [RCO]+), indicating that all were isomers
of TAG 7:0/7:0/7:0. Even to consider only the two more abundant C7 acids (iso- and anteiso), there are 8 possible TAG isomers so it is probable that the peaks represented mixed TAG of
these C7 acids. The shortest chain acids found were iso- and n-hexanoic acid, and a plot of
m/z 271 (corresponding to lyso 6:0/6:0) showed small peaks with evidence of C6 and C7 acyl
ions.
In some cases the TAG was a mixture of three different acids, for example in the
chromatogram of m/z 341 that shows many peaks for lyso 17:0. The mass spectrum of the
first peak (Fig. 1b) showed lyso ions corresponding to total fatty acyl carbons 15:0 (7:0/8:0),
16:0 (7:0/9:0 or 8:0/8:0) and 17:0 (8:0/9:0), indicating that the parent TAG contained the
saturated acids C7-9. Confirmation came from the acyl-related ions that were found for the
same three acids (listed in Fig 1b). As there are 8 positional isomers for the parent TAG
7:0/8:0/9:0, and different branched acid isomers, it is likely that several forms were coeluting
in this one peak. The group of peaks following 1A in the m/z 341 plot (Fig. 1a) showed the
same ions, and the different retention times indicate that the TAG comprised different mixes
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of the isomeric forms of the acids: iso-, anteiso- and n-, as well as probable regioisomerism of
the fatty acids within TAG molecular species.
In many cases there were more than three acyl groups present in an unresolved mixture of
TAG, especially with higher mass species (eg the chromatogram for m/z 369 in Fig. 1a). At
higher masses, the mass spectra became increasingly more complex and it was not possible to
confidently identify TAG with unsaturated acyl groups, although these were expected from
the fatty acids found after saponification. The last clearly identified TAG was 9:0/9:0/9:0 (KI
found were 3109, 3121 and 3132), which eluted at 32.6 – 32.9 min and gave the upper mass
limit (m/z 512) for this GC-MS analysis.
GC-MS Analyses: Glyceryl Ethers
In addition to fatty acids, GC-MS analysis of the saponified samples showed at least 35 1-Oalkylglycerol ethers as their TMS derivatives. As described previously [2], these compounds
were found by extracting the single ion chromatogram for m/z 205, formed by the loss of
CH2OR to give [CH(OTMS)- CH2(OTMS)]+, with confirmatory fragmentation ions at m/z
147, 113, 117 and 73. The molecular weights of the parent alkylglycerol ether TMS
derivatives were found from the ions for [M-15]+ and [M-147]+. The alkylglycerol ethers
comprised a series for alkyl groups from C11:0 – C22:0, and included n- and branched
isomers and possibly some which were unsaturated. KI values of n-alkylglycerols gave a
linear regression with Cn (slope 95% CI 95.0-95.9, N = 12), as found previously [2]. The
major components in samples from both sexes were the C13:0 – 18:0 alkyl glycerols, with
C14:0 and 16:0 compounds being the most abundant.
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Although it was expected that the unsaponified samples would contain parent diacylglycerol
ethers (DAGE), none was detected by GC-MS analysis. Based on the relative abundances of
C7:0 acids and C14:0 alkyl glycerol ethers, the smallest expected DAGE of significance was
expected to be 1-O-tetradecyl-2,3-diheptanoylglycerol (DAGE 28:0, MW 512). As this is
isobaric with TAG 27:0, one of the last-eluting TAG, it is likely than the putative DAGE
would be too large for GC analysis under the conditions used. Additionally, the GC-MS
analysis was dominated by the TAG and their fragmentation ions, making it difficult to detect
any distinctively ether-related ions.
GC-MS Analyses: FA and hFA
The LC-MS data indicated that hydroxy fatty acids were present, although they had not been
found amongst the TMS derivatized fatty acids and alcohols. A search was therefore made
for hydroxy fatty acids, using FAME plus TMS derivatization. There was good evidence for a
C18:1 hydroxy TMS fatty acid methyl ester (KI 2290): ions for M+ (m/z 384, 1.9%), [M –
15]+ (m/z 369, 19%), [M – 47]+ (m/z 337, 0.5%) and other ions related to (CH2-OTMS) at m/z
117 (8.5%) and 159 (38%), and (CH2-COOCH3) at m/z 43 (57%), 59 (35%), 73 (100%) and
87 (67%). Similarly, evidence for the corresponding C18:2 hydroxy fatty acid (KI 2276) was
found with ions for M+ at m/z 382 (0.2%), [M – 15]+ at m/z 367 (4.0%), and the other ions
m/z 159 (7.0%), 117 (3.2%), 89 (12%), 87 (15%), 73 (26%), 59 (26%) and 43 (26%). These
peaks were small and coeluted with other fatty acids and alcohols. Although there was
evidence of other hydroxy fatty acids, this was more ambiguous and is not reported here.
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The literature shows that the most abundant ions for hydroxy fatty acids (FAME + TMS
derivatives) are formed from cleavage of the carbon chain at the OTMS substituent[5-7], but
as these ions are not known in advance they cannot be used to find unknown compounds. The
other ions are losses from the molecular ion, or fragments formed from the CH2OTMS or
CH2COOCH3 moieties, which are not specific and are often of low abundance. Another
complication in finding hydroxy fatty acids was the complexity of the mixture and coelution
of many related compounds with the same functionality (fatty acids and alcohols). Others
have reported on similar difficulties in the analysis of hydroxy fatty acids in complex
mixtures [8, 9].
Legend to Figure
Fig. 1a GC-MS total ion and selected ion chromatograms showing molecular species of TAG
found in an extract of the paracloacal secretion of a female possum. The top panel shows the
full scan and the six lower panels show selected ion chromatograms characteristic of DAG
fragment ions 14:0 (m/z 299) to 19:0 (m/z 369) resulting from the loss of an acyl residue from
parent TAGs.
Fig. 1b The mass spectrum of the peak marked by an arrow in Fig. 1a, a probable mixture of
24:0 TAGs, that was identified as containing DAG 17:0 (m/z 341), 16:0 (m/z 327) and 15:0
(m/z 313) fragmentations for losses of 7:0, 8:0 and 9:0 acyl groups.
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Fig. 1 Attached as pdf
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