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Running Head: ‘Bicyclic Naphthenic Acids’
6
Bicyclic naphthenic acids in oil sands process
water: identification by comprehensive
multidimensional gas chromatography-mass
spectrometry
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8
Michael J. Wildea, Charles E. Westa,b, Alan G. Scarletta, David Jonesa, Richard
A. Frankc, L. Mark Hewittc and Steven J. Rowland*a
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a
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b
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c
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Fax: +44(0)1752 584710
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E-mail: srowland@plym.ac.uk
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Petroleum and Environmental Geochemistry Group, Biogeochemistry Research Centre, University of
Plymouth, Drake Circus, Plymouth, PL4 8AA, UK.
Present address: EXPEC Advanced Research Center, Saudi Aramco, Dhahran 31311, Saudi Arabia
Water Science and Technology Directorate, Environment Canada, 867 Lakeshore Road, Burlington, ON,
Canada L7R 4A6
*Corresponding Author:
Phone: +44 (0)1752 584557
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Although bicyclic acids have been reported to be the major naphthenic acids in oil sands process-
29
affected water (OSPW) and a well-accepted screening assay indicated that some bicyclics were the
30
most acutely toxic acids tested, none have yet been identified.
31
Here we show by comprehensive multidimensional gas chromatography-mass spectrometry (GCxGC-
32
MS), that >100 C8-15 bicyclic acids are typically present in OSPW. Synthesis or purchase allowed us
33
to establish the GCxGC retention times of methyl esters of numerous of these and the mass spectra
34
and published spectra of some additional types, allowed us to identify bicyclo[2.2.1]heptane,
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bicyclo[3.2.1]octane, bicyclo[4.3.0]nonane, bicyclo[3.3.1]nonane and bicyclo[4.4.0]decane acids in
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OSPW and a bicyclo[2.2.2]octane acid in a commercial acid mixture. The retention positions of
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authentic bicyclo[3.3.0]octane and bicyclo[4.2.0]octane carboxylic acid methyl esters and published
38
retention indices, showed these were also possibilities, as were bicyclo[3.1.1]heptane acids.
39
Bicyclo[5.3.0]decane and cyclopentylcyclopentane carboxylic acids were ruled out in the samples
40
analysed, on the basis that the corresponding alkanes eluted well after bicyclo[4.4.0]decane (latest
41
eluting acids).
42
Bicyclo[4.2.1]nonane,
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spiro[4.5]decane carboxylic acids could not be ruled out or in, as no authentic compounds or literature
44
data were available.
45
Mass spectra of the methyl esters of the higher bicyclic C12-15 acids suggested that many were simply
46
analogues of the acids identified above, with longer alkanoate chains and/or alkyl substituents. Our
47
hypothesis is that these acids represent the biotransformation products of the initially somewhat more
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bio-resistant bicyclanes of petroleum. Although remediation studies suggest that many bicyclic acids
49
can be relatively quickly removed from suitably treated OSPW, examination by GC×GC-MS may
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show which isomers are affected most. Knowledge of the structures will allow the toxicity of any
51
residual isomers to be calculated and measured.
bicyclo[3.2.2]nonane,
bicyclo[3.3.2]decane,
bicyclo[4.2.2]decane
and
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Key words: Naphthenic acids, Bicyclics, GCxGC-MS
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Highlights:
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
Bicyclic acids are known to be major components of oil sands process water
Organic acid extracts were examined from several different sources
In each sample more than 100 bicyclic acids were detected and separated by GC×GC-MS
Numerous bicyclic acids were identified for the first time
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1. INTRODUCTION
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‘Naphthenic acids’ occurring naturally in the oil sands of Alberta, Canada are concentrated
61
by processing, resulting in oil sands process-affected water (OSPW) which, after much re-
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use, is stored in large tailings ponds or lagoons, awaiting final reclamation [1]. Undiluted
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OSPW has been shown to be somewhat toxic in numerous biological assays, but with time in
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storage the composition and toxicity changes, the latter usually reducing [2]. Nonetheless,
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residual toxicity remains and this has promoted numerous studies of treatment methods with
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oxidants or ozone, or by photocatalysis or bioremediation [3].
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Numerous studies have shown that the major acids in different OSPW samples comprise, as a
68
group, unknown alicyclic bicyclic compounds [2, 4-7] and a well-accepted screening assay
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indicated that some synthetic alicyclic bicyclics were the most acutely toxic acids tested [8].
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However, almost nothing is known about the identities, or even the numbers, of bicyclic acids
71
present in OSPW.
72
Cyr and Strausz [9] isolated a C16 bicyclic acid from oil sands deposits in Alberta which had
73
a mass spectrum similar to that of drimane or labdane bicyclanes, but these have not yet been
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reported in OSPW acids (cf [10-12]). Bowman et al. [13] recently identified bicyclic
75
monoaromatic, indane and tetralin acids in a pore water sample from a composite tailings
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deposit, which combines fluid fine tailings from oil sands processing with gypsum to form a
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non-segregating deposit, but no alicyclic bicyclics were identified.
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Elucidation of acid structures also has geochemical significance, providing an insight into the
79
microbial degradation mechanisms of petroleum [14]. Some alicyclic bicyclic acids in crude
80
oils and commercial naphthenic acids preparations derived from refining petroleum, have
81
been identified [14-16], but several studies have noted the differences between the latter and
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OSPW acids, so perhaps nothing directly can be inferred from a comparison [17].
83
Furthermore, the few bicyclic acids identified in commercial naphthenic acids to date
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represent only a small fraction of those actually present, as the >100 compounds revealed by
85
comprehensive multidimensional gas chromatography-mass spectrometry of the methyl esters
86
(GCxGC-MS) of two commercial naphthenic acids mixtures attests [18].
87
Fortunately the bicyclic acids in OSPW seem to be quite prone to removal by ozone
88
treatment and bacterial action [3]. Nonetheless, it is important to establish the identities of
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these acids so that the toxicity of relevant isomers can be measured, the mechanisms of
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remediation treatments better understood and the products of remediation treatment
91
predicted.
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In the present study we examined several methylated OSPW acidic extracts and a commercial
93
acid mixture, by GCxGC-MS and identified several of the bicyclic acids present. Some
94
bicyclics previously assumed to be representative of OSPW constituents, were not common.
95
2. MATERIALS AND METHODS
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The naming of bicyclic compounds varies considerably throughout the literature. As an
97
attempt to keep the naming of the compounds discussed consistent, the IUPAC nomenclature
98
rules for polycyclic compounds based on the Von Baeyer system [19] have been used, with
99
numbering of the carbons within the bicyclic core starting at a bridgehead carbon (Figure 1A
100
and B). Alternative names for compounds commonly used by chemical suppliers and search
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engines (e.g. decalin or octahydro-pentalene) are given alongside the systematic names.
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Authentic bicyclo[2.2.1]heptane-2-ethanoic acid
(Figure 1A;
103
trimethylbicyclo[3.1.1]heptane-3-carboxylic
((+)-3-pinanecarboxylic
acid
Structure Ib), 2,6,6acid)
(IIa),
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bicyclo[2.2.2]octane-2-carboxylic acid (IVa), 4-pentylbicyclo[2.2.2]octane-1-carboxylic acid
105
(IVc),
106
carboxylic
107
bicyclo[3.3.1]nonane-1-carboxylic acid (VIIIa) were purchased from Sigma (Poole, UK).
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Authentic bicyclo[2.2.1]heptane-1-carboxylic acid (Ia), bicyclo[2.2.2]octane-1-carboxylic
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acid (IVb) and 5-methylbicyclo[3.3.1]nonane-1-carboxylic acid (VIIIc) were purchased from
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Molport (Riga, Latvia). Bicyclo[3.2.1]octane-6-carboxylic acid (Va) was synthesised from 2-
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hydroxybicyclo[3.2.1]octane-6-carboxylic acid (Sigma) by base catalysed dehydration
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followed by hydrogenation [20].
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synthesised essentially by the methods of Sasaki et al. [21] as modified by Peters et al. [22].
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Thus, reaction of adamantan-2-one in methanesulfonic acid in the presence of sodium azide
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produced the mesylate which was not isolated but heated with potassium hydroxide to give
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the unsaturated bicyclo[3.3.1]non-2-ene-7-carboxylic acid, obtained after extraction into
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acidified chloroform [21]. The corresponding saturated bicyclo[3.3.1]nonane-3-carboxylic
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acid (VIIIb) was obtained by hydrogenation [22] and the methyl esters by heating with
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BF3/methanol. Bicyclo[4.3.0]nonane-3-carboxylic (Xa) and 2-methylbicyclo[4.3.0]nonane-3-
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carboxylic acids (Xb) were obtained by catalytic hydrogenation (cf [15]) of the corresponding
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indane acids (Sigma). Bicyclo[4.4.0]decane-2-carboxylic (XIVa), 3-carboxylic acid (XIVb),
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2-ethanoic (XIVc), 3-ethanoic (XIVd), and 2-propanoic acids (XIVe; numbers refer to position
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of alkanoate substituents on bicyclic core) were synthesised as described previously [15]. 7-
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methylbicyclo[4.2.0]octane-7-carboxylic acid (VIIa) was prepared by hydrogenating 1-
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methyl-1,2-dihydrocyclobutabenzene-1-carboxylic acid methyl ester over a Raney Nickel
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catalyst at 100 °C and 100 bar using a H-Cube® (ThalesNano Nanotechnology Inc,
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Budapest).
bicyclo[3.3.0]octane-2-carboxylic
acid
acid
(VIa),
4-methylbicyclo[3.3.0]octane-2-
(3-methyl-octahydro-pentalene-1-carboxylic
acid)
(VIb)
and
Bicyclo[3.3.1]nonane-3-carboxylic acid (VIIIb) was
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Four different OSPW samples and a commercial naphthenic acids mixture were analysed
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(Table 1). The OSPW included two samples (#1 and #2) from industry A described in two
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previous studies ([23, 24]). Briefly, sodium salt concentrates of #1 and #2 were acidified to
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pH < 2 and the acids extracted with ethyl acetate before derivatisation with BF3/methanol
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[24]. Another OSPW (#3) was provided from industry B (Table 1) at a site with a high
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concentration of particulate matter. This water sample was filtered, acidified and then eluted
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through a 200 mg ENV+ SPE cartridge with acetonitrile before being dried under N2 and
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derivatised with BF3/methanol. A fourth OSPW acid extract (#4) from industry A was
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obtained by extracting a sample of raw OSPW, collected from a different tailings pond using
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the methods described previously [24]. The latter sample had undergone no pre-
139
treatment/clean-up prior to extraction and derivatisation.
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In addition to the above samples, a commercial naphthenic acids mixture (#5) was obtained
141
from Merichem Co. for comparison (Table 1) and
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previously used [24-26]. Derivatisation of the acids with BF3/methanol was followed by
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silver ion solid phase extraction (Ag+ SPE). Analysis herein focused on fraction 3 obtained
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by elution through the argentation solid phase extraction column with hexane, since this
145
contained the bicyclic acids (methyl esters).
fractionated
based on a method
146
147
Accurate mass measurements were made using a Thermofisher LTQ Orbitrap XL high
148
resolution mass spectrometer with electrospray ionisation. The mass range was m/z 120–
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2000; mass accuracy <3 ppm RMS with external calibration. For negative ionization the
150
instrument was externally calibrated using the above, sodium dodecyl sulfate and sodium
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taurocholate. For loop-injections a Thermo Scientific Surveyor MicroLC was used to provide
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solvent flow at 20μL/min., through a 2μL sample loop. Solvents used were H2O:MeOH (1:1).
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For nano-electrospray an Advion Triversa NanoMate was used to deliver samples diluted
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into MeOH ± 10% NH4OAc at a flow of approximately 0.25μL min-1. API source settings:
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Infusion NanoMate source temperature 275oC or 200oC, sheath gas flow 3 to 7 (arb. units) 2
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(arb. units), aux gas flow was not used capillary (ionising) voltage positive ionisation: + 3.2
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to 3.7kV negative ionisation: - 3.5 to – 4.0kV. Mass spectra were acquired at a minimum
158
resolution of 30,000 (at m/z 400). Theoretical masses and mass accuracies were calculated
159
using an online calculator tool [27].
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Comprehensive multidimensional gas chromatography–mass spectrometry (GCxGC-MS)
161
analyses were conducted as described previously [23, 28], using an Agilent 7890A gas
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chromatograph (Agilent Technologies, Wilmington, DE) fitted with a Zoex ZX2 GCxGC
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cryogenic modulator (Houston, TX, USA) interfaced with an Almsco BenchTOFdx™ time-
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of-flight mass spectrometer (Almsco International, Llantrisant, Wales, UK). The first-
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dimension column was a 100% dimethyl polysiloxane 60 m x 0.25 mm x 0.25 µm Rxi®-1ms
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(Restek, Bellefonte, USA), and the second-dimension column was a 50% phenyl
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polysilphenylene siloxane 2.5 m x 0.1 mm x 0.1 µm BPX50 (SGE, Melbourne, Australia).
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Helium was used as carrier gas and the flow was kept constant at 1.0 mL min-1. Samples
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(1µL) were injected at 300°C splitless. The oven was programmed from 40°C (hold for 1
170
min), then heated to 130°C at 10°C min-1 then at 2°C min-1 to 320°C (held for 15 min). The
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modulation period was 6s. The MS transfer line temperature was 290°C and ion source
172
300°C. Data processing was conducted using GC Image™ v2.1 (Zoex, Houston, TX, USA).
9
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3. RESULTS & DISCUSSION
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When examined by ESI Orbitrap high-resolution mass spectrometry with negative ion
175
electrospray ionisation, the bicyclic acids of the OSPW extracts produced ions which we
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attributed to the [M-H]- ions, mainly of C12-16 acids (m/z 195-251; ESI Orbitrap), although
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some samples had small amounts of lower carbon number species. Although OSPW is known
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to be a heterogeneous substrate, this is consistent with the data presented in numerous studies
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of different OSPW samples [2, 4-7]. The accurate masses of some of the more abundant acids
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in one of the OSPW samples (#1), for example were: 209.1544 (C13H21O2 requires 209.1547,
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mass accuracy 1.4 ppm; C13H21S requires 209.1369 and C11H13SO2 requires 209.0642),
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223.1699 (C14H23O2 requires 223.1704, mass accuracy 2.2 ppm; C14H23S requires 223.1526
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and C12H15SO2 requires 223.0798) and 237.1855 (C15H25O2 requires 237.1860, mass
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accuracy 2.1 ppm; C15H25S requires 237.1682 and C13H17SO2 requires 237.0955), indicating
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that the major ionised bicyclic species were acids fitting the formula CnH2n-4O2 and not, for
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example, nominally isobaric keto bicyclics or tricyclic hydroxy acids or sulphur compounds
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(at least in this OSPW sample).
188
When examined by GCxGC-MS as the methyl esters, selected ion mass chromatography of
189
the molecular ions produced by electron ionisation confirmed the presence of C 8-15 bicyclic
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acids in samples #3 and #4, with at least C11-15 bicyclics in all the samples (#1-#5; e.g. Figure
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2). Moreover, GCxGC-MS revealed the true complexity of the OSPW mixtures. The exact
192
ranges varied between samples. For example, one OSPW acid extract (#3) contained at least
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nineteen C9, twenty seven C10, forty C11 and numerous C11+ peaks within the chromatogram
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(Figure 2: B), whereas the extract from another OSPW tailings pond (#4) appeared even more
195
complex (Figure 2: C), possibly due to the lack of pre-treatment or clean-up, which may have
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196
removed some lower molecular weight compounds in the more treated samples (e.g. #1 and
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#2, Figure 2: A and Figure S1).
198
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Recently Damasceno et al. [18] analysed two commercial acid mixtures by GCxGC-MS,
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characterising groups of naphthenic acids by their ‘z’ value (i.e. hydrogen deficiency
201
attributed to the number of rings). They detected 124 and 132 individual bicyclic acids (z = -
202
4) in two samples (Sigma Aldrich and Miracema-Nuodex naphthenic acids) with carbon
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numbers ranging from C9-16 [18]. Similar numbers were detected in a sample of Merichem
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commercial acids herein (#5, Figure 2; D), so similar numbers of bicyclics appear to be
205
present in OSPW and commercial naphthenic acids. Such large numbers must represent many
206
different structural types of bicyclic acids, not just those routinely cited as examples [29-31].
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We attempted to calculate the likely maximum possible number present for the simplest (C8-
208
11)
209
be associated with the carboxylate/alkanoate chain. The latter is reasonable based on the
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identifications of ethanoate side chains of the co-occurring tricyclic and pentacyclic acids
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[20, 23, 32] and what is known of the biodegradation processes from which the acids
212
originate [33-35].
213
Thus, for a C11 acid, for example, at most ten carbons are left for formation of the bicyclic
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‘core’ of the acid. If any alkyl substituents were present, the number of carbon atoms in the
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‘core’ would be less than ten and more alkylation would be present. Since alkyl groups
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identified or tentatively established in OSPW acids to date have not exceeded those
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comprising four carbon atoms in total (e.g. a combination of ethyl and methyl groups), it is
acids. We assumed that at least one (necessarily), and sometimes two, carbon atoms would
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218
reasonable to assume that the smallest number of atoms in the bicyclic ‘core’ would probably
219
be six. We calculated that three structural types exist for acids with a C6 core. These have
220
cyclopropyl- or cyclobutyl rings; the former are present in carane- and thujane-type
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compounds and the latter present in bicyclo[3.1.1]heptanes (pinanes), bicyclo[4.2.0]octanes
222
and bicyclo[2.2.0]hexanes, which are known in the ladderane acids [36]. For the acids with a
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C7 core and the requisite substituents, there are four structural types (examples given in
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Figure 1B; I-III), for the C8 core acids, six (e.g. Figure 1B; IV-VII), for the acids with a C9
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core, seven (e.g. Figure 1B; VIII-XI) and for the acids with a C10 core, nine possible structures
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(where the rings are fused at two carbon atoms, e.g. Figure 1B; XII-XVI). Spiro- and non-
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fused structures were also considered, such as spiro[4.5]decane carboxylic acid (Figure 1B;
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XVII), fused at one carbon atom and the non-fused cyclopentylcyclopentane carboxylic acid
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(Figure 1B; XVIII). Thus, our calculations suggest that even the simplest acids in the OSPW
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sample might comprise over 30 structural types and for each of these, many stereoisomers
231
exist. Examples of some of these bicyclic structural types are given in Figure 1B; most ring
232
types have been identified within natural products. Thus, in theory, it is easy to account for
233
the >100 bicyclic acids we observed in the OSPW and commercial acids. The remaining
234
analytical challenge is to identify what at least some of these actually are.
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235
Examination by GCxGC-MS, of methyl esters of authentic purchased acids or those
236
synthesised herein, allowed us to establish GC retention regions of nine structural types
237
(Figure S1).
238
It was clear from the GC1 retention positions of pseudo-homologues within a given structural
239
type (e.g. the bicyclo[4.4.0]decane (decalin) carboxylic, ethanoic and propanoic acids; Figure
240
S1) that increasing molecular weight within a structural class increased the GC1 retention
241
times in an approximately linear fashion, as expected. Also, as expected, similar homologues
242
(e.g. C10) from different structural types, were generally quite well separated both in the GC1
243
and GC2 dimensions. For example, the methyl ester of 4-methylbicyclo[3.3.0]octane-2-
244
carboxylic acid (Figure S1; C10 acid VIb) was well separated (GC1) from those of the
245
bicyclo[3.3.1]nonane-1- and 3-carboxylic acids (Figure S1; C10 acids VIIIa and b). Within a
246
group of related stereoisomers (e.g. cis/trans, or positional isomers) of a particular acid (e.g.
247
isomers of bicyclo[4.4.0]decane acids) the relative retention positions produced a so-called
248
grouping or ‘tiling’ effect, with both GC1 and GC2 retention positions differing (by up to
249
about 50 retention index units in GC1) between isomers (Figure S1). The combined effects
250
produced chromatograms in which the profiles of the complex distributions of individual
251
OSPW bicyclic acids of carbon numbers C8-15 were apparent (Figures 2 and S1). In general,
252
the GC2 retention position seems to give a good separation of the different structural classes
253
of acids (Figure S1) but is also clear that differences in positional substitution (e.g. of the
254
bicyclo[3.3.1]nonane-3- and 1-carboxylic acids and decalin-1- and 2-carboxylic acids) can
255
produce a difference of about 0.25 seconds in the GC2 dimension. The quaternary-substituted
256
acid (VIIIa) eluted earlier in the GC2 dimension, as did the 3-substituted decalin acids
257
(Figure S1).
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258
We next undertook a systematic examination of the retention positions and mass spectra of
259
the >100 individual GCxGC peaks and compared these with those of reference compounds.
260
Since interpretation of the data for the lower homologues was likely to be simplest and might
261
give clues to the identities of the presumably more alkylated higher homologues, we began
262
with the C8-10 acids.
263
264
3.1 C8 bicyclic acids
265
C8 acids were present in the OSPW acid extracts #3 and #4. Figure 3 shows the GC×GC
266
retention positions of a series of peaks within #3 and #4, with the expected retention positions
267
and molecular ions (m/z 154) of C8 bicyclic acid methyl esters indicated. One peak which
268
was present within both OSPW acid extracts, #3 and #4 (Figure 3; A and B, peak 1a) was
269
identified as bicyclo[2.2.1]heptane-2-carboxylic acid methyl ester after comparison with a
270
NIST library spectrum (exo-bicyclo[2.2.1]heptane-2-carboxylic acid when compared with the
271
mass spectrum reported by Curcuruto et al. [37]) (Figure 3; C and D). Interpretation of the
272
mass spectrum of a second peak (Figure 3; A and B, peak 1b) resulted in the identification of
273
bicyclo[2.2.1]heptane-1-carboxylic acid methyl ester, confirmed by matching the GC×GC
274
retention time and mass spectrum with that of an authentic standard (Figure 3; E and F). The
275
other peaks within this series (Figure 3; A and B, peaks 1c and d) were believed to be isomers
276
possessing the same bicyclo[2.2.1]heptane core; either endo/exo isomers, or isomers with the
277
methyl carboxylate group substituted elsewhere on the ring (mass spectra detailed in
278
supplementary information Figure S2). Examination of the retention behaviour of purchased
279
bicyclo[2.2.1]heptane ethanoic acid (Figure 1A; Ib) compared with an OSPW acid extract
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280
which did not contain C8 acids (e.g. #1 and #2), also indicated that more alkylated
281
bicyclo[2.2.1]heptanes were present (Figure S1) and literature data were also available for
282
the retention indices of some C8-11 isomers of the latter on apolar and polar phases [38].
283
These also suggested that numerous bicycloheptane acids were possibilities for the unknowns
284
(Figure S1).
285
286
Compounds with the bicyclo[2.2.1]heptane skeletons ( Figure 1B; I, e.g. norbornane and
287
bornane), are well-known in nature and are most often encountered as derivatives of
288
camphor. Thus, there is precedence for the biosynthesis of compounds with this skeleton and
289
numerous analogues have been studied. Seifert and Teeter [39] suggested that naphthenic
290
acids from a Californian petroleum might include such structural types. GC retention indices
291
on apolar and polar phases and mass spectra or partial spectra of the methyl esters of isomers
292
of C8-11 acids have been published [37, 38, 40] and we obtained the mass spectrum of the
293
methyl ester of the C9 bicyclo[2.2.1]heptane ethanoic acid (Figure 1A; Ib, Figure S3).
294
Common spectral features seem to be small molecular ions (<10% abundant) and abundant
295
(often base peak) ions at m/z 95 (Figure S2; C and D). The abundance of the molecular ion
296
can vary dramatically, however, in different stereoisomers of the same acid type (vide infra).
297
The retention position of 2,6,6-trimethylbicyclo[3.1.1]heptane-3-carboxylic acid methyl ester
298
meant C8, as well as higher bicyclo[3.1.1]heptane acids (Figure S1), were also a possibility
299
for the identities of some of the unknowns, but no exact match was found in the spectra of the
300
OSPW acids (methyl esters). The most common compounds found with
301
bicyclo[3.1.1]heptane (Figure 1B; II) skeletons are pinenes; trimethyl- monterpenes produced
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Accepted by J. Chromatogr. A.
302
by plants, particularly abundant in resin from pine trees (i.e. turpentine oil). The mass
303
spectrum of 2,6,6-trimethylbicyclo[3.1.1]heptane-3-carboxylic acid methyl ester (3-
304
pinanecarboxylic acid methyl ester) is complex (Figure S4), perhaps as a result of the bridged
305
structure containing a cyclobutane ring within the core. The cyclobutane ring makes the
306
bicyclic acid liable to ring-opening and subsequent rearrangement. This is supported by an
307
extremely low molecular ion abundance (<2%) at m/z 196 (Figure S4). Distinguishable
308
features of the mass spectrum included a strong (95%) M-60 ion (m/z 136) corresponding to
309
loss of the methyl carboxylate moiety with a hydrogen transfer and a base peak at m/z 81, as
310
well as an intense ion at m/z 83 consistent with cyclic C6H9+ and C6H11+ ions respectively
311
(Figure S4).
312
313
3.2 C9 bicyclic acids
314
Next, we examined the GC retention positions of commercially available C9 (and C14)
315
bicyclo[2.2.2]octane acids (Figure 1A; IVa and c). When compared with the retention
316
positions of unknown bicyclic acids within OSPW acid extracts #1 and #2 (Figures S1and 2)
317
both reference acids had relatively long GC2 retention times but despite not being identical
318
to those of some unknowns, bicyclo[2.2.2]octane acids were considered as possible identities.
319
Furthermore, bicyclo[2.2.2]octane-1-carboxylic acid methyl ester (Figure 1A; IVb) was
320
identified within the Merichem acid extract (#5); an unknown peak had a matching retention
321
position and mass spectrum to that of the authentic reference compound (Figure 4; B and C,
322
peak 2a). Mass spectral interpretation of other peaks within the series indicated other isomers
323
were also present.
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Accepted by J. Chromatogr. A.
324
325
Compounds with the bicyclo[2.2.2]octane (Figure 1B; IV) skeleton are found as stable, cage-
326
like skeletons in natural products such as eremolactone [41, 42] isolated from Eremophila
327
fraseri and (–)-seychellene [43] found in patchouli oil, extracted from Pogostemon cablin. In
328
addition to bicyclo[2.2.2]octane -1-carboxylic acid (Figure 1A; IVb) we were able to purchase
329
bicyclo[2.2.2]octane-2-carboxylic acid (Figure 1A; IVa) and 4-pentylbicyclo[2.2.2]octane-1-
330
carboxylic acid (Figure 1A; IVc) and obtain the spectrum of the methyl esters (Figure S5; A
331
and B). Whilst the mass spectrum of bicyclo[2.2.2]octane-2-carboxylic acid methyl ester
332
(Figure 1A; IVa) was characterised by a small molecular ion (m/z 168) and base peak ion
333
(m/z 136) due to loss of methanol from the latter (Figure S5; A), the mass spectrum of
334
bicyclo[2.2.2]octane-1-carboxylic acid methyl ester (Figure 4; C) contained pronounced
335
molecular and M-29 ions, similar to those of some of the unknowns, as did the mass spectrum
336
of the 4-methyl-1-carboxylic acid isomer (free acid, NIST library) perhaps due to the loss of
337
–C2H5. The mass spectrum of the C14 4-pentylbicyclo[2.2.2]octane-1-carboxylic acid methyl
338
ester (Figure S5; B) also showed a fairly abundant molecular ion (m/z 238) and the loss of M-
339
29 and M-28 (m/z 209 and 210). Denisov et al. [44] reported the mass spectra of a range of
340
substituted bicyclo[2.2.2]octanes with many showing loss of an ethyl group (C2H5, 29
341
Daltons) from the molecular ion. They proposed a mechanism for the loss of ethyl from a
342
monocyclic intermediate brought about by the rupture of a bond at a bridgehead carbon
343
coupled with a hydrogen transfer [44].
344
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Accepted by J. Chromatogr. A.
345
When examined by GC×GC-MS, bicyclo[3.2.1]octane-6-carboxylic acid methyl ester was
346
identified within #3 and #4. The retention position and mass spectrum of the authentic
347
reference compound (Figure 1A; Va) matched those of an unknown peak present in both
348
samples (Figure 5; C-E, peak 3a).
349
350
Compounds with the bicyclo[3.2.1]octane-type skeleton are common in several natural
351
products [45]. However the hydrocarbon, bicyclo[3.2.1]octane and alkyl substituted
352
homologues have also long been known in petroleum [46, 47]. The mass spectrum of
353
bicyclo[3.2.1]octane-6-carboxylic acid (Figure 5; E) contained a small molecular ion (m/z
354
168), ion attributed to methanol loss (m/z 136) and a base peak ion typical of methyl esters
355
(m/z 87).
356
Similarly, cis-bicyclo[3.3.0]octane was identified in petroleum over 50 years ago [47].
357
Previously we identified 4-methylbicyclo[3.3.0]octane-2-carboxylic acid in a commercial
358
sample of naphthenic acids [15] by comparison of the mass spectrum with that of a
359
purchased reference sample. Since then we were able to purchase the C9 parent acid,
360
bicyclo[3.3.0]octane-2-carboxylic acid (two isomers) and comparison of the mass spectra and
361
GC×GC retention times has now led to the identification of the corresponding methyl esters
362
within a fraction of Merichem acid extract (#5, Figure S6; B-E). Another unknown compound
363
within the Merichem acid extract, also in the OSPW acid extract from industry B (#3) and a
364
different tailings from industry A (#4), had a very similar mass spectrum to that of the minor
365
bicyclo[3.3.0]octane-2-carboxylic acid methyl ester isomer (Figure S7, peak 7c). However,
366
the retention time of the unknown was different from the ester of the authentic acid and thus
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Accepted by J. Chromatogr. A.
367
the unknown was postulated to be a different isomer. The mass spectrum contained an ion at
368
m/z 150, attributed to the loss of water (M+-18; Figure S7). The loss of water is often
369
observed in the mass spectra of non-derivatised acids, keto- or hydroxy acids, but uncommon
370
in spectra of methyl esters. However, loss of water (M+-18) is observed in the mass spectra of
371
some bicyclo[4.4.0]decane acid methyl esters and again appears to be specific to certain
372
isomers [15]. The mass spectrum of the unknown displayed an ion at m/z 74 (Figure S7), also
373
a characteristic ion of methyl esters suggesting, it was not a non-methylated C10 acid. The
374
molecular ion did not show multiple isotopic peaks suggesting the compound did not contain
375
sulphur and the lack of tailing in the chromatogram often observed for non-derivatised or
376
more polar compounds indicated it was not a keto- or hydroxy acid and was most likely a
377
different isomer of bicyclo[3.3.0]octane-2-carboxylic acid.
378
379
3.3 C10 bicyclic acids
380
The retention positions of the synthetic bicyclo[3.3.1]nonane-1- and 3-carboxylic acid methyl
381
esters (Figure 1A; VIIIa and b) showed that these acids were absent from some OSPW
382
samples (Figure S1). However two unknown acids in #5 (Figure 6; peaks 4a and c) had
383
matching retention positions and mass spectra with those of bicyclo[3.3.1]nonane-3-
384
carboxylic acid methyl ester and bicyclo[3.3.1]nonane-1-carboxylic acid methyl ester (Figure
385
6; B, C, F and G).One unknown (Figure 6; peak 4b) had the same retention position and a
386
mass spectrum containing similar ions but with different intensities to that of a C11
387
homologue, 5-methylbicyclo[3.3.1]nonane-1-carboxylic acid methyl ester (Figure 6; D and
388
E). The GC×GC retention position and mass spectrum of authentic 5-
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Accepted by J. Chromatogr. A.
389
methylbicyclo[3.3.1]nonane-1-carboxylic acid methyl ester was similar to that of an unknown
390
present within the OSPW acid extract from industry B, #3 (Figure S8). Previously it has been
391
speculated that biodegradation of adamantanes might produce ring-opened acids with the
392
bicyclo[3.3.1]nonane skeleton, since this occurs in the biodegradation of adamantan-2-one
393
[23, 48]. Indeed this seems to be possible, at least for some of the present samples (viz: #3
394
and #5). The data suggest the acids in the OSPW extracts (and some commercial acids)
395
sometimes included bicyclo[3.3.1]nonane carboxylic acids.
396
397
Bicyclo[4.3.0]nonane carboxylic acids (e.g. Figure 1B; X) were also identified previously in
398
a commercial naphthenic acids mixture by comparison of the mass spectra with literature
399
mass spectra of the methyl esters of synthetic 2-carboxylic acid isomers [15]. In the present
400
study, we were able to synthesise the corresponding 3-carboxylic acids (Figure 1A; Xa,
401
Figure S9) and a 2-methyl-3-carboxylic acid isomer (Figure 1A; Xb, Figure S10). The GC2
402
retention times of the bicyclo[4.3.0]nonane acid standards were generally greater than those
403
of most of the unknowns (Figure S1). However, a few of the unknowns within an OSPW acid
404
extract (#3) possessed mass spectra very similar to those previously identified as C9
405
bicyclo[4.3.0]nonane carboxylic acids (methyl esters) in a commercial acid mixture [15, 37]
406
(Figure 7; C and D) as well as the synthesised standards (Figure 7; A, B and E, F and Figure
407
S9). It is even more likely that members of the C12-15 acids include this structural type, since
408
there are several more late-eluting peaks in these classes (Figures 2 and S1). These data
409
suggest the acids in the OSPW extracts sometimes include bicyclo[4.3.0]nonane carboxylic
410
acids.
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Accepted by J. Chromatogr. A.
411
The spectra of the bicyclo[4.3.0]nonane acids within the commercial acid mixture were
412
characterised by medium abundance molecular ions (ca 20%) and in the C10 parent acid,
413
bicyclo[4.3.0]nonane-2-carboxylic acid methyl ester, by ions due to loss of methanol (m/z
414
150) and m/z 87 [15, 49] . The mass spectra of the isomers of the synthesised
415
bicyclo[4.3.0]nonane-3-carboxylic acid methyl esters varied considerably (Figure S9). Thus
416
in the major isomer (69% of total resolved peaks) the molecular ion was abundant (80%;
417
Figure S9), whereas in more minor isomers the molecular ion was only <5% abundant
418
(Figure S9). The mass spectra were easily distinguished from those of the 2- isomers and we
419
can now assign the isomers present in commercial acids studied herein (#5) and previously
420
[15], to both 3-isomers and almost certainly 2-isomers, given the mass spectra (Figure 7).
421
422
The GC×GC retention position of 4-methylbicyclo[3.3.0]octane-2-carboxylic acid (Figure S1
423
and Figure 1A; VIb) was close to those of some of the unknowns within the OSPW acid
424
extracts (#1; Figure S1). However, there was no exact retention time or mass spectral match.
425
The mass spectrum of the C10 4-methylbicyclo[3.3.0]octane-2- carboxylic acid methyl ester
426
(Figure S11) was characterised by a quite strong (30%) molecular ion and characteristic
427
fragment ions, particularly at m/z 140 (70%) assumed to be due to loss of a propene moiety,
428
likely via a cycloreversion/retro-Diels-Alder rearrangement, typical of cyclic hydrocarbons
429
[50, 51]. The GC2 retention position of the C10 authentic acid methyl ester (Figure S1) and
430
the tentative identification of the C9 parent acid (Figure S7) suggested that some of the C11+
431
acids present in the OSPW might have this skeleton.
432
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Accepted by J. Chromatogr. A.
433
7-methylbicyclo[4.2.0]octane-7-carboxylic acid methyl ester eluted very closely to unknown
434
bicyclic acids in an OSPW acid extract (#1, Figure S1) but none matched exactly the
435
particular isomers present. Bicyclo[4.2.0]octane carboxylic acids contain a fused cyclobutane
436
ring (Figure 1B; VII), similar in structure to short-chain ladderane fatty acids previously
437
identified as degradation products of ladderane lipids [36]. Ladderane lipids are specific for
438
bacteria capable of anaerobic ammonium oxidation (anammox) and therefore the acids can be
439
used as biomarkers for anammox bacteria [52]. The mass spectra of both 7-
440
methylbicyclo[4.2.0]octane-7-carboxylic acid methyl ester isomers (Figure S12) displayed
441
weak molecular ions (m/z 182) as expected for alicyclic acids containing a highly strained,
442
fused cyclobutane ring. The base peak at m/z 101 was attributed to the fragmentation across
443
the cyclobutane ring.
444
445
3.4 C11+ bicyclic acids
446
Bicyclic naphthenic acids, believed to be products of biodegradation, have frequently been
447
assumed to possess bicyclo[4.4.0]decane structures (e.g. [29-31]) and this has been supported
448
by the occurrence of such acids identified within at least one commercial acid mixture [15].
449
The retention positions of the synthetic bicyclo[4.4.0]decane (decalin) carboxylic, ethanoic
450
and propanoic acid methyl esters substituted in either the 2- or 3- positions on the decalin
451
core showed that these acids were absent or had a very low abundance in some of the samples
452
of OSPW acids which we examined (#1 and #2), as demonstrated by the elution of these
453
acids late in the GC2 retention window (#1, Figure S1). A small number of
454
bicyclo[4.4.0]decane acids were tentatively identified within another OSPW (#3), based on
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Accepted by J. Chromatogr. A.
455
mass spectral comparison with those previously reported in commercial acid mixtures [15],
456
such as an isomer of bicyclo[4.4.0]decane-3-carboxylic acid methyl ester, as well as
457
bicyclo[4.4.0]decane-1-carboxylic acid methyl ester which was compared with a NIST
458
library mass spectrum (Figure 8).
459
Petroleum hydrocarbons and related compounds possessing bicyclo[4.4.0]decane cores such
460
as drimanes, cadinanes and eudesmanes have been well studied [12, 53, 54]. Fused
461
cyclohexyl rings are common in biologically derived compounds e.g. hopanes. Therefore,
462
bicyclic sesquiterpenes can be reasonably postulated to be biodegradation products of higher
463
terpenes [53].
464
465
Although we could obtain no samples of bicyclo[3.2.2]nonane (Figure 1A; IX),
466
bicyclo[4.2.1]nonane (XI), bicyclo[4.2.2]decane (XII), bicyclo[5.3.0]decane (XIII),
467
bicyclo[5.2.1]decane (XV), bicyclo[3.3.2]decane (XVI), spiro[4.5]decane (XVII) or
468
cyclopentylcyclopentane (XVIII) carboxylic acids, when we examined the reported NIST GC
469
retention indices of the hydrocarbons bicyclo[5.3.0]decane and cyclopentylcyclopentane, it
470
was clear that these eluted well after decalin (bicyclo[4.4.0]decane). Since the acid methyl
471
esters would be expected to have the same relative retention orders and bicyclo[4.4.0]decane
472
acids, when present, were the latest eluting acids; we can fairly confidently rule out these
473
acids in these samples of OSPW. Since we could find no sources of bicyclo[4.2.1]nonane,
474
bicyclo[3.2.2]nonane, bicyclo[4.2.2]decane, bicyclo[3.3.2]decane or spiro[4.5]decane
475
carboxylic acids to allow us to study the mass spectra or GC retention behaviour, and the
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Accepted by J. Chromatogr. A.
476
retention indices of the alkanes appear not to have been published, we cannot rule these out
477
as possibilities.
478
479
Examination of the mass spectral features observed for the authentic reference compounds
480
(Figure 1A) were used to postulate structural features of the unknown acids. For example,
481
methyl esters of acids in which the methylated carboxylic group is substituted onto the ring,
482
creating a tertiary carbon atom (e. g. in the mass spectra [15] of the esters of
483
bicyclo[4.4.0]decane-2- or 3- carboxylic acid (Figure 1A; XIVa and b, Figure 8) or
484
bicyclo[4.3.0]nonane-2 [15] or 3- carboxylic acids (Figure 1A; Xa, Figures 7 and S9),
485
commonly lose a neutral methanol molecule, or methoxy radical (M-31/32), (though the
486
spectra of stereoisomers vary; Figure S9).
487
In contrast, methyl esters of acids in which the methylated carboxylic group is substituted
488
onto the ring via a longer alkanoate chain, (e. g. in the mass spectra [15] of the esters of
489
bicyclo[4.4.0]decane-2- or 3-ethanoic or propanoic acids (Figure 1A; XIVc-e) or
490
bicyclo[2.2.1]heptane-2-ethanoic acid (Figure 1A; Ia, Figure S3)), commonly lose a
491
·CH2CO2CH3 radical (mass 73 and mass 74with occurrence of hydrogen transfer).
492
Methyl esters of acids in which the methylated carboxylic group is substituted onto the
493
bridgehead carbon, creating a quaternary carbon atom (e. g. in the NIST mass spectrum of the
494
esters of bicyclo[4.4.0]decane-4a-carboxylic acid or mass spectrum of bicyclo[3.3.1]nonane-
495
1-carboxylic acid (Figure 1A; VIIIa, Figure 6; C)), commonly lose a methylated carboxy
496
radical ·CO2CH3 radical (mass 59 and sometimes mass 60 with the occurrence of hydrogen
497
transfer).
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Accepted by J. Chromatogr. A.
498
Abundant lower mass fragment ions such as m/z 55, 67, 79 and 81 present in many of the
499
reference compound mass spectra are common ions observed in the mass spectra of
500
cycloalkanes/polycycloalkanes, particularly those containing substituted cyclohexyl and
501
cyclopentyl rings [55]. Therefore these ions were postulated to originate from fragmentations
502
within the bicyclic core via more complex mechanisms and rearrangements i.e.
503
corresponding to C4H7+, C5H7+, C6H7+ and C6H9+ fragment ions respectively. Fragmentation
504
within a bicyclic core requires fission of at least two bonds. Mass spectral studies of
505
cycloalkanes, specifically bicyclic hydrocarbons, suggest that electron ionisation results in
506
the fission of one of the bonds at a tertiary carbon bridgehead, followed by subsequent
507
rearrangement and fragmentation [44, 56, 57].
508
The mass spectra of the C11 unknowns in the OSPW samples exhibited some of the above
509
features. In general they were also characterised by abundant molecular ions (m/z 196) and
510
ions at m/z 81, 95 and 107 were often predominant (Figure S13). In some spectra, ions
511
which may indicate losses of ethyl (M-29) and other alkyl (e.g. M-57, butyl) substituents,
512
were present. To contain alkyl substituent groups of this size (e.g. C4), a C11 acid would
513
require a bicyclic core to contain only six carbons (e.g. C4-bicyclo[2.2.0]hexane carboxylic
514
acids). Spectra of the methyl esters of such acids are distinctive and do not match those
515
observed here [36]. Thus, we conclude that the apparent C3/C4 losses from the unknown
516
represent losses from the rings, as observed in the spectrum of the methyl ester of authentic
517
4-methylbicyclo[3.3.0]octane-2- carboxylic acid, which shows an ion due to loss of propene
518
(Figure 1A; VIb, Figure S11).
519
Although a number of structural features can be observed from the mass spectra of the methyl
520
esters of the C12-16 acids, including molecular ions, ions due to losses of methanol (M-32) and
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Accepted by J. Chromatogr. A.
521
to losses of alkyl groups or alkene moieties (e.g. M-15, M-28 and 29) from the molecular ion
522
and ions due to losses of ethanoate (M-73) and propanoate (M-87) side chains, no more
523
rigorous assignments of the structural types could be made than for the C11 acids. Thus we
524
assume these are mostly higher homologues of the bicyclo[2.2.1]heptane, bicyclo[2.2.2],
525
[3.2.1], [3.3.0]octane, bicyclo[3.3.1] and [4.3.0]nonane and some bicyclo[4.4.0]decane
526
skeleta, with possibly bicyclo[4.2.1]nonane, bicyclo[3.2.2]nonane, bicyclo[3.3.2]decane or
527
spiro[4.5]decane carboxylic acids represented also. We include the mass spectra of a few
528
unknown bicyclic acid methyl esters (Figures S13) in order that they may in future be
529
compared with those of synthesised acids as these become available.
530
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Accepted by J. Chromatogr. A.
531
4. CONCLUSIONS
532
Consideration of the GC retention behaviour, numbers of structural types and interpretation
533
of the electron ionisation mass spectra of the methyl esters of a number of synthetic and
534
purchased bicyclic carboxylic acids allowed identification of various bicyclic acids in OSPW
535
and commercial acids.
536
More than one hundred C8-15 bicyclic acids were typically present in OSPW. Synthesis or
537
purchase
538
bicyclo[4.3.0]octane and bicyclo[3.3.1]octane acids in OSPW and a bicyclo[2.2.2]octane acid
539
in a commercial acid mixture. The retention positions of authentic bicyclo[3.3.0]octane and
540
bicyclo[4.2.0]octane carboxylic acid methyl esters and published retention indices, showed
541
these were also possibilities, as were bicyclo[3.1.1]heptane acids. In most OSPW acid
542
extracts analysed the bicyclo[4.4.0]decane carboxylic (decalin) acids which have always been
543
assumed to be present in OSPW, were relatively minor components. Bicyclo[5.3.0]decane
544
and cyclopentylcyclopentane carboxylic acids were ruled out on the basis that the
545
corresponding alkanes eluted well after bicyclo[4.4.0]decane (latest eluting acids).
546
Bicyclo[4.2.1]nonane, bicyclo[3.2.2]nonane, bicyclo[3.3.2]decane, bicyclo[4.2.2]decane and
547
spiro[4.5]decane carboxylic acids could not be ruled out or in, as no authentic compounds or
548
literature data were available. Mass spectra of the methyl esters of the higher bicyclic C12-15
549
acids suggested that many were simply analogues of the above, with longer alkanoate chains
550
and/or alkyl substituents. Our hypothesis is that these acids represent the biotransformation
551
products of the initially somewhat more bio-resistant bicyclanes of petroleum. Remediation
552
studies suggest at least some bicyclic acids can be relatively quickly removed from suitably
553
treated OSPW [3], but a closer examination of which isomers are degraded will now be
allowed
us
to
identify
bicyclo[2.2.1]heptane,
bicyclo[3.2.1]octane,
27 Disclaimer: This is a pre-publication version. Readers are recommended to
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Accepted by J. Chromatogr. A.
554
possible using the methods demonstrated here. This may be deemed important as some
555
bicyclic acids are more acutely toxic than others [8]. Clearly many bicyclic acids remain to be
556
identified. Since a wider literature of mass spectra of bicyclic hydrocarbons (e.g. [44, 57-61])
557
is available than is extant for the acids, a useful approach may be to convert the acids to the
558
hydrocarbons (cf [62, 63]). Combining this older approach with the modern chromatography
559
methods (viz: GC×GC-MS) used here, may prove particularly valuable.
560
561
Acknowledgments
562
We are grateful for a Plymouth University PhD scholarship to DJ. Funding of this research
563
was provided by an Advanced Investigators Grant (no. 228149) awarded to SJR for project
564
OUTREACH, by the European Research Council, to whom we are also extremely grateful.
565
We thank Dr C. Anthony Lewis for his contributions, particularly in delimiting the number of
566
possible isomers as well as all his advice and input. We would like to acknowledge the
567
EPSRC National Mass Spectrometry Service Centre at Swansea University, UK for obtaining
568
the accurate mass data. We thank Professor O. Baudoin, Université Claude Bernard Lyon,
569
France, for providing a sample of 1-methyl-1,2-dihydrocyclobutabenzene-1-carboxylic acid
570
methyl ester.
571
572
573
574
575
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the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
576
Tables
577
Table 1: Details of samples studied with assigned sample numbers
578
Figures
579
580
Figure 1: (A) Structures of synthesised or purchased authentic bicyclic acids and (B) examples
of generalised structures and names of possible C11 bicyclic acids
581
582
583
584
585
Figure 2: Extracted ion chromatograms for molecular ions of C8-15 bicyclic acid methyl esters
(m/z 154, 168, 182, 196, 210, 224, 238 and 252) in (A) an OSPW acid extract from
industry A collected 2009, #2, (B) an OSPW acid extract from industry B, #3, (C) an
OSPW acid extract from industry A collected from a different tailings pond in 2013, #4
and (D) a fraction of Merichem acid extract, #5
586
587
588
589
590
591
592
593
Figure 3: Extracted ion chromatograms (m/z 154, 125, 95 and 87) of (A and B) two OSPW acid
extracts from industries A and B (#3 and #4), analysed by GC×GC-MS and mass
spectra of (C and E) unknown peaks 1a and 1b, identified by comparison with (D) a
NIST library spectrum of bicyclo[2.2.1]heptane-2-carboxylic acid methyl ester and (F)
a purchased reference standard of bicyclo[2.2.1]heptane-1-carboxylic acid methyl ester.
Unknown peaks labelled 1c and 1d were speculated to be bicyclo[2.2.1]heptane
carboxylic acid methyl ester isomers based on mass spectral interpretation (mass
spectra are given in supplementary information, Figure S2).
594
595
596
597
Figure 4: Extracted ion chromatogram (m/z 168) of (A) a fraction of Merichem acid extract (#5)
analysed by GC×GC-MS and mass spectrum of (B) unknown peak 2a, identified by
comparison with (C) that of authentic bicyclo[2.2.2]octane-1-carboxylic acid methyl
ester with the same GC×GC retention position
598
599
600
601
602
Figure 5: Extracted ion mass chromatograms (m/z 168 and 87) of (A and B) OSPW acid extracts
from Industries A and B (#3 and #4) analysed by GC×GC-MS and mass spectra (C and
D) of the same unknown (3a) present within both samples identified by comparison
with (E) the mass spectrum and retention position of authentic bicyclo[3.2.1]octane-6carboxylic acid methyl ester
603
604
605
606
607
608
609
Figure 6: Extracted ion chromatogram (m/z 196, 182, 151, 137 and 123) of (A) a fraction of
Merichem acid extract (#5) analysed by GC×GC-MS and mass spectra of (B, D and F)
two unknown peaks (4a and c) identified and one C11 unknown peaks (4b) tentatively
identified by comparison with the mass spectra and retention positions of (C) purchased
bicyclo[3.3.1]nonane-1-carboxylic acid methyl ester, (E) 5methylbicyclo[3.3.1]nonane-1-carboxylic acid methyl ester and (G)
bicyclo[3.3.1]nonane-3-carboxylic acid methyl ester
29 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
610
611
612
613
614
615
Figure 7: Electron ionisation mass spectra of (A,C and E) unknown peaks (5a-c) within an
OSPW acid extract (#3), tentatively identified by comparison with the mass spectra of
(B and F) two isomers of synthesised bicyclo[4.3.0]nonane-3-carboxylic acid methyl
ester and (D) an unknown within a fraction of Merichem acid extract (#5) previously
identified as an isomer of bicyclo[4.3.0]nonane carboxylic acid methyl ester [15] (most
likely 2- isomer, similar to mass spectrum reported by Curcuruto et al. [37])
616
617
618
619
620
621
622
623
Figure 8: Electron ionisation mass spectra of (A, C, D and E) unknown peaks within an OSPW
acid extract (#3), tentatively identified by comparison with the mass spectra of
previously identified bicyclo[4.4.0]decane acid methyl esters [15] e.g. (B)
bicyclo[4.4.0]decane-3-carboxylic acid methyl ester and (F) NIST library spectrum of
bicyclo[4.4.0]decane-1-carboxylic acid methyl ester
30 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
624
625
626
627
628
629
630
References
[1]
J.V. Headley, K.M. Peru, A. Janfada, B. Fahlman, C. Gu, S. Hassan, Characterization of
oil sands acids in plant tissue using Orbitrap ultra-high resolution mass spectrometry with
electrospray ionization, Rapid Communications in Mass Spectrometry 25 (2011) 459462.
631
632
633
[2]
D.M. Grewer, R.F. Young, R.M. Whittal, P.M. Fedorak, Naphthenic acids and other
acid-extractables in water samples from Alberta: What is being measured?, Science of
The Total Environment 408 (2010) 5997-6010.
634
635
636
[3]
J.W. Martin, T. Barri, X. Han, P.M. Fedorak, M.G. El-Din, L. Perez, A.C. Scott, J.T.
Jiang, Ozonation of Oil Sands Process-Affected Water Accelerates Microbial
Bioremediation, Environmental Science & Technology 44 (2010) 8350 - 8356.
637
638
639
[4]
S. Mishra, V. Meda, A.K. Dalai, J.V. Headley, K.M. Peru, D.W. McMartin, Microwave
treatment of naphthenic acids in water, Journal of Environmental Science and Health,
Part A 45 (2010) 1240-1247.
640
641
642
[5]
J.V. Headley, K.M. Peru, D.W. McMartin, M. Winkler, Determination of dissolved
naphthenic acids in natural waters by using negative-ion electrospray mass spectrometry,
Journal of AOAC International 85 (2002) 182-187.
643
644
645
[6]
M.P. Barrow, J.V. Headley, K.M. Peru, P.J. Derrick, Fourier transform ion cyclotron
resonance mass spectrometry of principal components in oilsands naphthenic acids,
Journal of Chromatography A 1058 (2004) 51-59.
646
647
648
649
[7]
J.W. Martin, X. Han, K.M. Peru, J.V. Headley, Comparison of high- and low-resolution
electrospray ionization mass spectrometry for the analysis of naphthenic acid mixtures in
oil sands process water, Rapid Communications in Mass Spectrometry 22 (2008) 1919 1924.
650
651
[8]
D. Jones, A.G. Scarlett, C.E. West, S.J. Rowland, Toxicity of Individual Naphthenic
Acids to Vibrio fischeri, Environmental Science & Technology 45 (2011) 9776-9782.
652
653
[9]
T.D. Cyr, O.P. Strausz, Bound carboxylic acids in the Alberta oil sands, Organic
Geochemistry 7 (1984) 127-140.
654
655
656
[10]
A. Dimmler, T.D. Cyr, O.P. Strausz, Identification of bicyclic terpenoid hydrocarbons in
the saturate fraction of Athabasca oil sand bitumen, Organic Geochemistry 7 (1984) 231238.
657
658
[11]
R.P. Philp, D.T. Gilbert, J. Friedrich, Bicyclic sesquiterpenoids and diterpenoids in
Australian crude oils, Geochimica et Cosmochimica Acta 45 (1981) 1173-1180.
659
660
[12]
R. Alexander, R.I. Kagi, R. Noble, J.K. Volkman, Identification of some bicyclic alkanes
in petroleum, Organic Geochemistry 6 (1984) 63-72.
31 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
661
662
663
664
[13]
D.T. Bowman, G.F. Slater, L.A. Warren, B.E. McCarry, Identification of individual
thiophene-, indane-, tetralin-, cyclohexane-, and adamantane-type carboxylic acids in
composite tailings pore water from Alberta oil sands, Rapid Communications in Mass
Spectrometry 28 (2014) 2075-2083.
665
666
[14]
C.M. Aitken, D.M. Jones, S.R. Larter, Anaerobic hydrocarbon biodegradation in deep
subsurface oil reservoirs, Nature 431 (2004) 291-294.
667
668
669
670
[15]
S.J. Rowland, C.E. West, A.G. Scarlett, D. Jones, Identification of individual acids in a
commercial sample of naphthenic acids from petroleum by two-dimensional
comprehensive gas chromatography/mass spectrometry, Rapid Communications in Mass
Spectrometry 25 (2011) 1741-1751.
671
672
673
674
675
[16]
C.E. West, J. Pureveen, A.G. Scarlett, S.K. Lengger, M.J. Wilde, F. Korndorffer, E.W.
Tegelaar, S.J. Rowland, Can two-dimensional gas chromatography/mass spectrometric
identification of bicyclic aromatic acids in petroleum fractions help to reveal further
details of aromatic hydrocarbon biotransformation pathways?, Rapid Communications in
Mass Spectrometry 28 (2014) 1023-1032.
676
677
678
679
[17]
S.J. Rowland, C.E. West, D. Jones, A.G. Scarlett, R.A. Frank, L.M. Hewitt, Steroidal
Aromatic ‘Naphthenic Acids’ in Oil Sands Process-Affected Water: Structural
Comparisons with Environmental Estrogens, Environmental Science & Technology 45
(2011) 9806-9815.
680
681
682
683
[18]
F.C. Damasceno, L.D. Gruber, A.M. Geller, M.C.V. de Campos, A.O. Gomes, R.C.
Guimarães, V.F. Péres, R.A. Jacques, E.B. Caramão, Characterization of naphthenic
acids using mass spectroscopy and chromatographic techniques: study of technical
mixtures, Analytical Methods 6 (2014) 807-816.
684
685
686
[19]
G. Moss, Extension and revision of the von Baeyer system for naming polycyclic
compounds (including bicyclic compounds), Pure and applied chemistry 71 (1999) 513529.
687
688
689
[20]
S.J. Rowland, R. Clough, C.E. West, A.G. Scarlett, D. Jones, S. Thompson, Synthesis
and mass spectrometry of some tri-and tetracyclic naphthenic acids, Rapid
Communications in Mass Spectrometry 25 (2011) 2573-2578.
690
691
692
[21]
T. Sasaki, S. Eguchi, T. Toru, Synthesis of adamantane derivatives. VIII. Novel
substitution reaction of adamantanone. A simple synthesis of bicyclo[3.3.1]non-2-ene-7carboxylic acid, Journal of the American Chemical Society 91 (1969) 3390-3391.
693
694
695
696
697
[22]
J.A. Peters, J.M. Van Der Toorn, H. Van Bekkum, 3,7-disubstituted
bicyclo[3.3.1]nonanes—III: Synthesis and conformation of bicyclo[3.3.1]nonane-3α,7αdicarboxylic acid, its dimethyl ester and some other 3,7-disubstituted
bicyclo[3.3.1]nonanes; adamantane as an integrated holding system, Tetrahedron 31
(1975) 2273-2281.
32 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
698
699
700
[23]
S.J. Rowland, A.G. Scarlett, D. Jones, C.E. West, R.A. Frank, Diamonds in the rough:
Identification of individual naphthenic acids in oil sands process water, Environmental
Science & Technology 45 (2011) 3154-3159.
701
702
703
704
[24]
A.G. Scarlett, H.C. Reinardy, T.B. Henry, C.E. West, R.A. Frank, L.M. Hewitt, S.J.
Rowland, Acute toxicity of aromatic and non-aromatic fractions of naphthenic acids
extracted from oil sands process-affected water to larval zebrafish, Chemosphere 93
(2013) 415-420.
705
706
707
708
[25]
H.C. Reinardy, A.G. Scarlett, T.B. Henry, C.E. West, L.M. Hewitt, R.A. Frank, S.J.
Rowland, Aromatic Naphthenic Acids in Oil Sands Process-Affected Water, Resolved by
GCxGC-MS, Only Weakly Induce the Gene for Vitellogenin Production in Zebrafish
(Danio rerio) Larvae, Environmental Science & Technology 47 (2013) 6614-6620.
709
710
711
[26]
D. Jones, C.E. West, A.G. Scarlett, R.A. Frank, S.J. Rowland, Isolation and estimation of
the ‘aromatic’ naphthenic acid content of an oil sands process-affected water extract,
Journal of Chromatography A 1247 (2012) 171-175.
712
713
[27]
http://homepages.warwick.ac.uk/staff/M.P.Barrow/massaccuracy.html Mass accuracy
and theoretical m/z calculations 27/11/2014
714
715
716
717
[28]
C.E. West, A.G. Scarlett, J. Pureveen, E.W. Tegelaar, S.J. Rowland, Abundant
naphthenic acids in oil sands process-affected water: studies by synthesis, derivatisation
and two-dimensional gas chromatography/high-resolution mass spectrometry, Rapid
Communications in Mass Spectrometry 27 (2013) 357-365.
718
719
[29]
F.M. Holowenko, M.D. MacKinnon, P.M. Fedorak, Naphthenic acids and surrogate
naphthenic acids in methanogenic microcosms, Water Research 35 (2001) 2595-2606.
720
721
[30]
J.S. Clemente, P.M. Fedorak, A review of the occurrence, analyses, toxicity, and
biodegradation of naphthenic acids, Chemosphere 60 (2005) 585-600.
722
723
724
[31]
J.V. Headley, K.M. Peru, M.P. Barrow, Mass spectrometric characterization of
naphthenic acids in environmental samples: A review, Mass Spectrometry Reviews 28
(2009) 121-134.
725
726
727
728
[32]
S.J. Rowland, C.E. West, A.G. Scarlett, D. Jones, R.A. Frank, Identification of individual
tetra- and pentacyclic naphthenic acids in oil sands process water by comprehensive twodimensional gas chromatography/mass spectrometry, Rapid Communications in Mass
Spectrometry 25 (2011) 1198-1204.
729
730
731
[33]
B.E. Smith, C.A. Lewis, S.T. Belt, C. Whitby, S.J. Rowland, Effects of Alkyl Chain
Branching on the Biotransformation of Naphthenic Acids, Environmental Science &
Technology 42 (2008) 9323-9328.
732
733
[34]
S.J. Rowland, D. Jones, A.G. Scarlett, C.E. West, L.P. Hin, M. Boberek, A. Tonkin, B.E.
Smith, C. Whitby, Synthesis and toxicity of some metabolites of the microbial
33 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
734
735
degradation of synthetic naphthenic acids, Science of The Total Environment 409 (2011)
2936-2941.
736
737
738
[35]
R.J. Johnson, B.E. Smith, P.A. Sutton, T.J. McGenity, S.J. Rowland, C. Whitby,
Microbial biodegradation of aromatic alkanoic naphthenic acids is affected by the degree
of alkyl side chain branching, ISME J 5 (2011) 486-496.
739
740
741
[36]
D. Rush, A. Jaeschke, E.C. Hopmans, J.A.J. Geenevasen, S. Schouten, J.S.S. Damsté,
Short chain ladderanes: Oxic biodegradation products of anammox lipids, Geochimica et
Cosmochimica Acta 75 (2011) 1662-1671.
742
743
744
[37]
O. Curcuruto, D. Favrctto, P. Traldi, D. Ajo, C. Cativiela, J.A. Mayoral, M.P. Lorez,
J.M. Fraile, J.I. Garcia, Mass spectrometry in stereochemical problems. 6 — The case of
mono and di-substituted norbornanes, Organic Mass Spectrometry 26 (1991) 977-984.
745
746
747
[38]
M. Heintz, M. Gruselle, A. Druilhe, D. Lefort, Relations entre structure chimique et
données de rétention en chromatographie en phase gazeuse VI-Alcools et esters
méthyliques de structures cycliques, Chromatographia 9 (1976) 367-372.
748
749
750
[39]
W.K. Seifert, R.M. Teeter, Preparative thin-layer chromatography and high-resolution
mass spectrometry of crude oil carboxylic acids, Analytical Chemistry 41 (1969) 786795.
751
752
[40]
S. Manabe, C. Nishino, Sex pheromonal activity of (+)-bornyl acetate and related
compounds to the American cockroach, Journal of Chemical Ecology 9 (1983) 433-448.
753
754
755
[41]
Y.-H. Kuo, C.-H. Chen, S.-L. Huang, A novel bicyclo [2. 2. 2] octane skeleton diterpene,
obtunone, from the heartwood of Chamaecyparis obtusa var. formosana, Chemical and
pharmaceutical bulletin 46 (1998) 181-183.
756
757
[42]
M. Asaoka, K. Ishibashi, N. Yanagida, H. Takei, Total synthesis of (±)-eremolactone,
Tetrahedron Letters 24 (1983) 5127-5130.
758
759
[43]
A. Srikrishna, G. Ravi, A stereoselective total synthesis of (−)-seychellene, Tetrahedron
64 (2008) 2565-2571.
760
761
762
[44]
Y.V. Denisov, N.S. Vorobeva, I.M. Sokolova, A.A. Petrov, Mass Spectrometric study of
hydrocarbons of the bicyclo[2.2.2]octane series (In Russian), Neftekhimiya 17 (1977)
186-191.
763
764
765
[45]
M. Presset, Y. Coquerel, J. Rodriguez, Syntheses and Applications of Functionalized
Bicyclo[3.2.1]octanes: Thirteen Years of Progress, Chemical Reviews 113 (2012) 525595.
766
767
768
[46]
I.M. Sokolova, S.S. Berman, N.N. Abryutina, A.A. Petrov, Natural concentrates of Biand tricyclic naphthenes, Chemistry and Technology of Fuels and Oils 25 (1989) 233235.
34 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
769
770
[47]
B.J. Mair, P.E. Eberly, K. Li, F.D. Rossini, Polycycloparaffin Hydrocarbons in
Petroleum, Industrial & Engineering Chemistry 50 (1958) 115-116.
771
772
773
[48]
S.A. Selifonov, Microbial oxidation of adamantanone by Pseudomonas putida carrying
the camphor catabolic plasmid, Biochemical and Biophysical Research Communications
186 (1992) 1429-1436.
774
775
776
[49]
V.S. Ranade, G. Consiglio, R. Prins, Functional-Group-Directed Diastereoselective
Hydrogenation of Aromatic Compounds. 21, The Journal of Organic Chemistry 65
(2000) 1132-1138.
777
[50]
C. Dass, Fundamentals of Contemporary Mass Spectrometry, Wiley, 2007.
778
779
[51]
F.W. McLafferty, F. Tureček, Interpretation of Mass Spectra, University Science Books,
1993.
780
781
782
783
[52]
J.S. Sinninghe Damsté, W.I.C. Rijpstra, J.A.J. Geenevasen, M. Strous, M.S.M. Jetten,
Structural identification of ladderane and other membrane lipids of planctomycetes
capable of anaerobic ammonium oxidation (anammox), FEBS Journal 272 (2005) 42704283.
784
785
786
[53]
R. Alexander, R. Kagi, R. Noble, Identification of the bicyclic sesquiterpenes drimane
and eudesmane in petroleum, Journal of the Chemical Society, Chemical
Communications (1983) 226-228.
787
788
789
790
[54]
G.N. Gordadze, T.V. Okunova, M.V. Giruts, O.G. Erdnieva, V.N. Koshelev, Petroleum
C15 polyalkyl substituted bicyclo[4.4.0]decanes (sesquiterpanes) as oil maturity
indicators (illustrated by the example of Jurassic and Cretaceous oils of Kalmykia),
Petroleum Chemistry 51 (2011) 117-122.
791
792
[55]
F.W. McLafferty, R.F. Gould, Mass Spectral Correlations: Advances in Chemistry,
Literary Licensing, LLC, 2012.
793
794
[56]
Y.V. Denisov, I.M. Sokolova, A.A. Petrov, Mass Spectrometric study of hydrocarbons of
the bicyclo[4.3.0]octane series (In Russian), Neftekhimiya 17 (1977) 491-497.
795
796
797
[57]
Y.V. Denisov, I.A. Matveyeva, I.M. Sokolova, A.A. Petrov, Mass-spectrometric study of
hydrocarbons of bicyclo[3.2.1]octane series, Petroleum Chemistry U.S.S.R. 17 (1977)
85-93.
798
799
[58]
Y.V. Denisov, N.S. Vorob'eva, A.A. Petrov, Mass Spectrometric study of hydrocarbons
of the bicyclo[3.3.0]octane series (In Russian), Neftekhimiya 17 (1977) 656-662.
800
801
802
[59]
Y.S. Brodskii, I.M. Lukashenko, I.A. Musayev, E.K. Kurashova, P.I. Sanin, Massspectra of dimethylbicyclo[4.4.0]decane stereoisomers (2,3-; 2,4-; 2,8- and 2,9dimethylbicyclo[4.4.0]decanes), Petroleum Chemistry U.S.S.R. 17 (1977) 77-84.
35 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
803
804
805
[60]
I.M. Lukashenko, Y.S. Brodskii, I.A. Musayev, E.K. Kurashova, V.G. Lebedevskaya,
P.I. Sanin, Mass spectra of methylbicyclo[4,4,0]decane stereoisomers, Petroleum
Chemistry U.S.S.R. 13 (1973) 38-44.
806
807
[61]
L.S. Golovkina, G.V. Rusinova, A.A. Petrov, Mass Spectrometry of Saturated
Hydrocarbons, Russian Chemical Reviews 53 (1984) 870-887.
808
809
[62]
N. Zelinsky, Über die chemische Natur des Naphthensäuren (I.), Berichte der deutschen
chemischen Gesellschaft (A and B Series) 57 (1924) 42-51.
810
811
812
813
814
[63]
W.K. Seifert, R.M. Teeter, W.G. Howells, M.J.R. Cantow, Analysis of crude oil
carboxylic acids after conversion to their corresponding hydrocarbons, Analytical
Chemistry 41 (1969) 1638-1647.
36 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
815
816
817
Figure 1: (A) Structures of synthesised or purchased authentic bicyclic acids and (B) examples
of generalised structures and names of possible C11 bicyclic acids
37 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
818
819
820
821
822
823
824
Figure 2: Extracted ion chromatograms for molecular ions of C8-15 bicyclic acid methyl esters
(m/z 154, 168, 182, 196, 210, 224, 238 and 252) in (A) an OSPW acid extract from industry A
collected 2009, #2, (B) an OSPW acid extract from industry B, #3, (C) an OSPW acid extract
from industry A collected from a different tailings pond in 2013, #4 and (D) a fraction of
Merichem acid extract, #5
38 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
825
826
827
828
829
830
831
832
833
Figure 3: Extracted ion chromatograms (m/z 154, 125, 95 and 87) of (A and B) two OSPW acid
extracts from industries A and B (#3 and #4), analysed by GC×GC-MS and mass spectra of (C
and E) unknown peaks 1a and 1b, identified by comparison with (D) a NIST library spectrum of
bicyclo[2.2.1]heptane-2-carboxylic acid methyl ester and (F) a purchased reference standard of
bicyclo[2.2.1]heptane-1-carboxylic acid methyl ester. Unknown peaks labelled 1c and 1d were
speculated to be bicyclo[2.2.1]heptane carboxylic acid methyl ester isomers based on mass
spectral interpretation (mass spectra are given in supplementary information, Figure S2).
39 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
834
835
836
837
838
839
Figure 4: Extracted ion chromatogram (m/z 168) of (A) a fraction of Merichem acid extract (#5)
analysed by GC×GC-MS and mass spectrum of (B) unknown peak 2a, identified by comparison
with (C) that of authentic bicyclo[2.2.2]octane-1-carboxylic acid methyl ester with the same
GC×GC retention position
40 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
840
841
842
843
844
845
Figure 5: Extracted ion mass chromatograms (m/z 168 and 87) of (A and B) OSPW acid extracts
from Industries A and B (#3 and #4) analysed by GC×GC-MS and mass spectra (C and D) of the
same unknown (3a) present within both samples identified by comparison with (E) the mass
spectrum and retention position of authentic bicyclo[3.2.1]octane-6-carboxylic acid methyl ester
41 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
846
847
848
849
850
Figure 6:
Extracted ion chromatogram (m/z 196, 182, 151, 137 and 123) of (A) a fraction of Merichem
acid extract (#5) analysed by GC×GC-MS and mass spectra of (B, D and F) two unknown peaks
(4a and c) identified and one C11 unknown (4b) tentatively identified by comparison with the
mass spectra and retention positions of (C) purchased bicyclo[3.3.1]nonane-1-carboxylic acid
42 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
851
852
methyl ester, (E) 5-methylbicyclo[3.3.1]nonane-1-carboxylic acid methyl ester and (G)
bicyclo[3.3.1]nonane-3-carboxylic acid methyl ester
853
854
855
856
857
858
859
Figure 7: Electron ionisation mass spectra of (A,C and E) unknown peaks (5a-c) within an
OSPW acid extract (#3), tentatively identified by comparison with the mass spectra of (B and F)
two isomers of synthesised bicyclo[4.3.0]nonane-3-carboxylic acid methyl ester and (D) an
unknown within a fraction of Merichem acid extract (#5) previously identified as an isomer of
bicyclo[4.3.0]nonane carboxylic acid methyl ester [15] (most likely 2- isomer, similar to mass
spectrum reported by Curcuruto et al. [37])
43 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
860
861
862
863
864
865
866
Figure 8: Electron ionisation mass spectra of (A, C, D and E) unknown peaks within an OSPW
acid extract (#3), tentatively identified by comparison with the mass spectra of previously
identified bicyclo[4.4.0]decane acid methyl esters [15] e.g. (B) bicyclo[4.4.0]decane-3carboxylic acid methyl ester and (F) NIST library spectrum of bicyclo[4.4.0]decane-1-carboxylic
acid methyl ester
44 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
867
45 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
868
869
870
871
872
Figure S1: Schematic of GCxGC-MS two dimensional extracted ion chromatogram (m/z 196,
210, 224, 238 and 252), illustrating the distributions of methyl esters of C11-15 bicyclic
acids in an OSPW acid extract (#1) plus retention positions of methyl esters of authentic
C9-14 acids
46 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
873
874
875
876
877
878
Figure S2: Extracted ion chromatograms (m/z 154, 125, 95 and 87) of (A and B) OSPW acid
extracts from Industries A and B (#3 and #4) analysed by GC×GC-MS with mass spectra (C and
D) of unknown peaks labelled 1c and d, speculated to be bicyclo[2.2.1]heptane carboxylic acid
isomers based on mass spectral interpretation
47 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
879 Figure S3: Electron ionisation mass spectrum of bicyclo[2.2.1]heptane-2-ethanoic acid methyl ester
880
881
882
883
884
885
886
887
888
48 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
889
890
891
892
893
894
895
896
897
898
899
900
Figure S4: Electron ionisation mass spectrum of 2,6,6-trimethylbicyclo[3.1.1]heptane-3carboxylic acid methyl ester ((+)-3-pinanecarboxylic acid methyl ester)
49 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
Figure S5: Electron ionisation mass spectrum of (A) bicyclo[2.2.2]octane-2-carboxylic acid
methyl ester and (B) 4-pentylbicyclo[2.2.2]octane-1-carboxylic acid methyl ester
50 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
919
920
921
922
923
924
Figure S6: Extracted ion chromatogram (m/z 168 and 136) of (A) a fraction of Merichem acid
extract (#5) analysed by GC×GC-MS and mass spectra of (B and D) unknown peaks 7a and b,
identified by comparison with (C and E) two isomers of an authentic bicyclo[3.3.0]octane-2carboxylic acid methyl ester standard
51 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
925
926
927
928
929
930
Figure S7: Extracted ion chromatograms (m/z 168 and 136) of (A) a fraction of Merichem acid
extract (#5) and (B) an OSPW acid extract (#3) analysed by GC×GC-MS and mass spectrum of
(D) an unknown peak (7c), postulated to be an isomer of (C) an authentic bicyclo[3.3.0]octane 2-carboxylic acid methyl ester standard
52 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946Figure S8: Extracted ion chromatogram (m/z 196, 182, 137 and 123) of (A) an OSPW acid extract, #3
947analysed by GC×GC-MS and mass spectrum of (B) an unknown peak (4b) tentatively identified by
948comparison with (C) the mass spectrum and GC×GC retention position of purchased 5949methylbicyclo[3.3.1]nonane-1-carboxylic acid methyl ester
950
53 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
951
952
953
Figure S9: Structure and electron ionisation mass spectra of three isomers of synthesised
bicyclo[4.3.0]nonane-3-carboxylic acid methyl ester
54 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
954
955
956
957
958
Figure S10: Structure and electron ionisation mass spectra of four isomers of synthesised 2methylbicyclo[4.3.0]nonane-3-carboxylic acid methyl ester
55 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
959
960
961
962
Figure S11: Electron ionisation mass spectrum of 4-methylbicyclo[3.3.0]octane-2-carboxylic
acid methyl ester
56 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
963
964
965
966
967
968
969
970
971
972
973
974
975
976
977
978
Figure S12: Electron ionisation mass spectra of two isomers of synthesised 7methylbicyclo[4.2.0]octane-7-carboxylic acid methyl ester
57 Disclaimer: This is a pre-publication version. Readers are recommended to consult
the full published version for accuracy and citation.
Accepted by J. Chromatogr. A.
979
980
981
982
983
984
985
Figure S13: Extracted ion chromatogram (m/z 196), 3D chromatogram and (A-F) electron
ionisation mass spectra of methyl esters of six C11 unknown acids in an OSPW acid extract (#1)