Development of a sensitive and quantitative method for the identification of two major furan fatty acids in human plasma

Article abstract of DOI:10.1194/jlr.D119000514

This article focuses on the establishment of an accurate and sensitive quantitation method for the analysis of furan fatty acids. In particular, the sensitivity of GC/MS and UPLC/ESI/MS/MS was compared for the identification and quantification of furan fatty acids. Different methylation methods were tested with respect to GC/MS analysis. Special attention needs to be paid to the methylation of furan fatty acids, as acidic catalysts might lead to the degradation of the furan ring. GC/MS analysis in full-scan mode demonstrated that the limit of quantitation was 10 μM. UPLC/ESI/MS/MS in multiple reaction monitoring mode displayed a higher detection sensitivity than GC/MS. Moreover, the identification of furan fatty acids with charge-reversal derivatization was tested in the positive mode with two widely used pyridinium salts. Significant oxidation was unexpectedly observed using N-(4-aminomethylphenyl) pyridinium as a derivatization agent. The formed 3-acyl-oxymethyl-1-methylpyridinium iodide derivatized by 2-bromo-1-methylpyridinium iodide and 3-carbinol-1-methylpyridinium iodide improved the sensitivity more than 2,000-fold compared with nonderivatization in the negative mode by UPLC/ESI/MS/MS. This charge-reversal derivatization enabled the targeted quantitation of furan fatty acids in human plasma. Thus, it is anticipated that this protocol could greatly contribute to the clarification of pathological mechanisms related to furan fatty acids and their metabolites.

Full text of DOI:10.1194/jlr.D119000514

Development of a sensitive and quantitative method for the
identification of two major furan fatty acids in human plasma
1
2
3
4
5
6*
Long Xu , Changfeng Hu , Yongguo Liu , Siming Li , Walter Vetter , Huiyong Yin and
Yonghua Wang1*
1School of Food Science and Engineering, South China University of Technology, Guangzhou
510640, China
2College of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou,
Zhejiang 310053, China
3Beijing Advanced Innovation Centre for Food Nutrition and Human Health, Beijing Key
Laboratory of Flavor Chemistry, Beijing Technology and Business University, Beijing 100048,
China
4Analytical Applications Center, Analytical Instruments Division, Shimadzu (China) Co., LTD.
Guangzhou Branch, Guangzhou 510656, China
5Institute of Food Chemistry, Department of Food Chemistry (170b), University of Hohenheim,
D-70593 Stuttgart, Germany
6CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of
Nutrition and Health, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of
Sciences (CAS), Shanghai 200031, China
*
Please address correspondence to:
hyyin@sibs.ac.cn
yonghw@scut.edu.cn
1
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Running title: Development of a sensitive measurement for furan fatty acids
Abbreviations: AMMP, 3-acyl-oxymethyl-1-methylpyridinium iodide; AMPP, N-(4-
aminomethylphenyl) pyridinium; BMP, 2-bromo-1-methylpyridinium iodide; CMP, 3-
carbinol-1-methylpyridinium iodide; CMPF, 3-carboxy-4-methyl-5-propyl-2-furanopropanoic
acid; CMPentylF, 3-carboxy-4-methyl-5-pentyl-2-furanopropanoic acid; 11D3, 11-(3,4-
dimethyl-5-propylfuran-2-yl)undecanoic acid; 11D3-d
)furan-2-yl)undecanoic acid; 11D5, 11-(3,4-dimethyl-5-pentylfuran-2-yl)undecanoic acid;
1D5-d 11-(3,4-dimethyl-5-(pentyl-4,4,5,5,5-d )furan-2-yl)undecanoic acid; DHA,
5
, 11-(3,4-dimethyl-5-(propyl-2,2,3,3,3-
d
5
1
5
,
5
docosahexaenoic acid; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EI, electron-
impact ionization; EPA, eicosapentaenoic acid; ESI, electrospray ionization; FAMEs: fatty acid
methyl esters; HOBt: N-hydroxybenzotriazole; IS, internal standards; LC-GC-PID-FID, high-
performance liquid chromatography coupled on-line with capillary gas chromatography with a
photoionization detector mounted in series with a flame ionization detector; LOD, limit-of-
detection; LOQ, limit-of-quantitation; MRM, multiple reaction monitoring; MS, mass
spectrometry; MW: molecular weight; PIS: precursor ion scan; RSD, relative standard
deviation; RT: retention time; SNR, single-to-noise ratio; T2D, type 2 diabetes; TEA,
triethylamine; TIC, total ion chromatogram.
2
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Abstract
This paper focuses on the establishment of an accurate and sensitive quantitation method
for analysis of furan fatty acids. Particularly, the sensitivity of GC-MS and UPLC-ESI-MS/MS
was compared for the identification and quantification of furan fatty acids. Different
methylation methods were tested with respect to GC-MS analysis. Special attention needs to be
paid to the methylation of furan fatty acids as acidic catalysts might lead to the degradation of
furan ring. GC-MS analysis in full scan mode demonstrated that the limit-of-quantitation (LOQ)
was 10 μM. UPLC-ESI-MS/MS in multiple reaction monitoring (MRM) mode displayed a
higher detection sensitivity than GC-MS. Moreover, identification of furan fatty acids with
charge-reversal derivatization was tested in positive mode with two widely used pyridinium
salts. Unexpectedly, significant oxidation was observed using N-(4-aminomethylphenyl)
pyridinium (AMPP) as
a
derivatization agent. The formed 3-acyl-oxymethyl-1-
methylpyridinium iodide (AMMP) derivatized by 2-bromo-1-methylpyridinium iodide and 3-
carbinol-1-methylpyridinium iodide improved the sensitivity more than 2000 fold, compared
with non-derivatization in negative mode by UPLC-ESI-MS/MS. This charge reversal
derivatization enabled the targeted quantitation of furan fatty acids in human plasma. Thus, it
is anticipated that this protocol could greatly contribute to the clarification of pathological
mechanisms related to furan fatty acids and their metabolites.
Keywords: Charge-reversal derivatization; Furan fatty acids; Multiple reaction monitoring;
UPLC-ESI-MS/MS; Precursor ion scan
3
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Introduction
The current state-of-the-art lipidomics and metabolomics enable the discovery of various
lipids associated with the development and progression of type 2 diabetes (T2D) (1-3), cancer
4) and cardiovascular diseases (5, 6). Recent studies showed that a dibasic urofuran acid, 3-
(
carboxy-4-methyl-5-propyl-2-furanopropanoic acid (CMPF), is found in high concentrations
in subjects diagnosed with prediabetics, gestational diabetes and T2D (7, 8). The elevated
CMPF has been recognized as a potential biomarker in diabetes development. CMPF is
believed to increase oxidative stress, impair insulin granule maturation and secretion (9). It has
been claimed that CMPF is a metabolite of furan fatty acids (Figure 1), however, it is
documented that furan fatty acids have a line of homologs in nature. Intriguingly, very recent
human studies demonstrated that CMPF formed as a significant metabolite of fish oil composed
of only eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ethyl esters without any
furan fatty acids identified (10-12). Hence, the exact origin of CMPF remains controversial and
related metabolic pathways remain elusive.
An accurate and sensitive quantitation method of furan fatty acids in food and biological
samples would greatly contribute to a clarification of the ongoing debate. Gas chromatography-
flame ion detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS) are
generally considered as the most preferable methods for furan fatty acid profiling. For this,
methylation of the carboxylate group is inevitable (13-15). However, the similar
chromatographic behavior of the furan ring with some common monounsaturated fatty acids at
high concentrations renders the peak identification of furan fatty acids ambiguous and laborious
(
16). It has been documented that hydrogenation of furan fatty acid methyl esters to more polar
4
澳
澳

tetrahydrofurancarboxylic acid methyl esters could facilitate the chromatographic separations
17). Moreover, some improved analytical methods including multi-dimensional GC-MS and
(
high-performance liquid chromatography coupled on-line with capillary gas chromatography
with a photoionization detector mounted in series with a flame ionization detector (LC-GC-
PID-FID) contributes greatly to the identification of furan fatty acids (18, 19). Very recently,
1
the quantification of furan fatty acids has also been achieved by H NMR (20). Accordingly, it
has been reported that a variety of food sources especially fish and fish oil contain furan fatty
acids in a sub-mg/g level (21, 22). Nonetheless, little evidence for the existence of furan fatty
acids in human tissues exists so far and extremely low levels of less than 50 ng/mL have been
reported (17, 23, 24). A complicated enrichment from large volumes of plasma/serum (e.g., >
2
0 mL) is imperative before analysis (24). Additionally, lack of commercial furan fatty acid
standards and deuterated analogs represents further key limiting factors for the accurate
quantitation in biological samples.
Generally, natural furan fatty acids can be divided into two major classes. The first class
is represented by a propyl moiety whereas the second is dominated by a pentyl moiety at α
2
position of the furan ring (19, 25). In addition to CMPF, 3-carboxy-4-methyl-5-pentyl-2-
furanopropanoic acid (CMPentylF) has also been identified in human plasma and urine (10,
2
6). It has been suggested that the metabolic fate of furan fatty acids is associated with this
difference (propyl or pentyl) (27). The most abundant furan fatty acid homologs detected in
food matrices and human plasma are 11-(3,4-dimethyl-5-propylfuran-2-yl)undecanoic acid
(
11D3) and 11-(3,4-dimethyl-5-pentylfuran-2-yl)undecanoic acid (11D5), representing the
aforementioned two classes (17, 28). Therefore, a targeted quantitation of these two classes of
5
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furan fatty acids especially 11D3 and 11D5 in human diets and biological tissues represents a
promising approach to clarify the relationship of furan fatty acids with CMPF.
Shot-gun lipidomics and liquid chromatography-tandem mass spectrometry (LC-
MS/MS)-based lipidomics have also been well developed for fatty acid analysis (29, 30).
However, an accurate and sensitive protocol for quantitative analysis of furan fatty acids by
shot-gun lipidomics or LC-MS/MS has not been developed yet. Up to now, only one report
exists on the qualitative analysis of furan fatty acids in fish lipids by HPLC/ESI-Q-TOF-MS
(
31).
Herein, we explored an approach for identification and quantitation of furan fatty acids in
biological samples by comparing the sensitivity of GC-MS and LC-MS/MS, with a special
focus on the levels of 11D3 and 11D5. Special attentions regarding the derivatization of furan
fatty acids were proposed for the first time in our investigation. Notably, the abundant fragments
derived from the derivatized moiety could be employed for quantitative analysis of individual
furan fatty acid species. We believe that this approach could facilitate the clarification of the
metabolic precursor of CMPF and contribute to investigate the biological relevance of furan
fatty acids.
6
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Experimental methods
Reagents and materials
Acetonitrile and methanol of LC-MS grade were purchased from Merck (Germany).
®
Ammonium acetate, 2-bromopyridine, 3-carbinolpyridine, Diazald , formic acid and
triethylamine (TEA) were purchased from Sigma-Aldrich (USA). 1,2-Dihenarachidoyl-sn-
glycero-3-phosphocholine (PC-21:0/21:0) were obtained from Nu-Chek Prep, Inc (USA).
Diethylene glycol monoethyl ether was obtained from TCI (Shanghai) development Co., Ltd
(
China). Iodomethane was purchased from Acros Organics (Belgium). Water was prepared
using a Milli-Q system (Millipore, USA).
1-(3,4-dimethyl-5-propylfuran-2-yl)undecanoic acid (purity > 98%) and 11-(3,4-
dimethyl-5-pentylfuran-2-yl)undecanoic acid (purity > 98%) were synthesized by Shanghai
Medicilon Inc., China. 11-(3,4-dimethyl-5-(propyl-2,2,3,3,3-d )furan-2-yl)undecanoic acid
chemical purity > 99.5%, deuterium purity > 99.0%) and 11-(3,4-dimethyl-5-(pentyl-4,4,5,5,5-
1
5
(
d
5
)furan-2-yl)undecanoic acid (chemical purity > 99.5%, deuterium purity > 99.0%) were
+
®
synthesized by Syncom, Netherlands. AMP MaxSpec Kit (50 tests, Catalog #710000),
containing N-(4-aminomethylphenyl) pyridinium (AMPP), 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC) hydrochloride aqueous solution, N-
hydroxybenzotriazole (HOBt), and acetonitrile/N,N-dimethylformamide (4:1, v/v) solution,
were purchased from Cayman Chemical Co. (Ann Arbor, MI).
Nomenclature
7
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Furan fatty acids are abbreviated according to the simplified “number-letter-number”
notation (21). The first number is indicative of the length of carboxyalkyl chain at α position.
A letter of “M” or “D” represents one or two methyl moieties at β positions. The second letter
donates the length of alkyl chain at α position. Accordingly, 11-(3,4-dimethyl-5-propylfuran-
-yl)undecanoic acid and 11-(3,4-dimethyl-5-pentylfuran-2-yl)undecanoic acid are denoted as
1D3 and 11D5, respectively. The corresponding deuterated analogs, 11-(3,4-dimethyl-5-
propyl-2,2,3,3,3-d )furan-2-yl)undecanoic acid and 11-(3,4-dimethyl-5-(pentyl-4,4,5,5,5-
)furan-2-yl)undecanoic acid, are abbreviated as 11D3-d and 11D5-d , respectively.
1
2
2
1
(
5
d
5
5
5
Preparation of furan fatty acid mixtures
The stock solutions of furan fatty acids and deuterated internal standards (IS) were
prepared in pure acetonitrile with a concentration of 10 μg/mL. The concentration of each stock
solution was determined gravimetrically. The mixtures of furan fatty acids were prepared from
individual stock solutions.
Preparation of human plasma samples
This study was approved by Ethics Committee of Shanghai General Hospital, affiliated
with Shanghai Jiao Tong University School of Medicine, Shanghai, China (Approval Number:
Expedited Review [2018] 136). Fasting blood samples were collected from healthy subjects
o
and stored at −80 C according to a standardized procedure as previously described (32).
Methylation of furan fatty acids
8
澳
澳

The methylation of furan fatty acids was conducted according to three different methods:
ethereal diazomethane, methanolic sulfuric acid (H SO -MeOH) and boron trifluoride-
methanol (BF -MeOH).
Firstly, PC-21:0/21:0 (5 μg) was mixed with furan fatty acids standards (11D3 and 11D5)
and dried under N . The methyl esters were prepared according to the method described by
2
4
3
2
Uchida et al with slight modifications (31). The mixed standards were dissolved in toluene (0.2
mL) and mixed with 0.5 M sodium methoxide in methanol (0.4 mL). The solution was
o
maintained at 50 C for 10 min. Glacial acetic acid (20 μL) is then added, followed by ddH
2
O
(
1 mL). The required methyl esters and non-esterified free furan fatty acids were extracted into
n-hexane (3 × 1 mL) and dried under N . The following methylation of free fatty acids was
2
performed by ethereal diazomethane. The final products were resuspended in n-hexane (50 μL),
then subjected to GC/MS analysis.
Secondly, furan fatty acid standards (11D3 and 11D5) with PC-21:0/21:0 (5 μg) as IS were
derivatized with 2 mL of methanol containing 2% sulfuric acid for 2 h at 80 °C in sealed
borosilicate glass tubes. Subsequently, 1 mL of aqueous saturated NaCl solution were added,
and the generated furan fatty acid methyl esters were extracted three times using 1 mL of n-
hexane. The combined organic layer was dried under N
2
and re-suspended in 50 μL of n-hexane,
then subjected to GC/MS analysis.
Thirdly, furan fatty acid standards (11D3 and 11D5) with PC-21:0/21:0 (5 μg) as IS were
derivatized with 2 mL of ~15% boron trifluoride in methanol for 1 h at 90 °C in sealed
borosilicate glass tubes. Subsequently, 1 mL of aqueous saturated NaCl solution were added,
and the generated furan fatty acid methyl esters were extracted three times using 1 mL of n-
9
澳
澳

hexane. The combined organic layer was dried under N
2
and re-dissolved in 50 μL of n-hexane
before subjected to GC/MS analysis.
Synthesis of 2-bromo-1-methylpyridinium iodide (BMP) and 3-carbinol-1-methylpyridinium
iodide (CMP)
Iodomethane (50 mmol, 3.11 mL) was added to 2-bromopyridine (10 mmol, 0.97 mL) or
3
-carbinolpyridine (10 mmol, 0.96 mL) and stirred at room temperature for 1 h. The yellow
crystals were washed with cold acetone (5 mL) for three times and dried in vacuum.
Derivatization of furan fatty acids with AMPP and BMP & CMP
The derivatization of furan fatty acids with AMPP was carried out according to the
manufacturer’s instruction. Briefly, 100 μL of 11D3 and 11D5 (1 μg/mL in acetonitrile) was
dried under N
2
and resuspended in 20 μL of cold acetonitrile/DMF (4:1, v/v). Then cold EDC
(
20 μL) and HOBt (10 μL) were sequentially added to the solution. The vial was briefly mixed
+
on a vortex mixer and placed on ice. Finally, the AMP solution (30 μL) was added, mixed and
o
heated at 60 C for 30 min.
The derivatization of furan fatty acids with BMP and CMP was conducted according to
the method of Yang et al with slight modifications (33). 10 μL of 11D3 and 11D5 (10 μg/mL in
acetone), BMP (7.5 mg/mL in acetonitrile, 20 μL) and CMP (10 mg/mL in acetonitrile, 20 μL)
were added and briefly mixed on a vortex mixer. Subsequently, TEA (1 μL) was added to the
o
solution, mixed and heated at 50 C for 30 min. After derivatization, the solution was dried
under N
2
2
and resuspended in 100 μL of acetonitrile/H O (7:3, v/v). The formed 3-acyl-
10澳
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oxymethyl-1-methylpyridinium iodides were abbreviated as 11D3-AMMP and 11D5-AMMP.
The scheme of furan fatty acid derivatization is shown in Figure S1.
GC-MS analysis
Furan fatty acids were analyzed as fatty acid methyl esters (FAMEs), which were separated
on an Agilent HP-INNOWax column (30 m × 0.25 mm i.d., 0.25 μm film thickness; USA) and
analyzed by gas chromatography triple quadrupole tandem mass spectrophotometry (GC-
TQ/MS, Agilent, Santa Clara, CA) based on an electron-impact (EI) ion source. The EI
ionization at 70 eV was performed, with a full scan range of m/z 50-500. All measurements
were carried out according to the following oven temperature program: the initial temperature
of 60 °C was held for 2 min, and raised to 160 °C with a ramp of 20 °C/min. Then the
temperature increased to 240 °C with a ramp of 5 °C/min and was held for 7 min, resulting in
a total run time of 30 min.
UPLC-ESI-MS/MS analysis
The analysis of free and derivatized furan fatty acids including furan fatty acids-AMPP
and furan fatty acids-AMMP was performed in ultra-high performance liquid chromatography-澳
tandem mass spectrometry system (UPLC-MS/MS 8050, Shimadzu, Kyoto, Japan) composed
of a Shimadzu 30 AD liquid chromatography system (an LC-30 A binary pump, an SIL-30AC
autosampler and a CTO-30AC column oven) and an 8050 triple quadrupole mass spectrometer
equipped with a heated electrospray ionization (ESI) source.
Analysis of free furan fatty acids was performed in negative mode. Separation of analytes
11澳
澳

was achieved with an Agilent ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, i.d. 1.8
μm) at a flow rate of 0.3 mL/min. The mobile phase was consisted of solvent A (H O with 10
2
mM CH
3
4
COONH ) and solvent B (MeOH with 10 mM CH
3
COONH ). Samples were applied
4
to the column at 60% B and eluted with a linear increase in B (60%-80% B from 1.0 to 6.5 min)
that reached 100% at 7.5 min and was held for 6 min. Then the elution was decreased to 80%
in B at 14.0 min and was held for 3.5 min. Finally, the elution was returned to the initial
condition at 18.0 min for 2 min of equilibration. The MS parameters were: nebulizing gas (N
2
)
flow rate, 3 L/min; drying gas (N ) flow rate, 15 L/min; desolvation line (DL) temperature, 250
2
o
o
o
C; heat block temperature, 400 C; interface temperature, 350 C.
Analysis of derivatized furan fatty acids was performed in positive mode. Separation of
®
TM
analytes was achieved with a Waters ACQUITY UPLC CSH C18 column (2.1 mm × 100
mm, i.d. 1.7 μm) at a flow rate of 0.4 mL/min. The mobile phase was consisted of solvent A
(
2
H O with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). Samples were
applied to the column at 10% B, then raised to 30% B from 1.0 to 2.0 min. The elution was
increased linearly in B (30%-80% from 2.0 to 5.0 min) that reached 100% at 6.0 min and was
held for 1 min before returning to initial conditions for 3 min of equilibration.
Validation procedure
The peak area ratios of furan fatty acid standards to corresponding IS against the
concentration ratios were plotted to construct the standard curves in pooled plasma. The
concentrations of standards are shown in Table S1.
A 7-point dilution series of each furan fatty acid species was prepared from the stock
12澳
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solution for external calibration. The above calibrator solutions were selected according to the
range of physiological levels. Three aliquots of one low-concentrated plasma sample spiked
with different furan fatty acid concentrations were used to determined precision and accuracy.
The intra-assay and inter-assay precision were evaluated by the relative standard deviation
(
RSD) of triplicate injections on the same day and on three consecutive days, respectively. The
accuracy was assessed according to the following formula:
measured concentration െ basal concentration
Accuracy ሺ%ሻ=
×100
added concentration
Additionally, five standard solutions with a concentration range of 0.005-50 ng/mL were
prepared to evaluate the limit-of-detection (LOD) and lower limit-of-quantitation (LLOQ). The
LOD was determined as the lowest concentration with single-to-noise ratio (SNR) > 3, while
LLOQ was determined according to the calculated precision (< 20% CV) and accuracy (80-
1
20%) by measurement of each concentration five times.
Analysis of furan fatty acids in human plasma
The furan fatty acid extraction was carried out by the following protocol. 11D3-d and
5
1
1D5-d (each of 10 ng) and plasma (20 μL) were added to a glass vial. Then the vial was sealed
5
o
with a Teflon/silicone disk and incubated at 60 C for 2 h after 1 M KOH in 95% ethanol (500
μL) was mixed with the plasma. Subsequently, pH was adjusted to 3~4 with 1 M HCl when the
mixture was cooled to room temperature. Free fatty acids were extracted by 300 μL of n-hexane
for three times and dried under N . The following derivatization with BMP and CMP was
2
carried out according to the above protocol.
13澳
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Statistical analysis
Significant differences of furan fatty acid concentrations between female and male
subjects were evaluated by student's unpaired t-tests. Data analysis was performed using
GraphPad Prism Software 8.0. A value of p < 0.05 was considered as statistical significance.
14澳
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Results and discussion
Comparison of different methylation methods of furan fatty acids for GC-MS analysis
Generally, fatty acids are analyzed by GC-FID or GC-MS as their volatile non-polar
derivatives. Methyl esters are the preferred derivates and can be derived by acid- or base-
catalyzed methylation or using diazomethane and related reagents (34). Acid- and base-
catalyzed methylation are suitable for the most common fatty acids such as straight-chain and
branched-chain fatty acids. However, special attention has to be paid to some unusual fatty
acids (e.g. fatty acids containing cyclopropene, cyclopropane or epoxy groups) as they are
susceptible to chemical attack by acidic catalyst (34, 35).
To compare the efficiency of different methylation protocols for furan fatty acids, 11D3
and 11D5 standards were derivatized with H
diazomethane using PC-21:0/21:0 as IS. The typical GC-MS chromatograms are shown in
Figure 2. While BF -MeOH was inefficient for the methylation of 11D3 and 11D5 (no methyl
2
SO
4
-MeOH, BF
3
-MeOH and ethereal
3
ester peaks were observed), both H
2
SO
4
-MeOH and CH
3
ONa-MeOH + CH
2
N yielded the
2
desired methyl products at approximately the same levels. Possibly, BF
degradation of the furan moiety (36). It should be noted that also using H
3
induced hydrolytic
2
SO -MeOH can lead
4
to degreased yields of furan fatty acid methyl esters if performed at elevated reaction
o
temperatures (> 90 C) or in the presence of elevated concentrations (> 2%) (data not shown).
These findings cast some doubt on the accuracy of some furan fatty acid determinations in food
samples using the BF
derivatization protocols should be used for the quantification of furan fatty acids.
5澳
3
-MeOH method (21). Overall, our results strongly indicate that basic
1
澳

The LOQ and LOD for the diazomethane and methanolic sulfuric acid method were 10
μM and 3.33 μM, respectively. The level of furan fatty acids within human blood is less than
0
.30 μM (17). As a consequence, tedious isolation and concentration of large volumes of
plasma/serum would be necessary, which means that GC-MS in full scan mode is not ideally
suited for the identification of furan fatty acids in human plasma or serum.
Analysis of furan fatty acids by UPLC-ESI-MS/MS in the negative mode
LC-MS/MS is widely used in lipidomics due to its high sensitivity and accuracy. We
therefore also analyzed free furan fatty acids (11D3 and 11D5, both at 1 μg/mL) with LC-
MS/MS in multiple reaction monitoring (MRM) mode (Figure S2). To optimize the detection
of 11D3 and 11D5, parameters such as dwell time, Q1 voltage, CE and Q3 voltage were
investigated in details (Table S2). The main fragmentations were: 11D3 (m/z 321→71, 321→99,
3
21→141) and 11D5 (m/z 349→71, 349→127, 349→141) (Figure S2). The most intensive
fragments for both 11D3 and 11D5 derived from the cleavage of the alkyl carboxyl chains. It is
2
worth noting that the abundance of fragments in MS spectra was 1000-fold less than the
1
molecular ion in MS . The fragmentation of furan fatty acids in the collision cell seemed to be
relatively random rather than specific. Results showed that LOQ and LOD were 50 ng/mL and
1
6.67 ng/mL, respectively.
Analysis of furan fatty acids by UPLC-ESI-MS/MS in the positive mode
It is generally acknowledged that in the electrospray ionization source cations exhibit a
better ionization efficiency than anions (37). Therefore, charge reversal derivatization is a
16澳
澳

powerful protocol to improve the sensitivity of mass spectrometric detection of fatty acids (29).
Particularly, pyridinium salts such as N-[4-(aminomethyl)phenyl]pyridinium, N-[4-
(
aminomethyl)benzyl]-2,4,6-trimethylpyridinium,
N-(benzenemethylamine)-2,4,6-
trimethylpyridinium, BMP and CMP can result in remarkable enhancements of the detection
sensitivity of fatty acids (29, 33). Amongst these pyridinium salts, AMMP and AMPP showed
the highest derivatization efficiency and therefore enhanced the detection sensitivity most
efficiently (29, 33). In addition, fragmentation patterns of most of the other pyridinium
derivates are more complex (29). Therefore, in the present study, derivatizations of furan fatty
acids with AMMP and AMPP were investigated. The chemical structures of the derivatives (-
AMPP and -AMMP) are illustrated in Figure 3.
+
In case of AMPP-derivatization of furan fatty acids, oxidation products ([M + 16] and [M
+
o
+
32] ) were detected even if the derivates had been stored at −80 C overnight. Especially m/z
+
3
67 was very prominent in the MS/MS spectra of [11D3-AMPP + 32] (m/z 521) and [11D5-
+
1
AMPP + 32] (m/z 549) (Figure S3). In MS scanning, the oxidation product peaks exhibit a
low abundance and therefore can easily be overseen.
Based on the fragmentation pattern, we hypothesize that the activated methylene groups
+
at the furan moiety are oxidized. [M − 18] was observed in MS/MS spectra of both 11D3-
+
AMPP and 11D5-AMPP. Presumably, [M − H
2
O] stems from the dehydration of the aromatic
side chain of the oxidation product. Interestingly, similar hydroxylated furan fatty acids had
been tentatively identified at very low concentrations in fish lipids (31). In the light of our
findings, this interpretation, however, is questionable and further analyses will be necessary to
confirm or disprove the natural occurrence of these products. In any case, we concluded that
17澳
澳

AMPP derivatization is not ideally suited for the derivatization of furan fatty acids.
Fortunately, no such artifacts (oxidation of the methylene group) was observed upon
derivatization with BMP and CMP. Charge remote fragmentation could be clearly observed in
the MS/MS spectra of both 11D3-AMMP and 11D5-AMMP (Figure 4). The fragmentation of
the N-pyridylcarbinol moiety yielded product ions with m/z 107 and m/z 124. The fragment at
m/z 178 was derived from the heterolytic cleavage between the β- and γ-C in the alkylcarboxyl
chain (33). Additionally, a series of fragments at m/z 192, 206, 220, 234 and 248 were observed
+
in the MS/MS spectra, indicating a difference of —CH
2
— (14 amu). Transition ion of M →124
+
was selected for quantitative analysis of 11D3-AMMP and 11D5-AMMP, with M →107 and
+
M →178 as qualitative references. Key parameters such as dwell time, Q1 voltage, CE and Q3
voltage for monitoring the above three transition ions were optimized. The results are shown in
Table S3.
Separation efficiency
Obviously, introduction of pyridinium group contributes to the polarity of fatty acids. Fatty
acids derivatized with AMMP (from C12:0-AMMP to C20:0-AMMP) could be separated well
on a C18 column (Figure 5). Although the peak of 11D3-AMMP (m/z 428) overlapped to some
extend with the peaks of C18:2-AMMP (m/z 386) and C15:0-AMMP (m/z 348), the first
quadruple (Q1) could discriminate the parent molecular ions clearly due to the different m/z
(
differences of mass-to-charge ratio ب 0.7).
Method development and validation
18澳
澳

The esterification of free fatty acids with AMMP and derivative yields has been studied
extensively (33, 38). Mechanistically, the final 3-acyloxymethyl-1-methylpyridinum iodide
could be formed via a nucleophilic attack by CMP on the 2-acyloxy1-methylpyridinum iodide
(
the esterification product of free fatty acid and BMP). Using equimolar amounts of starting
materials and a two-fold molar excess of TEA, the desired products have been obtained in good
yields (> 80%) (38). Based on these observations, derivatization was carried out in a one-pot
strategy by simply mixing 11D3/11D5, BMP, CMP and TEA. The derivatized 11D3 and 11D5
were quantified by UPLC-ESI-MS/MS with deuterium labeled 11D3 and 11D5, respectively,
as IS. Standard curves were established based on the peak area ratios of derivatized furan fatty
+
acids and the corresponding IS with transition ions (M Ĭ124). As shown in Table 1, good
linearities were obtained in dynamic ranges of 0.5 - 320 ng/mL and 2.5 - 1600 ng/mL for 11D3-
AMMP and 11D5-AMMP, respectively.
The precision and accuracy of this UPLC-ESI-MS/MS approach were analyzed on seven
physiological concentrations covering the range of measured levels in subjects. As can be seen
from Table 2 and Table 3, most intra-day and inter-day RSDs were lower than 12%, while the
accuracy was between 84% and 115%. Moreover, our results demonstrated that 11D3-AMMP
and 11D5-AMMP had an LLOQ of 0.05 ng/mL. The detection sensitivity was 1000-fold higher
than in the negative mode of ionization used with free 11D3 and 11D5. Therefore, this
derivatization method is accurate for the quantitation of furan fatty acids in plasma or other
biological samples.
Targeted quantification of furan fatty acids in human plasma
19澳
澳

11D3 and 11D5 were quantified in the plasma of 27 healthy Chinese subjects (18 males
and 9 females) using the AMMP derivatization methods. The UPLC-ESI-MS/MS
chromatograms of 11D3, 11D5 in human plasma and corresponding IS are shown in Figure 6.
Although some unidentified peaks were observed in these samples, the detection and
identification of furan fatty acids was not impaired. Therefore, targeted quantitation of 11D3
and 11D5 in human plasma could be achieved. The concentrations of 11D3 and 11D5 in the
individuals are shown in Figure 7. The concentration of 11D3 was generally low, usually below
2
0 ng/mL. In contrast, 11D5 was found at concentrations above 100 ng/mL. Interestingly, in
female subjects, the average level of furan fatty acids was significantly (p < 0.05) higher than
in male subjects. Also, the contents of furan fatty acids in female cohorts showed larger
deviations than in male cohorts.
Fatty acid profiles in human plasma have been extensively investigated by GC-MS (39),
multidimensional mass-spectrometry-based shotgun lipidomics (29) and LC-MS/MS (33).
However, to the best of our knowledge, furan fatty acids have been reported in three
publications so far investigating the fatty acid content of German individuals (17, 23, 24). In
these studies, unusually high sample volumes (> 20 mL of plasma or 30 mL of whole blood)
were used and concentrated prior analysis. This explains, why ever since then, furan fatty acids
have not been reported. In addition, the ‘unusual’fragmentation pattern of furan fatty acids may
have prevented their identification.
Comparing our results on furan fatty acids in human blood with the previous study (17),
we found parallel levels of 11D3 and significantly higher levels of 11D5. Furthermore, it is
interesting to note that the sex-specific differences had not been reported previously. Future
20澳
澳

investigations will have to clarify these striking differences. Overall, it can be asserted that the
sensitivity of our new UPLC-ESI-MS/MS protocol (not necessitating isolation or enrichment)
is approximately 1000-fold higher than the previously established GC-MS protocols.
The metabolic precursor of CMPF yet remains to be identified. According to previous
findings, β-oxidation does not occur on the alkyl chain at α
Therefore, it was proposed that furan fatty acids with a propyl group at α
precursors of CMPF. Accordingly, homologs bearing a pentyl group at α
2
position of furan fatty acids (40).
2
positions serve as
position serve as
2
precursors of CMPentylF (27). It is well-documented that the concentrations of CMPF in males
is generally higher than in females (7, 10). Our results and previous findings indicate that higher
levels of CMPF coincides with lower levels of 11D3. Therefore, this sex-specific difference in
11D3 and CMPF concentrations in human plasma may indicate that 11D3 serves as metabolic
precursor for CMPF. This assumption also warrants further specific studies.
Non-targeted analysis of furan fatty acid analogues
The above-described charge reversal derivatization method enables the identification of
+
+
furan fatty acids (11D3 and 11D5) according to the transition ions of M →107, M →124 and
+
M →178. Several saturated and unsaturated furan fatty acids have previously been identified
in other biological samples (27). To investigate the occurrence of 11D3 and 11D5 analogues in
human plasma, precursor ion scan (PIS) was used for the identification of furan fatty acids in
pooled samples based on the product ions of m/z 107, 124 and 178 at the collision energy of
−
48, −40 and −52 eV, respectively. The total ion chromatogram (TIC) of PIS (precursor of m/z
24) is shown in Figure 8. Precursors of m/z 107, 124 and 178 were compared for
1澳
1
2
澳

comprehensive identification of all fatty acids in human plasma (Figure S4 ~ S13). However,
some homologs including 7-(3,4-dimethyl-5-pentylfuran-2-yl)heptanoic acid, 9-(3,4-dimethyl-
5
-propylfuran-2-yl)nonanoic acid, 9-(3-methyl-5-pentylfuran-2-yl)nonanoic acid and 9-(3,4-
dimethyl-5-pentylfuran-2-yl)nonanoic acid (former three, < 0.5 ng/mL; the last, ~ 4.0 ng/mL),
which had been identified in German individuals (17), were not found in our subjects. These
differences might be related to race, diet and pathophysiological conditions. Therefore, future
investigations will be necessary to confirm this observation.
22澳
澳

Conclusions
In the present study, we have demonstrated the superiority of UPLC-ESI-MS/MS over the
established GC/MS methods. Acid-catalyzed ring opening of the furan moiety and oxidation of
the activated methylene moieties were identified as major side reactions during sample
preparation and derivatization. The optimized protocol reported here largely avoid these side
reactions thereby allowing for more accurate, quantitative determination of furan fatty acids
from biological samples. Moreover, the non-targeted strategy using PIS shows great potential
for the detection and identification of furan fatty acid analogues.
To the best of our knowledge, this is the first study using this strategy for characterization
and quantification of furan fatty acids in human tissues. We believe that the UPLC-ESI-MS/MS
approach will greatly contribute to the illumination of the CMPF precursor and the related
metabolic pathway.
23澳
澳

Acknowledgements
This work was financially supported by National Science Fund for Distinguished Young
Scholars of China (31725022), National Natural Science Foundation of China (31601398),
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing
Technology and Business University (BTBU) (20171049), Innovation and Entrepreneurship
Team of “Blue Ocean Talent Plan” in Nanhai District, Foshan, Guangdong, China
(
201811070001).
Additionally, the authors would like to thank Mingjiang Zhu (Zhejiang Hisun
Pharmaceutical Co., Ltd.), Shuyuan Guo (SCIEX, Analytical Instrument Trading Corporation),
Zheng Yan (Chinese Academy of Sciences), Xia Shen (ShanghaiTech University), Ningning
Liang (Chinese Academy of Sciences), Shanshan Zhong (Chinese Academy of Sciences) and
Luxiao Li (Chinese Academy of Sciences) for their contributions to mass spectrometric analysis.
Conflict of interest
The authors declare that they have no conflict of interest.
24澳
澳

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27澳
澳

Table 1. Linearity range of furan fatty acid derivatives
Furan fatty acid
IS
R2
Linearity (ng/mL)
derivatives
1
1D3-AMMP
11D3-d
5
-AMMP
-AMMP
0.9995
0.9991
0.5-320
11D5-AMMP
11D5-d
5
2.5-1600
28澳
澳

Table 2. Precision of furan fatty acid derivatives
STD spiked 1
STD spiked 2
STD spiked 3
STD spiked 4
STD spiked 5
STD spiked 6
STD spiked 7
Furan fatty
acid
Intra-
assay
Inter-
assay
(%)
Intra-
assay
(%)
Inter-
assay
(%)
Intra-
assay
(%)
Inter-
assay
(%)
Intra-
assay
(%)
Inter-
assay
(%)
Intra-
assay
(%)
Inter-
assay
(%)
Intra-
assay
(%)
Inter-
assay
(%)
Intra-
assay
(%)
Inter-
assay
(%)
derivatives
(%)
1
1D3-
AMMP
1D5-
AMMP
6
1
.31
.99
12.01
7.86
3.49
2.92
6.17
4.82
4.35
1.14
3.19
6.74
3.78
3.87
7.61
3.35
3.37
4.16
4.08
4.60
5.73
3.88
4.90
1.71
2.12
2.17
2.65
2.61
1
a
Precision of 11D3: aliquots of one plasma sample with a low level of 5.01 ± 0.17 ng/mL were spiked with 7 different concentrations (No. 1~7: 5, 10, 15,
20, 25, 30, 35 ng/mL);
b
Precision of 11D5: aliquots of one plasma sample with a low level of 104.41 ± 0.62 ng/mL were spiked with 7 different concentrations (No. 1~7: 20, 40,
6
c
0, 80, 100, 120, 140 ng/mL);
Data shown as the relative standard deviation (RSD).
29澳
澳

Table 3 Accuracy of furan fatty acid derivatives
Furan fatty acid
derivatives
STD spiked 1
(%)
STD spiked 2
(%)
STD spiked 3 STD spiked 4 STD spiked 5 STD spiked 6 STD spiked 7
Range
(%)
(%)
(%)
(%)
(%)
(%)
1
1D3-AMMP
102.51 ± 10.98
104.37 ± 4.27
104.11 ± 3.86
90.72 ± 5.23
98.13 ± 4.80
99.52 ± 3.85
100.57 ± 5.31 102.07 ± 4.72
90.34 ± 6.28
93.42 ± 2.37
96.36 ± 2.70
84 ~ 115
86 ~ 108
11D5-AMMP
102.77 ± 4.47 101.25 ± 2.93 102.25 ± 4.30
a
Accuracy of 11D3: aliquots of one plasma sample with a low level (5.01 ± 0.17 ng/mL) were spiked with 7 different concentrations (No. 1~7: 5, 10, 15,
20, 25, 30, 35 ng/mL);
b
Accuracy of 11D5: aliquots of one plasma sample with a low level (104.41 ± 0.62 ng/mL) were spiked with 7 different concentrations (No. 1~7: 20, 40,
6
c
0, 80, 100, 120, 140 ng/mL);
Data shown as mean ± SD.
30澳
澳