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

2020; Elsevier BV; Volume: 61; Issue: 4 Linguagem: Inglês

10.1194/jlr.d119000514

1539-7262

Long Xu, Changfeng Hu, Yongguo Liu, Siming Li, Walter Vetter, Huiyong Yin, Yonghua Wang,

Fatty Acid Research and Health

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. 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. The current state of the art in lipidomics and metabolomics enables the discovery of various lipids associated with the development and progression of T2D (1Razquin C. Toledo E. Clish C.B. Ruiz-Canela M. Dennis C. Corella D. Papandreou C. Ros E. Estruch R. Guasch-Ferré M. Plasma lipidomic profiling and risk of type 2 diabetes in the PREDIMED trial.Diabetes Care. 2018; 41: 2617-2624Crossref PubMed Scopus (72) Google Scholar, 2Balgoma D. Pettersson C. Hedeland M. Common fatty markers in diseases with dysregulated lipogenesis.Trends Endocrinol. Metab. 2019; 30: 283-285Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 3Allalou A. Nalla A. Prentice K.J. Liu Y. Zhang M. Dai F.F. Ning X. Osborne L.R. Cox B.J. Gunderson E.P. 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Recent studies have shown 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 (7Prentice K.J. Luu L. Allister E.M. Liu Y. Jun L.S. Sloop K.W. Hardy A.B. Wei L. Jia W. Fantus I.G. The furan fatty acid metabolite CMPF is elevated in diabetes and induces β cell dysfunction.Cell Metab. 2014; 19: 653-666Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 8Liu Y. Prentice K.J. Eversley J.A. Hu C. Batchuluun B. Leavey K. Hansen J.B. Wei D.W. Cox B. Dai F.F. et al.Rapid elevation in CMPF may act as a tipping point in diabetes development.Cell Reports. 2016; 14: 2889-2900Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The elevated CMPF has been recognized as a potential biomarker in diabetes development. CMPF is believed to increase oxidative stress and impair insulin granule maturation and secretion (9Koppe L. Poitout V. CMPF: a biomarker for type 2 diabetes mellitus progression?.Trends Endocrinol. Metab. 2016; 27: 439-440Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). It has been claimed that CMPF is a metabolite of furan fatty acids (Fig. 1); however, it has been 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 EPA and DHA ethyl esters without any furan fatty acids identified (10Prentice K.J. Wendell S.G. Liu Y. Eversley J.A. Salvatore S.R. Mohan H. Brandt S.L. Adams A.C. Wang X.S. Wei D. CMPF, a metabolite formed upon prescription omega-3-acid ethyl ester supplementation, prevents and reverses steatosis.EBioMedicine. 2018; 27: 200-213Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 11Mohan H. Brandt S.L. Kim J.H. Wong F. Lai M. Prentice K.J. Al Rijjal D. Magomedova L. Batchuluun B. Burdett E. et al.3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) prevents high fat diet-induced insulin resistance via maintenance of hepatic lipid homeostasis.Diabetes Obes. Metab. 2019; 21: 61-72Crossref PubMed Scopus (7) Google Scholar, 12Sinclair A. Xu L. Wang Y. 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF): a metabolite identified after consumption of fish oil and fish.Nutr. Bull. 2018; 43: 153-157Crossref Scopus (3) Google Scholar). 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. GC-flame ion detection (FID) and GC/MS are generally considered as the most preferable methods for furan fatty acid profiling. For this, the methylation of the carboxylate group is inevitable (13Müller M. Hogg M. Ulms K. Vetter W. Concentrations, stability and isolation of the furan fatty acid 9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid from disposable latex gloves.J. Agric. Food Chem. 2017; 65: 7919-7925Crossref PubMed Scopus (11) Google Scholar, 14Chvalová D. Špička J. Identification of furan fatty acids in the lipids of common carp (Cyprinus carpio L.).Food Chem. 2016; 200: 183-188Crossref PubMed Scopus (8) Google Scholar, 15Wendlinger C. Vetter W. High concentrations of furan fatty acids in organic butter samples from the german market.J. Agric. Food Chem. 2014; 62: 8740-8744Crossref PubMed Scopus (23) Google Scholar). 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 (16Wahl H.G. Liebich H.M. Hoffmann A. Identification of fatty acid methyl esters as minor components of fish oil by multidimensional GC-MSD: new furan fatty acids.J. High Resolut. Chromatogr. 1994; 17: 308-311Crossref Scopus (20) Google Scholar). It has been documented that the hydrogenation of furan fatty acid methyl esters to more polar tetrahydrofurancarboxylic acid methyl esters could facilitate the chromatographic separations (17Puchta V. Spiteller G. Weidinger H. F-Säuren: Eine bisher unbekannte Komponente der Phospholipide des Humanblutes.Liebigs Ann. Chem. 1988; 1988 (German.): 25-28Crossref Scopus (28) Google Scholar). Moreover, some improved analytical methods, including multidimensional GC/MS and HPLC coupled on-line with capillary gas chromatography with a photoionization detector mounted in series with a flame ionization detector, contribute greatly to the identification of furan fatty acids (18Boselli E. Grob K. Lercker G. Determination of furan fatty acids in extra virgin olive oil.J. Agric. Food Chem. 2000; 48: 2868-2873Crossref PubMed Scopus (34) Google Scholar, 19Wahl, H. G., A., Chrzanowski, H. M., Liebich, and A., Hoffmann, . 1994. Identification of furan fatty acids in nutritional oils and fats by multi-dimensional GC-MSD. Accessed February 10, 2020, at https://www.gerstel.com/pdf/p-gc-an-1994-06-ar.pdf.Google Scholar). The quantification of furan fatty acids has also recently been achieved by 1H NMR (20Gottstein V. Mueller M. Günther J. Kuballa T. Vetter W. Direct 1H NMR quantitation of valuable furan fatty acids in fish oils and fish oil fractions.J. Agric. Food Chem . 2019; 67: 11788-11795Crossref PubMed Scopus (8) Google Scholar). Accordingly, it has been reported that a variety of food sources, especially fish and fish oil, contain furan fatty acids below the mg/g level (21Vetter W. Laure S. Wendlinger C. Mattes A. Smith A.W.T. Knight D.W. Determination of furan fatty acids in food samples.J. Am. Oil Chem. Soc. 2012; 89: 1501-1508Google Scholar, 22Kirres C. Vetter W. Furan fatty acid content and homologue patterns of fresh green matrices.J. Food Compos. Anal. 2018; 67: 63-69Crossref Scopus (3) Google Scholar). 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 (17Puchta V. Spiteller G. Weidinger H. F-Säuren: Eine bisher unbekannte Komponente der Phospholipide des Humanblutes.Liebigs Ann. Chem. 1988; 1988 (German.): 25-28Crossref Scopus (28) Google Scholar, 23Puchta V. Spiteller G. Struktur der F-Säuren enthaltenden Plasmalipide.Liebigs Ann. Chem. 1988; 1988 (German.): 1145-1147Crossref Scopus (14) Google Scholar, 24Wahl H.G. Chrzanowski A. Müller C. Liebich H.M. Hoffmann A. Identification of furan fatty acids in human blood cells and plasma by multi-dimensional gas chromatography-mass spectrometry.J. Chromatogr. A. 1995; 697: 453-459Crossref Scopus (10) Google Scholar). A complicated enrichment from large volumes of plasma/serum (e.g., >20 ml) is imperative before analysis (24Wahl H.G. Chrzanowski A. Müller C. Liebich H.M. Hoffmann A. Identification of furan fatty acids in human blood cells and plasma by multi-dimensional gas chromatography-mass spectrometry.J. Chromatogr. A. 1995; 697: 453-459Crossref Scopus (10) Google Scholar). Additionally, a 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 the α2 position of the furan ring (19Wahl, H. G., A., Chrzanowski, H. M., Liebich, and A., Hoffmann, . 1994. Identification of furan fatty acids in nutritional oils and fats by multi-dimensional GC-MSD. Accessed February 10, 2020, at https://www.gerstel.com/pdf/p-gc-an-1994-06-ar.pdf.Google Scholar, 25Spiteller G. Furan fatty acids: occurrence, synthesis, and reactions. Are furan fatty acids responsible for the cardioprotective effects of a fish diet?.Lipids. 2005; 40: 755-771Crossref PubMed Scopus (116) Google Scholar). In addition to CMPF, 3-carboxy-4-methyl-5-pentyl-2-furanopropanoic acid has also been identified in human plasma and urine (10Prentice K.J. Wendell S.G. Liu Y. Eversley J.A. Salvatore S.R. Mohan H. Brandt S.L. Adams A.C. Wang X.S. Wei D. CMPF, a metabolite formed upon prescription omega-3-acid ethyl ester supplementation, prevents and reverses steatosis.EBioMedicine. 2018; 27: 200-213Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 26Tovar J. Mello V.D. Nilsson A. Johansson M. Paananen J. Lehtonen M. Hanhineva K. Björck I. Reduction in cardiometabolic risk factors by a multifunctional diet is mediated via several branches of metabolism as evidenced by nontargeted metabolite profiling approach.Mol. Nutr. Food Res. 2017; 61: 1600552Crossref Scopus (23) Google Scholar). It has been suggested that the metabolic fate of furan fatty acids is associated with this difference (propyl or pentyl) (27Xu L. Sinclair A.J. Faiza M. Li D. Han X. Yin H. Wang Y. Furan fatty acids – beneficial or harmful to health?.Prog. Lipid Res. 2017; 68: 119-137Crossref PubMed Scopus (40) Google Scholar). 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 (17Puchta V. Spiteller G. Weidinger H. F-Säuren: Eine bisher unbekannte Komponente der Phospholipide des Humanblutes.Liebigs Ann. Chem. 1988; 1988 (German.): 25-28Crossref Scopus (28) Google Scholar, 28Vetter W. Wendlinger C. Furan fatty acids - valuable minor fatty acids in food.Lipid Technol. 2013; 25: 7-10Crossref Scopus (36) Google Scholar). Therefore, a targeted quantitation of these two classes of furan fatty acids, especially 11D3 and 11D5, in human diets and biological tissues represents a promising approach for clarifying the relationship of furan fatty acids with CMPF. Shotgun lipidomics and LC/MS/MS-based lipidomics have also been well developed for fatty acid analysis (29Wang M. Han R.H. Han X. Fatty acidomics: global analysis of lipid species containing a carboxyl group with a charge-remote fragmentation-assisted approach.Anal. Chem. 2013; 85: 9312-9320Crossref PubMed Scopus (109) Google Scholar, 30Bian X. Sun B. Zheng P. Li N. Wu J-L. Derivatization enhanced separation and sensitivity of long chain-free fatty acids: application to asthma using targeted and non-targeted liquid chromatography-mass spectrometry approach.Anal. Chim. Acta. 2017; 989: 59-70Crossref PubMed Scopus (32) Google Scholar). However, an accurate and sensitive protocol for the quantitative analysis of furan fatty acids by shotgun lipidomics or LC/MS/MS has not been developed yet. To date, there is only one report on the qualitative analysis of furan fatty acids in fish lipids by HPLC/ESI/Q-TOF/MS (31Uchida H. Itabashi Y. Watanabe R. Matsushima R. Oikawa H. Suzuki T. Hosokawa M. Tsutsumi N. Ura K. Romanazzi D. et al.Detection and identification of furan fatty acids from fish lipids by high-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry.Food Chem. 2018; 252: 84-91Crossref PubMed Scopus (10) Google Scholar). Herein, we explored an approach for the 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. We paid particular attention to the derivatization of furan fatty acids. Notably, the abundant fragments derived from the derivatized moiety could be used for the quantitative analysis of individual furan fatty acid species. We believe this approach could facilitate the clarification of the metabolic precursor of CMPF and contribute to a greater understanding of the biological relevance of furan fatty acids. Acetonitrile and methanol of LC/MS grade were purchased from Merck. Ammonium acetate, 2-bromopyridine, 3-carbinolpyridine, Diazald®, formic acid, and triethylamine (TEA) were purchased from Sigma-Aldrich. 1,2-Dihenarachidoyl-sn-glycero-3-phosphocholine (PC-21:0/21:0) were obtained from Nu-Chek Prep, Inc. Diethylene glycol monoethyl ether was obtained from TCI (Shanghai) Development Co., Ltd. Iodomethane was purchased from Acros Organics. Water was prepared using a Milli-Q system (Millipore). 11-(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. 11-(3,4-Dimethyl-5-(propyl-2,2,3,3,3-d5)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-d5)furan-2-yl)undecanoic acid (chemical purity >99.5%; deuterium purity >99.0%) were synthesized by Syncom. The AMP+ MaxSpec® Kit (50 tests, Catalog #710000) containing N-(4-aminomethylphenyl) pyridinium (AMPP), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous solution, N-hydroxybenzotriazole, and acetonitrile/N,N-dimethylformamide (4:1; v/v) solution was purchased from Cayman Chemical Company. Furan fatty acids are abbreviated according to the simplified number-letter-number notation (21Vetter W. Laure S. Wendlinger C. Mattes A. Smith A.W.T. Knight D.W. Determination of furan fatty acids in food samples.J. Am. Oil Chem. Soc. 2012; 89: 1501-1508Google Scholar). The first number indicates the length of the carboxyalkyl chain at the α1 position. The letter "M" or "D" represents one or two methyl moieties at β positions. The second letter donates the length of the alkyl chain at the α2 position. Accordingly, 11-(3,4-dimethyl-5-propylfuran-2-yl)undecanoic acid and 11-(3,4-dimethyl-5-pentylfuran-2-yl)undecanoic acid are denoted as 11D3 and 11D5, respectively. The corresponding deuterated analogs, 11-(3,4-dimethyl-5-(propyl-2,2,3,3,3-d5)furan-2-yl)undecanoic acid and 11-(3,4-dimethyl-5-(pentyl-4,4,5,5,5-d5)furan-2-yl)undecanoic acid, are abbreviated as 11D3-d5 and 11D5-d5, respectively. The stock solutions of furan fatty acids and deuterated internal standards (ISs) 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. This study was approved by the Ethics Committee of Shanghai General Hospital, which is affiliated with Shanghai Jiao Tong University School of Medicine. Fasting blood samples were collected from healthy subjects and stored at −80°C according to a standardized procedure as previously described (32Han X. Rozen S. Boyle S.H. Hellegers C. Cheng H. Burke J.R. Welsh-Bohmer K.A. Doraiswamy P.M. Kaddurah-Daouk R. Metabolomics in early Alzheimer's disease: identification of altered plasma sphingolipidome using shotgun lipidomics.PLoS One. 2011; 6: e21643Crossref PubMed Scopus (282) Google Scholar). The methylation of furan fatty acids was conducted according to three different methods: ethereal diazomethane, methanolic sulfuric acid (H2SO4-MeOH), and boron trifluoride-methanol (BF3-MeOH). First, 5 μg PC-21:0/21:0 was mixed with furan fatty acid standards (11D3 and 11D5) and dried under N2. The methyl esters were prepared according to the method described by Uchida et al. (31Uchida H. Itabashi Y. Watanabe R. Matsushima R. Oikawa H. Suzuki T. Hosokawa M. Tsutsumi N. Ura K. Romanazzi D. et al.Detection and identification of furan fatty acids from fish lipids by high-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry.Food Chem. 2018; 252: 84-91Crossref PubMed Scopus (10) Google Scholar) with slight modifications. 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 maintained at 50°C for 10 min. Glacial acetic acid (20 μl) was then added, followed by ddH2O (1 ml). The required methyl esters and nonesterified free furan fatty acids were extracted into n-hexane (3 × 1 ml) and dried under N2. The following methylation of free fatty acids was performed by ethereal diazomethane. The final products were resuspended in 50 μl n-hexane and then subjected to GC/MS analysis. Second, furan fatty acid standards (11D3 and 11D5) with 5 μg PC-21:0/21:0 as ISs were derivatized with 2 ml methanol containing 2% sulfuric acid for 2 h at 80°C in sealed borosilicate glass tubes. A saturated aqueous solution of NaCl (1 ml) was then added, and the generated furan fatty acid methyl esters were extracted three times using 1 ml n-hexane. The combined organic layer was dried under N2 and resuspended in 50 μl n-hexane and then subjected to GC/MS analysis. Finally, furan fatty acid standards (11D3 and 11D5) with 5 μg PC-21:0/21:0 as ISs were derivatized with 2 ml of ∼15% boron trifluoride in methanol for 1 h at 90°C in sealed borosilicate glass tubes. A saturated aqueous solution of NaCl (1 ml) was then added, and the generated furan fatty acid methyl esters were extracted three times using 1 ml n-hexane. The combined organic layer was dried under N2 and redissolved in 50 μl n-hexane and then subjected to GC/MS analysis. 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 5 ml cold acetone three times and dried in a vacuum. The derivatization of furan fatty acids with AMPP was carried out according to the manufacturer's instructions. Briefly, 100 μl 11D3 and 11D5 (1 μg/ml in acetonitrile) was dried under N2 and resuspended in 20 μl cold acetonitrile/N,N-dimethylformamide (4:1; v/v). Cold 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (20 μl) and N-hydroxybenzotriazole (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 heated at 60°C for 30 min. The derivatization of furan fatty acids with BMP and CMP was conducted according to a previously described method (33Yang W-C. Adamec J. Regnier F.E. Enhancement of the LC/MS analysis of fatty acids through derivatization and stable isotope coding.Anal. Chem. 2007; 79: 5150-5157Crossref PubMed Scopus (135) Google Scholar) with slight modifications. 11D3 and 11D5 (10 μg/ml in acetone, 10 μl), 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. TEA (1 μl) was then added to the solution, mixed, and heated at 50°C for 30 min. After derivatization, the solution was dried under N2 and resuspended in 100 μl acetonitrile-H2O (7:3; v/v). The formed 3-acyl-oxymethyl-1-methylpyridinium iodides (AMMPs) are abbreviated as 11D3-AMMP and 11D5-AMMP. The scheme of furan fatty acid derivatization is shown in supplemental Fig. S1. Furan fatty acids were analyzed as fatty acid methyl esters, which were separated on an Agilent HP-INNOWax column [30 m × 0.25 mm inner diameter (ID), 0.25 μm film thickness] and analyzed by triple quadrupole GC/MS (Agilent Technologies) based on EI. EI 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; the temperature was then increased to 240°C with a ramp of 5°C/min and held for 7 min, resulting in a total run time of 30 min. The analysis of free and derivatized furan fatty acids, including furan fatty acids-AMPP and furan fatty acids-AMMP, was performed on a Shimadzu UPLC/MS/MS 8050 system composed of a 30AD LC 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 ESI source. The analysis of free furan fatty acids was performed in the negative mode. Analytes were separated with an Agilent ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm ID) at a flow rate of 0.3 ml/min. The mobile phase consisted of solvent A (H2O with 10 mM CH3COONH4) and solvent B (MeOH with 10 mM CH3COONH4). Samples were applied 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. The elution was then decreased to 80% in B at 14.0 min and 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 as follows: nebulizing gas (N2) flow rate, 3 l/min; drying gas (N2) flow rate, 15 l/min; desolvation line temperature, 250°C; heat block temperature, 400°C; and interface temperature, 350°C. The analysis of derivatized furan fatty acids was performed in the positive mode. Analytes were separated with a Waters ACQUITY UPLC CSH C18 column (2.1 mm × 100 mm, 1.7 μm ID) at a flow rate of 0.4 ml/min. The mobile phase consisted of solvent A (H2O with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). Samples were applied to the column at 10% B and 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. The peak area ratios of furan fatty acid standards to corresponding ISs against the concentration ratios were plotted to construct the standard curves in pooled plasma. The concentrations of standards are shown in supplemental Table S1. A seven-point dilution series of each furan fatty acid species was prepared from the stock 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 determine precision and accuracy. Intra- and interassay precision was 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: Additionally, five standard solutions with a concentration range of 0.005 to 50 ng/ml were prepared to evaluate the LOD and lower limit of quantitation (LLOQ). The LOD was determined as the lowest concentration with a single-to-noise ratio >3, while the LLOQ was determined according to the calculated precision (coefficient of variation <20%) and accuracy (80–120%) by measuring each concentration five times. The furan fatty acid extraction was carried out by the following protocol. 11D3-d5 and 11D5-d5 (each 10 ng) and plasma (20 μl) were added to a glass vial. The vial was then sealed 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. The pH was then adjusted to between 3 and 4 with 1 M HCl when the mixture was cooled to room temperature. Free fatty acids were extracted by 300 μl n-hexane three times and dried under N2. The following derivatization with BMP and CMP was carried out according to the above protocol. Significant differences of furan fatty acid concentrations between female and male subjects were evaluated by Student's unpaired t-tests. Data were analyzed using GraphPad Prism version 8.0. P < 0.05 was considered to be statistically significant. Generally, fatty acids are analyzed by GC-FID or GC/MS as their volatile nonpolar derivatives. Methyl esters are the preferred derivates and can be derived by acid- or base- catalyzed methylation or using diazomethane and related reagents (34Christie W.W. Preparation of ester derivatives of fatty acids for chromatographic analysis.Adv. Lipid. Method. 1993; 2: e111Google Scholar). 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 must 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 catalysts (34Christie W.W. Preparation of ester derivatives of fatty acids for chromatographic analysis.Adv. Lipid. Method. 1993; 2: e111Google Scholar, 35Christie W.W. Han X. Lipid Analysis: Isolation, Separation, Identification and Lipidomic Analysis. 4th edition. Woodhead Publishing, Philadelphia, PA2010Crossref Scopus (275) Google Scholar). To compare the efficiency of different methylation protocols for furan fatty acids, 11D3 and 11D5 standards were derivatized with H2SO4-MeOH, BF3-MeOH, and ethereal diazomethane using PC-21:0/21:0 as the IS. The typical GC/MS chromatograms are shown in Fig. 2. While BF3-MeOH was inefficient for the methylation of 11D3 and 11D5 (no methyl ester peaks were observed), both H2SO4-MeOH and CH3ONa-MeOH + CH2N2 yielded the desired methyl products at approximately the same levels. BF3 possibly induced the hydrolytic degradation of the furan moiety (36Jie M.L.K. Sinha S. Fatty acids, part 21: ring opening reactions of synthetic and natural furanoid fatty esters.Chem. Phys. Lipids. 1981; 28: 99-109Crossref Scopus (16) Google Scholar). It should be noted that also using H2SO4-MeOH can lead to degreased yields of furan fatty acid methyl esters if performed at elevated reaction temperatures (>90°C) or in the presence of elevated concentrations (>2%) (data no