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

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.

Firstly, PC-21:0/21:0 (5 μg) was mixed with furan fatty acids standards (11D3 and 11D5) and dried under N2. The methyl esters were prepared according to the method described by 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 maintained at 50 o C for 10 min. Glacial acetic acid (20 μL) is then added, followed by ddH2O (1 mL). The required methyl esters and non-esterified 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 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 nhexane. The combined organic layer was dried under N2 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-by guest, on May 7, 2020 www.jlr.org Downloaded from hexane. The combined organic layer was dried under N2 and re-dissolved in 50 μL of n-hexane before subjected to GC/MS analysis.

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 N2 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 heated at 60 o 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 solution, mixed and heated at 50 o C for 30 min. After derivatization, the solution was dried under N2 and resuspended in 100 μL of acetonitrile/H2O (7:3, v/v). The formed 3-acyl-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 chromatographytandem 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

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 basecatalyzed methylation or using diazomethane and related reagents (34). Acid-and basecatalyzed 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 These findings cast some doubt on the accuracy of some furan fatty acid determinations in food samples using the BF3-MeOH method (21). Overall, our results strongly indicate that basic derivatization protocols should be used for the quantification of furan fatty acids.
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, 321→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 worth noting that the abundance of fragments in MS 2 spectra was 1000-fold less than the molecular ion in MS 1 . 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 16.67 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 by guest, on May 7, 2020 www.jlr.org

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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 + 124). 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
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 20 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. 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 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 α2 position of furan fatty acids (40).

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.