alexa Detection of N-(1-deoxy-D-fructos-1-yl) Fumonisin C1, C2 and C3 in Corn Powder by LC - Orbitrap MS

ISSN: 2161-0525

Journal of Environmental & Analytical Toxicology

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Detection of N-(1-deoxy-D-fructos-1-yl) Fumonisin C1, C2 and C3 in Corn Powder by LC - Orbitrap MS

Yosuke Matsuo1, Kentaro Takahara2, Hidemi Hatabayashi1 and Hiroyuki Nakagawa1,3*
1National Agriculture and Food Research Organization (NARO), Food Research Institute, 2-1-12 Kannon-dai, Tsukuba-shi, Ibaraki 305-8642, Japan
2Thermo Fisher Scientific, C-2F, 3-9 Moriya-cho, Yokohama-shi, Kanagawa, 221-0022, Japan
3National Agriculture and Food Research Organization (NARO), Advanced Analysis Center, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
*Corresponding Author: Hiroyuki Nakagawa, National Agriculture and Food Research Organization (NARO), Food Research Institute, 2-1-12 Kannon-dai, Tsukuba- Shi, Ibaraki 305-8642, Japan, Tel: +81298388080, Email: [email protected]

Received Date: Dec 22, 2017 / Accepted Date: Dec 26, 2017 / Published Date: Jan 02, 2018

Abstract

Detection of N-(1-deoxy-D-fructos-1-yl) fumonisin C1, C2 and C3 (NDfrc-FCs) in a reference material of corn powder were performed with LC-Orbitrap MS. The peaks of NDfrc-FCs were eluted 0.1 ~ 0.3 min earlier than those of fumonisin C1, C2 and C3 (FCs), from the C18 column, probably due to their hydrophilic structures having the carbohydrate residues. At negative ionization mode scan with LC-MS analysis, the fragment ions of the tricarballylic acid (TCA) and characteristic fumonisin ions lacking TCA were detected at the identical retention times with those of respective parent NDfrc-FCs. Mass fragmentation patterns of NDfrc-FCs were confirmed to be almost in consistent with those of FCs. This study is the first report of natural occurrence of NDfrc-FC1, FC2, and FC3 in corn powder.

Keywords: Corn; N-(1-deoxy-D-fructos-1-yl) fumonisin C; Fusarium; LC-Orbitrap MS

Introduction

Fusarium mycotoxins occur worldwide in foods and feeds such as wheat and maize [1]. Among Fusarium mycotoxins, fumonisins are a group of mycotoxins that are typically produced by Fusarium verticillioides and F. proliferatum [2]. The fumonisin analogs thus far identified are separated into four main groups, such as the fumonisin A, B, C, and P types [3]. The fumonisin B (FB), FB1, FB2, and FB3, are the most abundant naturally occurring fumonisins, with FB1 frequently being detected at the highest levels. FB1 typically accounts for 70 to 80% of the total fumonisins produced, while FB2 usually makes up 15 to 25% and FB3 usually 3 to 8% [2,4]. For this reason, FB is under intense investigation, while other types of fumonisins are less recognized.

FB1 is a causative compound of equine leukoencephalomalacia [5] and porcine pulmonary edema syndrome [6], and has also been confirmed to be hepatotoxic and hepatocarcinogenic for rats and mice [7]. In 2001, JECFA established a provisional maximum tolerable daily intake (PMTDI) as 2 μg kg-1 bw day-1 for FB1, FB2, and FB3, either alone or in combination [8].

On the other hand, the glycosylated derivatives of several mycotoxins such as zearalenone and trichothecenes were found [9-12]. These glycosylated derivatives are referred to as “modified (masked) mycotoxins” [12,13]. Hydrolysis of modified mycotoxins to their aglycons has also been reported [14], therefore it has been suggested that they present an additional potential risk to consumers. As one of the modified mycotoxins derived from fumonisins, N-(1-deoxy-D-fructos- 1-yl) fumonisin B1 (NDfrc-FB1) was reported to be formed through the conjugation of the primary amine residue of FB1 with D-glucose [15]. Since NDfrc-FB1 was partly converted back to FB1 in the gastrointestinal tract of rats [16], it also may have to be included as a part of fumonisins from the standpoint of mycotoxin risk. Actually, in 2014, the Panel on Contaminants in the Food Chain (CONTAM Panel) reported that it is appropriate to assess human exposure to modified forms of the various toxins in addition to the parent compounds. In order to evaluate the total fumonisin in foods and feeds properly, there is a need to clarify the presence of modified fumonisins including NDfrc-FB1. Previously, authors detected NDfrc-FB2 and NDfrc-FB3 in a corn powder reference material [17], thereafter fumonisins C (FCs) were further detected in the same reference material in the following study [18]. Although the toxicological knowledge of FCs is limited, fumonisin C1 (FC1) has been reported to exhibit phytotoxicity and cytopathy at concentrations similar to those of FB1 [19]. Considering the structural similarity (Figure 1), authors inferred that FCs may be conjugated with D-glucose non-enzymatically via the free primary amine harbored in its structure. In this study, the detection of N-(1-deoxy-D-fructos-1-yl) fumonisin Cs (NDfrc-FCs) in the corn powder reference material was performed.

environmental-analytical-toxicology-Fumonisin-structures

Figure 1: Fumonisin structures.

Materials and Methods

Sample preparation

The screening for the N-(1-deoxy-D-fructos-1-yl) fumonisin FCs was conducted in a similar manner with author’s previous studies [17,18]. A reference material of corn powder (batch number MTC- 9999C (Trilogy)) that was confirmed to be contaminated with the fumonisins (FBs and FCs) was used [17,18]. This material was a crop that was naturally contaminated with the above toxins. There was no spike of any other toxins artificially. The manufacturer-warranted concentrations of FB1, FB2, and FB3 were 19.8 ± 4.8 mg / kg (FB1), 6.6 ± 2.1 mg / kg (FB2), and 2.2 ± 1.6 mg / kg (FB3), respectively. This material was stored at -20°C in the dark until analysis. For the extraction of mycotoxins, the corn powder (8 g) and 40 mL of methanol/water (75:25, v/v) were homogenized and centrifuged. A portion of the supernatant (5 mL) was loaded onto a strong anion exchange column (Sep-Pak® Accell Plus QMA) for the purification with no conditioning. After washing the column with 5 mL methanol/water (3:1, v/v) and 5 mL methanol successively, the fumonisins were eluted with 5 ml of methanol-acetic acid (98:2, v/v). All of the eluate was collected in a glass tube, and the solvent was evaporated under a nitrogen gas stream at 50°C. The residue was dissolved in 0.25 mL of acetonitrile/water/acetic acid (5:94:1, v/v/v), and subjected to LC-MS analysis.

For confirming that the compounds harboring the fumonisin residue were actually recovered, purification with any of the following commercially available immuno-affinity columns (IACs), FUMONIPREP® (r-biopharm® Darmstadt, Germany), FumoniStar™ (Romer Labs® Tulln, Austria) and FumoniTest™ (VICAM® Milford, MA, USA), was optionally adopted for the eluent of the strong anion exchange column. The evaporated residue was dissolved in 2 mL of phosphate buffer saline (PBS) containing 0.01% (v/v) Tween 20. The resulting solution (2 mL) was put on the respective IAC formerly conditioned with 3 mL of PBS containing 0.01% (v/v) Tween 20. The IAC was successively washed with 6 mL of PBS containing 0.01% (v/v) Tween 20, and 6 mL of 10 mM ammonium acetate aqueous solution. Thereafter, air stream was put through the column with a syringe so that all of the PBS remaining in the IAC cartridge was pushed out, and 5 ml of methanol-acetic acid (98:2, v/v) was put on the IAC for elution. All of the eluate was collected in a glass tube, and the solvent was evaporated under a nitrogen gas stream at 50°C. The residue was re-dissolved in 0.25 mL of acetonitrile/water/acetic acid (5:94:1, v/v/v), and subjected to LC-MS analysis.

LC-MS analysis

Detection of NDfrc-FCs was conducted basically in accordance with author’s previous studies with the LC-Orbitrap MS instrument, “Exactive™” (Thermo Fisher Scientific) with slight modifications [17,18]. The chromatographic separation was carried out on a HyPURITY™ C18 column (250 × 3 mm i.d., 5 μm particle size) (Thermo) with an injection volume of 20 μL. LC was performed by using 0.5 mmol / L ammonium acetate and 0.1% acetic acid aqueous solution as solvent A and 0.1% acetic acid in acetonitrile as solvent B. The flow rate was set to 0.4 mL/min, and the column temperature was maintained at 40 °C. The ionization was conducted with a heated electrospray ionization source (HESI-II) in negative mode with a spray voltage of 4.50 kV. As the common fragment ion of fumonisins, ketene form of tricarballylic acid (TCA) ion [TCA -H2O -H]- ([TCAK-H]-) was detected with high sensitivity in negative ion mode than positive ion mode. The screening was performed in the range of 150-1500 m/z at a resolving power of 100,000 FWHM (full width at half maximum) (m/z 200), with an accurate mass/high resolution (AM/HR) full scan (scan event 1) and all ion fragmentation spectrum acquisition with collision energy in a single run. Fragmentation was achieved with optional CID (collisioninduced dissociation) equipment, using a collision energy of 60 eV (scan event 2), that was optimized with the chemical standard of FB1, although some of the fragment ions were also observed even at scan event 1 without CID.

O-Phtalaldehyde (OPA) treatment

For confirmation of the D-glucose binding position in the fumonisin molecule structures, treatment with the OPA reagent was optionally performed, as reported previously [17]. Since OPA reacts specifically with primary amines by forming a complex [20], this reagent is often used for the derivatization of fumonisin molecules. For the preparation of the OPA reagent, 8 mg of OPA was dissolved in 0.2 mL of methanol, and diluted with 1 mL of 100 mM sodium tetraborate aqueous solution and 0.01 mL of 2-mercaptoethanol [20]. If the structures of NDfrc-FCs are similar to those of NDfrc-FB2 and NDfrc-FB3 (glucose bound to the primary amine of the fumonisin molecule) [19], it is considered that OPA would not react with these species. Hence, the corn powder extract prepared as described above (25 μL) was treated with OPA reagent (50 μL) by mixing, and immediately subjected to LC-MS analysis.

Results

Detection of NDfrc-FC1

Firstly, the existence of FB1 and FC1 was confirmed in the corn powder extract, based on the full scan results using the calculated masses. In the full scan data (scan event 1), peaks corresponding to the monitor ions [FB1 -H]- (720.3812) and [FC1 -H]- (706.3655) were detected at 14.28 min and 14.15 min, respectively (Figure 2A). The FB1 peak was confirmed with the standard FB1 injected into the LCMS system. As fragment ions of FB1, [FB1 -TCAK -H]- (562.3597) and [FB1 -2xTCAK -H]- (404.3379) were observed (scan event 1) (Table 1). Regarding the detection of FC1, abundant [FC1 -H]- (706.3660) ion was detected with a deviation of 0.48 mmu (0.69 ppm). As fragment ions of FC1, [FC1 -TCAK -H]- (548.3445) and [FC1 -2xTCAK -H]- (390.3225) were observed (scan event 1) (Table 1), respectively.

  FB1 (RT: 14.28 min) FC1 (RT: 14.15 min) NDfrc-FB1 (RT: 14.09 min) NDfrc-FC1 (RT: 13.98 min)
Formula Cal. Mass (m/z)a Obs. Mass (m/z)b Error (mmu(ppm)) Formula Cal. Mass (m/z)a Obs. Mass (m/z)b Error (mmu(ppm)) Formula Cal. Mass (m/z)a Obs. Mass (m/z)b Error (mmu(ppm)) Formula Cal. Mass (m/z)a Obs. Mass (m/z)b Error (mmu(ppm))
[TCA-H2O -H]- C6H6O5 157.0142 157.0137 -0.54 (-3.47) C6H6O5 157.0142 157.0138 -0.44 (-2.79) C6H6O5 157.0142 157.0138 -0.44 (-2.79) C6H6O5 157.0142 157.0139 -0.35 (-2.20)
([TCAK-H]-) 157.0135c -0.73 (-4.63) 157.0136c -0.68 (-4.34) 157.0135c -0.70 (-4.44) 157.0138c -0.47 (-2.98)
[M-2xTCAK-Glc-H]- - - - - - - - - C22H47NO5 404.3381 404.3383  0.20 (0.50) C21H45NO5 390.3225 390.3236 1.08 (2.77)
404.3381c -0.04 (-0.11) 390.3223c -0.23 (-0.59)
[M-2xTCAK-H]- C22H47NO5 404.3381 404.3379 -0.29 (-0.71) C21H45NO5 390.3225 390.3225 -0.02 (-0.05) C28H57NO10 566.391 ND C27H55NO10 552.3753 ND
404.3381c -0.07 (-0.18) 390.3246c 2.12 (5.43) ND ND
[M-TCAK-Glc-H]- - - - - - - - - C28H53NO10 562.3597 562.3601  0.44 (0.78) C27H51NO10 548.344 548.3445 0.52 (0.96)
562.3599c  0.19 (0.34) 548.3447c 0.65 (1.18)
[M-TCAK-H]- C28H53NO10 562.3597 562.3597  0.07 (0.12) C27H51NO10 548.344 548.3445 0.46 (0.84) C34H63NO15 724.4125 724.4135 1.02 (1.41) C33H61NO15 710.3968 710.3976 0.80 (1.13)
562.3597c  0.01 (0.02) 548.3439c -0.09 (-0.16) 724.4131c 0.59 (0.82) 710.3975c 0.69 (0.96)
[M-Glc-H]- - - - - - - - - C34H59NO15 720.3812 720.3821 0.95 (1.32) C33H57NO15 706.3655 706.3664 0.85 (1.20)
720.3816c 0.40 (0.55) 706.3663c 0.73 (1.03)
[M-H]- C34H59NO15 720.3812 720.3807 -0.52 (-0.72) C33H57NO15 706.3655 706.366 0.48 (0.69) C40H69NO20 882.434 882.4343 0.31 (0.35) C39H67NO20 868.4184 868.4197 1.31 (1.51)
720.3813c 0.09 (0.13) 706.3660c 0.42 (0.60) 882.4348c 0.74 (0.84) 868.4197c 1.31 (1.51)

aMass values calculated based on elemental formulas, bMass values detected by full scan (scan event 1), cMass values detected with the all ions fragmentation with collision energy (scan event 2)

Table 1: Exact mass values of FB1, FC1, NDfrc-FB1 and NDfrc-FC1 and relative fragment ions (calculated and observed) at negative polarity.

environmental-analytical-toxicology-Detection-NDfrc

Figure 2: Detection of NDfrc-FC1 with scan results (scan event 1) (A) and full mass spectrum obtained at 13.95 min (scan event 1) (B).

Secondly, the existence of NDfrc-FB1 and NDfrc-FC1 was searched, based on the full scan results using the calculated masses. In the full scan data (scan event 1), peaks corresponding to the monitor ions [NDfrc-FB1 -H]- (882.4340) and [NDfrc-FC1 -H]- (868.4184) were detected at 14.09 min and 13.98 min, respectively. Regarding detection of NDfrc-FB1, abundant [NDfrc-FB1 -H]- (882.4343) ion was detected with a deviation of 0.31 mmu (0.35 ppm) (scan event 1) (Table 1). In addition, the fragment ions [NDfrc-FB1 -TCAK -H]- (724.4135), [NDfrc-FB1 -Glc -H]- (720.3821), [NDfrc-FB1 -Glc -TCAK -H]- (562.3601), and [NDfrc-FB1 -Glc -2xTCAK -H]- (404.3383) were observed, with deviations of 1.02 mmu (1.41 ppm), 0.95 mmu (1.32 ppm), 0.44 mmu (0.78 ppm) and 0.20 mmu (0.50 ppm), respectively (scan event 1) (Table 1). Based on the loss of TCA from fumonisin structure in the ketene form (TCAK), the calculated mass values of [NDfrc-FC1 -TCAK -H]- (710.3968), [NDfrc-FC1 -Glc -H]- (706.3655), [NDfrc-FC1 -Glc -TCAK -H]- (548.3440) and [NDfrc-FC1 -Glc -2xTCAK -H]- (390.3225) ions (Table 1) were used for the screening on the result of both scan (scan event 1 and 2). Consequently, the fragment ions [NDfrc-FC1 -TCAK -H]- (710.3976), [NDfrc-FC1 -Glc -H]- (706.3664), [NDfrc-FC1 -Glc -TCAK -H]- (548.3445) and [NDfrc- FC1 -Glc -2xTCAK -H]- (390.3236) were observed with deviations of 0.80 mmu (1.13 ppm), 0.85 mmu (1.20 ppm), 0.52 mmu (0.96 ppm) and 1.08 mmu (2.77 ppm), respectively (scan event 1) (Figures 2A and 2B), whereas [NDfrc-FC1 -Glc -TCA -H]- (552.3753) suggested as a fragment of NDfrc-FC1 was not detected (Table 1). The observed mass values and their respective mass deviations from the calculated values were summarized in Table 1.

Detection of NDfrc-FC2 and NDfrc-FC3

Figure 3 shows the results of screening for NDfrc-FC2 and NDfrc- FC3 in the corn powder extract. Using the same procedure as adopted for FC1, the existence of FC2 and FC3 was confirmed based on the full scan results (scan event 1) using the calculated mass of [FC2 -H]- (690.3706). A major peak corresponding to [FC2 -H]- was detected at 15.84 min, as shown in Figure 3A. As fragment ions of FC2, [FC2 -TCAK -H]- (532.3497) and [FC2 -2xTCAK -H]- (374.3287) were observed with deviations of 0.63 mmu (1.17 ppm) and 1.09 mmu (2.92 ppm), respectively (scan event 1) (Table 2). When the full-scan results (scan event 1) were scrutinized with the calculated mass of [NDfrc- FC2 -H]- (852.4235), a major peak was detected at 15.57 min (Figure 3A), and abundant [NDfrc-FC2 -H]- (852.4247) was detected with a mass deviation of 1.23 mmu (1.44 ppm) (Figure 3B). In addition, as the fragment ions of [NDfrc-FC2 -H]-, [NDfrc-FC2 -TCAK -H]- (694.4028), [NDfrc-FC2 -Glc -H]- (690.3716), [NDfrc-FC2 -TCAK -Glc -H]- (532.3496) were observed with deviations of 0.84 mmu (1.21 ppm), 0.95 mmu (1.38 ppm) and 0.50 mmu (0.95 ppm), respectively (scan event 1) (Figure 3B; Table 2). In contrast, one fragment ion [NDfrc-FC2 -2xTCAK -Glc -H]- (374.3281) was observed only in the scan event 2 with deviations of 0.54 mmu (1.45 ppm) (Table 2). In the case of screening for NDfrc-FC3, a major peak for [NDfrc-FC3 -H]- was observed at 14.79 min (scan event 1) (Figure 3A), and a fragmentation pattern similar to that of NDfrc-FC2 was also observed (details shown in Table 3). Based on the data described above, authors were convinced that both NDfrc-FC2 and NDfrc-FC3 were contained in the corn powder extract.

  FB2 (RT: 16.04 min) FC2 (RT: 15.87 min) NDfrc-FB2 (RT: 15.72 min) NDfrc-FC2 (RT: 15.57 min)
Formula Cal. Mass (m/z)a Obs. Mass (m/z)b Error (mmu(ppm)) Formula Cal. Mass (m/z)a Obs. Mass (m/z)b Error (mmu(ppm)) Formula Cal. Mass (m/z)a Obs. Mass (m/z)b Error (mmu(ppm)) Formula Cal. Mass (m/z)a Obs. Mass (m/z)b Error (mmu(ppm))
[TCA-H2O -H]- C6H6O5 157.014 157.014 -0.59 (-3.37) C6H6O5 157.014 157.014 -0.33 (-2.11) C6H6O5 157.014 157.014 -0.39 (-2.49) C6H6O5 157.014 157.014 -0.32 (-2.01)
([TCAK-H]-) 157.0136c -0.68 (-4.34) 157.0138c -0.51 (-3.27) 157.0136c -0.61 (-3.85) 157.0139c -0.39 (-2.49)
[M-2xTCAK-Glc-H]- - - - - - - - - C22H47NO4 388.343 388.344 0.40 (1.02) C21H45NO4 374.328 ND
388.3434c 0.21 (0.55) 374.3281c 0.54 (1.45)
[M-2xTCAK-H]- C22H47NO4 388.343 388.343 0.18 (0.47) C21H45NO4 374.328 374.329 1.09 (2.92) C28H57NO9 550.396 ND C27H55NO9 536.38 ND
388.3437c 0.43 (1.10) 374.3274c -0.19 (-0.51) ND ND
[M-TCAK-Glc-H]- - - - - - - - - C28H53NO9 546.365 546.365 0.42 (0.77) C27H51NO9 532.349 532.35 0.50 (0.95)
546.3652c 0.48 (0.88) 532.3500c 0.87 (1.63)
[M-TCAK-H]- C28H53NO9 546.365 546.365 0.23 (0.43) C27H51NO9 532.349 532.35 0.63 (1.17) C34H63NO14 708.418 708.418 0.82 (1.15) C33H61NO14 694.402 694.403 0.84 (1.21)
546.3651c 0.36 (0.65) 532.3494c 0.26 (0.49) 708.4182c 0.57 (0.81) 694.4030c 1.03 (1.48)
[M-Glc-H]- - - - - - - - - C34H59NO14 704.386 704.387 1.09 (1.15) C33H57NO14 690.371 690.372 0.95 (1.38)
704.3869c 0.62 (0.89) 690.3716c 0.95 (1.38)
[M-H]- C34H59NO14 704.386 704.386 -0.29 (-0.42) C33H57NO14 690.371 690.371 0.40 (0.59) C40H69NO19 866.439 866.44 0.60 (0.69) C39H67NO19 852.424 852.425 1.23 (1.44)
704.3868c 0.50 (0.71) 690.3717c 1.08 (1.56) 866.4402c 1.08 (1.25) 852.4258c 2.39 (2.80)

aMass values calculated based on elemental formulas, bMass values detected by full scan (scan event 1), cMass values detected with the all ions fragmentation with collision

Table 2: Exact mass values of FB2, FC2, NDfrc-FB2 and NDfrc-FC2 and relative fragment ions (calculated and observed) at negative polarity.

  FB3 (RT: 15.35 min) FC3 (RT: 15.02 min) NDfrc-FB3 (RT: 14.96 min) NDfrc-FC3 (RT: 14.79 min)
Formula Cal. Mass (m/z)a Obs. Mass (m/z)b Error (mmu(ppm)) Formula Cal. Mass (m/z)a Obs. Mass (m/z)b Error (mmu(ppm)) Formula Cal. Mass (m/z)a Obs. Mass (m/z)b Error (mmu(ppm)) Formula Cal. Mass (m/z)a Obs. Mass (m/z)b Error (mmu(ppm))
[TCA-H2O -H]- C6H6O5 157.0142 157.0138 -0.41 (-2.59) C6H6O5 157.0142 157.014 -0.24 (-1.52) C6H6O5 157.0142 157.014 -0.28 (-1.81) C6H6O5 157.0142 157.0139 -0.33 (-2.11)
([TCAK-H]-) 157.0137c -0.62 (-3.95) 157.0139c -0.32 (-2.01) 157.0138c -0.45 (-2.88) 157.0139c -0.36 (-2.30)
[M-2xTCAK-Glc-H]- - - - - - - - - C22H47NO4 388.3432 ND C21H45NO4 374.3276 ND
388.3445c 1.25 (3.22) ND
[M-2xTCAK-H]- C22H47NO4 388.3432 388.3435 0.30 (0.78) C21H45NO4 374.3276 374.3278 0.24 (0.64) C28H57NO9 550.3961 ND C27H55NO9 536.3804 ND
388.3430c -0.28 (-0.71) ND ND ND
[M-TCAK-Glc-H]- - - - - - - - - C28H53NO9 546.3648 546.3651 0.36 (0.65) C27H51NO9 532.3491 532.3487 -0.41 (-0.77)
546.3652c 1.03 (1.88) 532.3498c 0.69 (1.29)
[M-TCAK-H]- C28H53NO9 546.3648 546.3654  0.66 (1.21) C27H51NO9 532.3491 532.3497 0.57 (1.06) C34H63NO14 708.4176 708.4191 1.49 (2.10) C33H61NO14 694.4019 ND
546.3652c  0.48 (0.88) 532.3502c 1.05 (1.98) 708.4183c 0.69 (0.98) 694.4031c 1.15 (1.65)
[M-Glc-H]- - - - - - - - - C34H59NO14 704.3863 704.3873 1.05 (1.49) C33H57NO14 690.3706 690.3713 0.65 (0.94)
704.3876c 1.30 (1.84) 690.3731c 2.48 (3.59)
[M-H]- C34H59NO14 704.3863 704.3864  0.07 (0.11) C33H57NO14 690.3706 690.3719 1.26 (1.82) C40H69NO19 866.4391 866.4397 0.60 (0.69) C39H67NO19 852.4235 852.4247 1.23 (1.44)
704.3870c  0.75 (1.06) 690.3722c 1.56 (2.27) 866.4402c 1.08 (1.25) 852.4254c 1.96 (2.30)

aMass values calculated based on elemental formulas, bMass values detected by full scan (scan event 1), cMass values detected with the all ions fragmentation with collision energy (scan event 2)

Table 3: Exact mass values of FB3, FC3, NDfrc-FB3 and NDfrc-FC3 and relative fragment ions (calculated and observed) at negative polarity.

environmental-analytical-toxicology-Detection-NDfrc

Figure 3: Detection of NDfrc-FC2 and NDfrc-FC3 with scan results (scan event 1 and 2) (A) and full mass spectrum obtained at 15.60 min (scan event 1) (B).

Structure configuration

Figure 4 shows the LC-MS chromatograms of the NDfrc-FBs and NDfrc-FCs detected in the corn powder extract before and after treatment with OPA reagent (scan event 1). It was considered that OPA would not react with NDfrc-FBs and NDfrc-FCs. As shown in Figure 4, the signal intensities of FBs and FCs were significantly decreased (approximately one thousandth) by the OPA treatment, whereas those of NDfrc-FBs and NDfrc-FCs were not. The peak area ratios of NDfrc- FCs, before to after the OPA reaction, were 1.02-1.40. On the other hand, these ratios of FCs, before to after the OPA reaction were 837.9- 1836.0. These results indicated that the primary amine residue was occupied by glucose conjugation in the molecules of NDfrc-FCs.

environmental-analytical-toxicology-Detection-NDfrc

Figure 4: Chromatograms of NDfrc-FBs, FBs, NDfrc-FCs and FCs in corn powder extract before and after the treatment with OPA reagent (scan event 1).

For confirmation whether the fumonisin residue structures was contained in the detected compound’s molecule or not, the residue obtained through the strong anion exchange column was further purified with commercially available IACs, and subjected to LC-MS analysis. Figure 5 shows the LC-MS chromatograms of the purified residues through the IACs at scan event 1. As shown in Figure 5, similar peaks as observed in Figures 2A and 3A were equally observed even after the purification with three different IACs. These results supports that the targeted compound’s detected in this study should have fumonisin residue structures in their molecules, suggesting that NDfrc-FCs as well as FCs were detected in the corn powder reference material.

environmental-analytical-toxicology-Purified-fumonisins

Figure 5: Purified fumonisins by anion exchange (QMA) and immuno affinity columns.

Disscusion

This research is the first report on the detection of naturallyoccurring NDfrc-FC1, FC2, and FC3 in corn powder. Poling et al. suggested that NDfrc-FBs were formed through a non-enzymatic reaction between FBs and glucose [15,17]. Hence, authors presumed that FCs would also react with D-glucose non-enzymatically to form glucose conjugates such as NDfrc-FCs. The earlier elution of NDfrc- FCs from the C18 column compared with FCs suggested that NDfrc- FCs were more polar than FCs by the glucose conjugation. Although this conjugation is similar to Maillard reaction [21,22], NDfrc-FB1 was reported as not to be reduced to FB1, in contrast to Maillard reaction products of amino acid and D-glucose [23].

With the alkali treatment, TCAs residues in FB molecules are suggested to be cleaved, releasing linear structure (hydrolyzed FB (HFB)) molecules. Therefore, fumonisin derivatives via TCA residues such as Lysil-FB, FB+methoxyglucose [24] and bound fumonisin [25] are considered to be summarized to HFB by the alkali treatment. Although the use of HFB obtained with either alkaline or enzymatic treatment has been proposed for quantitation of the total fumonisins in starch [24], NDfrc-FB1 could not be determined with these methods. Accordingly, NDfrc-FCs would not be summarized to hydrolyzed fumonisins, either.

Supposing that ionization efficiency of each type of fumonisin with LC-MS in this study was equivalent, the amount of NDfrc-FBs was estimated as approximately 3.9~4.8% of FBs. On the one hand, the amount of NDfrc-FCs was estimated as approximately 11.9~19.4% of FCs (Figure 4). According to the fumonisin biosynthesis pathway [26], FB structures are constructed through a conjugation with alanine, whereas FCs are constructed with glycine [27]. For the Maillard reaction efficiency of alanine and glycine, it was reported as that glycine was more responsive than alanine with glucose [28]. Hence, it was suggested that FCs was more responsive with glucose than FBs.

From the standpoint of toxicity, NDfrc-FB1 has been reported as less toxic, compared to FB1 [29]. N-substituted fumonisins such as FA1 and N-Carboxymethyl-FB1 [30] have been reported as less toxic, compared to FB1. Therefore, the primary amine residue in the fumonisin molecule appears to be necessary for its biological activity. Although FC1 has been reported to exhibit phytotoxicity and cytopathy at concentrations similar to those of FB1 [19], NDfrc-FCs are suggested as less toxic compared to FCs, from the above considereation. On the other hand, it was also reported that NDfrc-FB1 was partly reduced to FB1 in the gastrointestinal tract of rats [16]. Therefore, NDfrc-FCs would be possibly reduced to FCs in the gastrointestinal tract.

Although FBs are the most abundant naturally occurring fumonisins, a part of F. verticillioides produce secondarily FCs as well as FBs [31], whereas a part of F. oxysporum strains were reported to produce FCs more than FBs [27,32]. In addition, F. oxysporum strains are often reported to cause serious diseases for various vegetables [33]. From these observations, authors insist the need for the extensive analysis of FCs and NDfrc-FCs.

Acknowledgements

A part of this work was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Research project for improving food safety and animal health). Authors thank Dr. Masayo Kushiro for suggestions and editing.

References

Citation: Matsuo Y, Takahara K, Hatabayashi H, Nakagawa H (2017) Detection of N-(1-deoxy-D-fructos-1-yl) Fumonisin C1, C2 and C3 in Corn Powder by LC - Orbitrap MS. J Environ Anal Toxicol 7: 536. DOI: 10.4172/2161-0525.1000536

Copyright: © 2017 Matsuo Y, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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