Accurate Identification of Degraded Products of Aflatoxin B1 Under UV Irradiation Based on UPLC-Q-TOF-MS/MS and NMR Analysis

Analysis, purification, and characterization of AFB1 degraded products are vital steps for elucidation of the photocatalytic mechanism. In this report, the UPLC-Q-TOF-MS/MS technique was first coupled with purification and NMR spectral approaches to analyze and characterize degraded products of AFB1 photocatalyzed under UV irradiation. A total of seventeen degraded products were characterized based on the UPLC-Q-TOF-MS/MS analysis, in which seven ones (1–7) including four (stereo) isomers (1,2, 5, and 6) were purified and elucidated by NMR experiments. According to the structural features of AFB1 and degraded products (1–7), the possible photocatalytic mechanisms were suggested. Furthermore, AFB1 and degraded products (1–7) were evaluated against different cell lines. The results indicated that the UPLC-Q-TOF-MS/MS technique combined with purification, NMR spectral experiments, and biological tests was an applicable integrated approach for analysis, characterization, and toxic evaluation of degraded products of AFB1, which could be used to evaluate other mycotoxin degradation processes.


INTRODUCTION
Aflatoxins (AFBs), a group of mycotoxins (including AFB 1 , AFB 2 , AFBG 1 , AFG 2 , and other derivatives) with highly toxic, mutagenic, and carcinogenic activities, are mainly produced by Aspergillus flavus and A. parasiticus (Massey et al., 1995;Rustom, 1997). These two fungi could infect plants, grains, food, and animals which could lead to significant food safety problems and economic losses. The core skeleton of AFBs is dihydrofuro [2,3-b]furan combined with a coumarin ring, in which the double bond on the furan ring is the key toxic group. The double bond (C-8/C-9) could be transformed to epoxide in the human body, which then quickly combines with DNA, glutathione S-transferase, or N7 guanine to form highly toxic adducts (Garner et al., 1971;Essigmann et al., 1977;Lin et al., 1977;Croy et al., 1978).
Aflatoxin B 1 is the most notorious type with potential teratogenic, mutagenic, and hepatocarcinogenic toxicity, and it is classified as a group I carcinogen by the International Agency for Research in Cancer (IARC) (Cancer, 1993). Thus, degradation or reduction of AFB 1 becomes a hot spot worldwide. Diverse approaches including physical, chemical, and biological methods are used to degrade or reduce AFBs (Alberts et al., 2009;Mendez-Albores et al., 2009;Liu et al., 2011;Luo et al., 2014;Kumar et al., 2017;Peng et al., 2018). Physical methods mainly include high temperature, irradiation, adsorption, and ultrasonic methods, among which UV irradiation is often employed as an effective method to degrade or reduce AFBs based on the photosensitive characteristics (Calado et al., 2014). Liu investigated the photodegradation of AFB 1 in water/ acetonitrile solution and characterized three degraded products based on UPLC-Q-TOF MS data . Later, they analyzed AFB 1 photodegradation in peanut oil under UV irradiation and concluded that the mutagenic effects of UVtreated samples were completely lost compared with those of untreated samples (Liu et al., 2011). Mao analyzed the degraded products of AFB 1 in peanut oil using the UPLC-Q-TOF-MS/MS technique (Mao et al., 2016). Wang investigated the degraded products using the LC-MS/MS approach and postulated toxicity of AFB 1 in methanol-water solution irradiated with Co 60 gamma-rays . Recently, Li's group investigated the photodegraded inactivation mechanism of the hypertoxic site in aflatoxin B 1 by HPLC-MS (Mao et al., 2019).
Obtaining pure AFB 1 -degraded products and elucidating their structures are very important to establish the photodegradation mechanism and toxic evaluation. Usually, due to limited amounts, purification of AFB 1 -degraded products was significantly difficult. Thus, most mycotoxin-degraded products were mainly characterized by LC-MS/MS techniques without further separation. The LC-MS/MS technique is a high-efficient and sensitive approach for analysis and structural characterization of different metabolites in mixtures, which is often used to dereplicate or detect new compounds from extracts or characterize mycotoxindegraded products. Yet, this technique could not differentiate (stereo) isomers easily. The nuclear magnetic resonance (NMR) spectral technique is a standard and universal approach for structural elucidation (Wang et al., 2018;Song et al., 2019;Li et al., 2020;Wang et al., 2020). In this study, UPLC-Q-TOF-MS/ MS analysis combined with purification and NMR spectral experiments was used to characterize AFB 1 -degraded products under UV irradiation. The possible photocatalytic mechanism was elucidated, and toxicities of AFB 1 and degraded products (1-7) were evaluated, which provided a thought for other mycotoxin degradation processes.

Chemicals and Reagents
Aflatoxin B 1 was purchased from Pribolab (Qingdao, China). Chromatographic-grade methanol and acetone were obtained from Tianjin Saifu Rui Technology Company (Tianjin, China). Analytical-grade methanol, acetone, and DMSO were obtained from Chron Chemicals (Chengdu, China). For NMR analysis, all deuterium reagents were purchased from Sigma (St. Louis, MO, USA).
Standard solutions of AFB 1 were placed in a 2-ml sealed centrifugal tube, prepared in methanol-DMSO (9:1 v/v), and fully dissolved in methanol using an ultrasound device from Beijing Tianlin Hengtai Technology Company (Beijing, China), and then, it was submitted to be degraded.

UV Irradiation
To investigate the degradation of AFB 1 , a UV lamp (20 W, 72 μws/cm 2 , GGZ250-1, Shanghai Jiming Special Lighting Appliance Factory) at 365 nm wavelength was used to perform the irradiation experiments. 18 mg of pure AFB 1 was added to acetone solvent, and 10 mg of pure AFB 1 was added to methanol solvent, and both of them were placed in a sealed centrifugal tube and illuminated at room temperature for 45 h .

UPLC-Q-TOF MS Analysis
AFB 1 and degraded products were analyzed using a UPLC-Q-TOF-MS/MS system (Waters, United States). Chromatographic analysis was carried out with a Waters Acquity UPLC-PDA system equipped with an analytical reverse-phase C-18 column (2.1 × 100 mm, 1.7 μm, Acquity BEH, Waters, United States) with an absorbance range of 200-400 nm. The column temperature was maintained at 40°C. 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) were used as the mobile phase. The gradient conditions were as follows: 0-10 min, 10 %-60% B; 10-12.5 min, 60 %-95% B; and 12.6-15 min, 10% B. The flow rate from the UPLC system into the ESI-Q-TOF-MS detector was 0.3 ml/min. The auto-injected volume was 3 μl. Time-of-flight MS detection was performed with a Waters SYNAPT G2 HDMS (Waters Corp., Manchester, United Kingdom) TOF mass spectrometer combined with an ESI source in the positive ion scan mode. The desolvation temperature was set at 400°C with desolvation gas flow at 600 L/h, and the source temperature was 100°C. The lock mass in all analyses was leucine-enkephalin ([M + H] + 556.2771), used at a concentration of 0.5 g/ml and infused at a flow rate of 10 L/min. Raw data were acquired using the centroid mode, and the mass range was set from m/ z 50 to 1200. The capillary voltage was set at 3.0 kV with 40 and 4.0 V of the sample and extraction cone voltage. The collision energy was set as 20 eV for low-energy scan and a ramp from 20 to 30 eV for high-energy scan. The instrument was controlled by MassLynx 4.1 software.

Toxic Evaluation of Degraded Products and AFB 1
All the degraded products and AFB 1 were tested for their cytotoxicity against human normal hepatocytes LO-2 and cancer cell lines Hep-G2 and MCF-7. Cells were incubated in a DMEM high glucose medium (Gibco, USA), added with 10% fetal bovine serum (Gibco, United States) and cultured in a 5% CO 2 incubator at 37°C. The cytotoxicity tests were performed using the MTS (Promega, United States) (Ahmed et al., 2019).

UPLC-Q-TOF-MS/MS Base Peak Intensity and the UPLC Chromatogram of Degraded Products
The UPLC-Q-TOF-MS/MS BPI of AFB 1 and its degraded products in methanol-H 2 O and acetone-H 2 O solvents are shown in Figures 1 and 2. The retention time and molecular weight of AFB 1 were 6.09 min and m/z 313 ([M+1]), respectively. A series of degraded products with different retention times (RTs) and molecular weights are shown in Table 1. Some ion peaks as (stereo) isomers possessed the same molecular weights (such as m/z 345) but with different RTs.

Structural Analysis of Degraded Products Based on Exact Molecular Weights and Fragment Ions
Different free radicals such as reactive hydroxyl (OH • ), hydrated electrons (eaq − ), hydrogen atoms (H • ), and methoxy species (OCH 3 • ) were produced when methanol-H 2 O and acetone-H 2 O solvents were irradiated under UV (White, 2001;Azrague et al., 2005). These free radicals could attack the AFB 1 structure to form different degraded products. The double bond C 8 -C 9 in AFB 1 was broken easily by these free radicals via addition reactions. Ten and seven main degraded products in methanol-H 2 O and acetone-H 2 O solvents were characterized based on molecular weights and fragment ions of compounds (Supplementary Tables S1, S2). The other degraded product fragmentation rules are provided in supporting information, considering a similar fragmentation pathway with AFB 1 (Figure 3 and Supplementary Figures S2-S9).
Four ions as (stereo) isomers (m/z 345, C 18 H 16 O 7 ) appeared at t R 4.70, 5.40, 5.84 and 5.99 min in methanol-H 2 O solvent with 32 Da (CH 4 O) more than that of AFB 1 (Supplementary Figure  S2). After a neutral loss of CH 3 OH from ion (m/z 345), the fragmentation pathways of these four ions were nearly the same as those of AFB 1 . It is suggested that these four degraded compounds might be addition products of CH 3 OH with AFB 1  Figure S3), suggesting one more oxygen atom connected on C 8 or C 9 . Both of them were suggested to be the addition products from free radical hydrogen atoms (OH • ) and methoxy species (OCH 3 • ) with C 8 / C 9 or C 9 /C 8 of AFB 1 under UV irradiation. The possible fragmentation pathway of the ion (m/z 361) is shown in Supplementary Figure S3.
Three ions at m/z 359 ([M+1], t R 4.94, 7.08 and 7.30 min) gave the molecular formula as C 19 H 18 O 7 based on HR-ESI-MS. The neutral loss of -CO, -CH 3 OH, and -C 2 H 2 was observed in the MS/MS profiles (Supplementary Figure S4). The possible structure and fragmentation pathway of these three ions is suggested in Supplementary Figure S4.
The molecular formula of the degraded product at m/z 391 ([M+1], t R 6.41 min) was determined to be C 20 H 22 O 8 based on HR-ESI-MS (Supplementary Figure S5). Sequential losses of two -CH 3 OH (391→359→327), one -CH 2 O (327→297), and one -CH 2 (297→283) implied that four methoxyls might be present in degraded products. Two methoxyls might be   connected on C-8/C-9 and the keto-carboxyl group might be transformed to another methoxyl through reduction and addition reactions, and the remaining -OMe was anchored on the aromatic ring. The possible fragmentation pathway of these three ions is suggested in Supplementary Figure S5. The molecular formula of ions at m/z 331 ([M+1, C 17 H 14 O 7 ] in acetone-H 2 O at 4.06 and 4.18 min) possessed 18 Da (H 2 O) more than that of AFB 1 , which indicated these two degraded products were (stereo) isomers (Supplementary Figure S6). The fragmentation pathways of these two ions were nearly the same as those of AFB 1 after the loss of a molecule of H 2 O, which implied that two degraded products were the adducts of H 2 O with the double bond C-8/C-9. Though the molecular formulas and fragmentation pathways of these two degraded products were the same, the retention time and abundance of fragment ions were different. A higher abundance of ion at m/z 313 (t R 4.18 min) was observed than the other (t R 4.06 min). This suggested that the position of OH on the furan ring was different in two degraded products. The possible fragmentation pathway of two ions is suggested in Supplementary Figure S6.
The molecular formula of the ion at m/z 347 ([M+1], t R 3.48 min) was determined to be C 18 H 14 O 8 based on HR-ESI-MS with 16 Da (O) more than that of degraded products (m/z 331), indicating two hydroxyl groups connected on C 8 and C 9 , respectively (Supplementary Figure S7). The possible fragmentation pathway is suggested in Supplementary Figure S7.
The molecular formula of the ion at m/z 371 ([M+1], t R 4.34 and 5.63 min) was determined to be C 20 H 18 O 7 based on HR-ESI-MS with 58 Da (CH 3 COCH 3 ) more than that of AFB 1 (m/z 313) (Supplementary Figure S8), which implied that one molecule of acetone attacked on C-8 or C-9 under UV irradiation. The possible fragmentation pathway of them is suggested in Supplementary Figure S8.
The molecular formulas of ions at m/z 401 ([M+1], t R 4.86 and 6.54 min) were determined to be C 21 H 20 O 8 based on HR-ESI-MS. The loss of 32 Da from m/z 401 to m/z 369 and the loss of 58 Da from m/z 401 to m/z 343 suggested that a methoxyl and acetone were connected on C-8/C-9 or C-9/C-8 (Supplementary Figure S9). The possible fragmentation pathway of these two ions is suggested in Supplementary Figure S9.
Though seventeen degraded products were characterized by molecular formula and fragment ions, the planar structures and configurations of some degraded products could not be

Purification and Elucidation of Seven Degraded Products Structures
Seven main degraded products with limited amounts were purified by HPLC and then elucidated by NMR spectra (Figure 4). Compounds 1-3 were isolated as the photochemical adducts of 6-methoxydifurocoumarone, which were analyzed based on the 1 H-NMR spectrum (Waiss and Wiley, 1969). In this study, the structures of these three degraded products were elucidated in detail by analyzing 1 H, 13 C, and 2D-NMR spectra ( Figure 5). The 1 H-NMR data of 1-3 and 13 C-NMR data of 1 and 3 are shown in Table 2 and Table 3. The relative configurations of 1 and 3 were determined by NOESY correlations ( Figure 5). Compound 4 was a new degraded product isolated from methanol solution. The molecular formula of 4 was determined to be C 18 H 17 O 8 on the basis of HR-ESI-MS with 16 more daltons than that of 1, implying that an additional hydroxyl group was present in 4, which was supported by the NMR spectra ( Table 2 and Table 3). The 1 H-1 H COSY and HMBC correlations confirmed that the additional hydroxyl group was connected on C-9 ( Figure 5). The NOESY correlations determined the relative configuration of 8-OMe and 9-OH to be β and α configuration, respectively ( Figure 5). Compounds 5 and 6 were obtained as an inseparable mixture through HPLC with various stationary and mobile phases, whereas well-resolved NMR spectra determined the structures of 5 and 6 as isomers. The 1 H and 13 C spectra data of 5 were reported, and 6 was a new degraded product reported for the first time (Cox and Cole, 1977;Wang et al., 2011;Wang et al., 2012;Stanley et al., 2020). The molecular formula of 5 and 6 was determined to be C 17 H 14 O 7 on the basis of HR-ESI-MS, with 18 more daltons than that of AFB 1 , implying that 5 and 6 might be transformed from AFB 1 through an addition reaction with H 2 O on the double bond (C-8/C-9). The planar and relative configurations of 5 and 6 were established based on 2D-NMR data ( Figure 5). Compound 7 was a new degraded product isolated from acetone solvent. The molecular formula of 7 was established to be C 20 H 19 O 7 based on HR-ESI-MS. In the 1 H NMR spectrum, an additional methyl (δ H 2.12 ppm) and an additional methylene unit (δ H 2.72 ppm) were observed compared with that of AFB 1 , which indicated that one molecule of acetone might be connected on C-8 or C-9. The 1 H-1 H COSY and HMBC correlations confirmed that the acetonyl group was connected with C-9 ( Figure 5). The NOESY correlations from H-9a (δ H 3.93 ppm) to H-1' (δ H 2.72 ppm) determined the acetonyl group to be α-configuration ( Figure 5). Considering that the stereochemistry of C-6a and C-9a were not changed in the photocatalytic reaction, the absolute configurations of (1-7) are shown in Figure 4.

Elucidation of the Photodegraded Mechanism of Degraded Products
According to the structural features of AFB 1 and degraded products (1-7), the possible photocatalytic reactions were suggested: 1) addition reactions happened between MeOH, H 2 O, or acetone with AFB 1 under UV irradiation to produce compounds such as 1-3 and 5-7; 2) compound 4 might be originated from the oxygen free radical attacking the double bond (C-8/C-9) to form an epoxide, which was further attacked by OMe • or OH • (Figure 6) (Waiss and Wiley, 1969;Iyer et al., 1994). The photocatalytic mechanism was suggested: MeOH, H 2 O, or CH 3 COCH 3 formed potential free radicals (H • , OH • , OMe • , or CH 3 COCH 2 • ) under UV irradiation. Then, H • attacked on the double bond (C-8 or C-9) leading to form carbon-free radicals, which was then coupled with OH • , OMe • , or CH 3 COCH 2 • to shape degraded products 1-3 and 5-7 (Waiss and  , 1969;Iyer et al., 1994;Jamil et al., 2017;Mao et al., 2019). In addition, O 2 in the air under UV irradiation could form O 2
From the structural features of degraded products (1-7), an interesting phenomenon was also observed that the group of C-9 (in 3, 4, 6, and 7) was α-configuration, whereas the group of C-8 in 1 and 2 was αor β-configuration. This demonstrated that steric hindrance (from right part of AFB 1 structure) might exist and prevent different groups (OH • , OMe • , or CH 3 COCH 2 • ) attacking C-9 from the positive face (β-position), whereas C-8 could be attacked from two sides (α-or β-configuration) without steric hindrance. The crystal structure of AFB 1 (Cheung and Sim, 1964;van Soest and Peerdeman, 1970) revealed that the right part of the AFB 1 structure was indeed closer to C-9 than C-8 in space, which might preclude different groups to attack C-9 from the FIGURE 6 | Possibly photocatalytic mechanism of AFB 1 in MeOH and acetone.
FIGURE 7 | Possible catalytic reaction happened at C-8 and C-9 positive face (β-position) due to spatial hindrance. The photocatalytic reactions are depicted in Figure 7.

Toxic Evaluation of Degraded Products
The cytotoxicity of AFB 1 and seven degraded products (1-7) was evaluated against human normal hepatocytes LO-2 and cancer cell lines Hep-G2 and MCF-7 using the MTS method, with cis-platinum as the positive control; the results are shown in Table 4. AFB 1 displayed stronger cytotoxicity to three cell lines than the degraded products, further supporting that the double bond (C-8/C-9) in the furan ring was the key toxic group, and the toxicity was markedly reduced after the double bond was broken.

CONCLUSION
In this work, the degraded products of AFB 1 under UV irradiation were analyzed through UPLC-Q-TOF-MS/MS, and seventeen degraded products were characterized. Seven degraded products were purified and elucidated by NMR experiments. The double bond (C-8/C-9) of all degraded products was broken, which was coupled with different groups such as OH • , H • , and OCH 3 • through addition reactions under UV irradiation. The cytotoxic evaluation revealed that the toxicity of AFB 1 -degraded products was markedly reduced after their double bond in the furan ring was cleaved. The results demonstrated that the UPLC-Q-TOF-MS/MS technique coupled with purification NMR analysis and biological tests was an applicably integrated approach for the analysis, characterization, and toxic evaluation of degraded products of AFB 1 , which can also be used to evaluate other mycotoxin degradation processes.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.