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 AFB1, AFB2, AFBG1, AFG2, 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 AFB-8,9-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 B1 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 AFB1 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., 2010; 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 AFB1 in water/acetonitrile solution and characterized three degraded products based on UPLC-Q-TOF MS data (Liu et al., 2010). Later, they analyzed AFB1 photodegradation in peanut oil under UV irradiation and concluded that the mutagenic effects of UV-treated samples were completely lost compared with those of untreated samples (Liu et al., 2011). Mao analyzed the degraded products of AFB1 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 AFB1 in methanol–water solution irradiated with Co60 gamma-rays (Wang et al., 2011). Recently, Li’s group investigated the photodegraded inactivation mechanism of the hypertoxic site in aflatoxin B1 by HPLC-MS (Mao et al., 2019).

Obtaining pure AFB1-degraded products and elucidating their structures are very important to establish the photodegradation mechanism and toxic evaluation. Usually, due to limited amounts, purification of AFB1-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 mycotoxin-degraded 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 AFB1-degraded products under UV irradiation. The possible photocatalytic mechanism was elucidated, and toxicities of AFB1 and degraded products (1–7) were evaluated, which provided a thought for other mycotoxin degradation processes.

Experimental

Chemicals and Reagents

Aflatoxin B1 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 AFB1 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 AFB1, a UV lamp (20 W, 72 μws/cm2, GGZ250-1, Shanghai Jiming Special Lighting Appliance Factory) at 365 nm wavelength was used to perform the irradiation experiments. 18 mg of pure AFB1 was added to acetone solvent, and 10 mg of pure AFB1 was added to methanol solvent, and both of them were placed in a sealed centrifugal tube and illuminated at room temperature for 45 h (Liu et al., 2010).

HPLC Operation

The degraded products were analyzed and isolated by semipreparative HPLC on SEP LC-52 with an MWD UV detector (Separation (Beijing) Technology Co. Ltd., Beijing, China) using a 250 mm × 10 mm i. d., 5 μm, ODS-A column (YMC, Kyoto, Japan). The mixture in methanol was purified by semipreparative HPLC (55–60% CH3OH in H2O, v/v, 2 ml/min, 30 min) and yielded 4 (0.6 mg, t R = 18.6 min), 3 (0.5 mg, t R = 22.3 min), 1 (0.5 mg, t R = 23.8 min), and 2 (0.4 mg, t R = 27.0 min), respectively. The mixture in acetone was isolated by semipreparative HPLC (40% CH3OH in H2O, v/v, 2 ml/min, 3 min; 40–100% CH3OH in H2O, v/v, 2 ml/min, 20 min) and yielded 5, 6 (7.0 mg, t R = 18.7 min), and 7 (1.5 mg, t R = 21.2 min).

Determination of Degraded Products

The degraded products were identified by NMR experiments. Compounds were analyzed by UPLC-Q-TOF-MS/MS in positive ion mode. 1D and 2D-NMR spectra were acquired using solvent signals (CD3OD: δ H 3.31/δ C 49.9; C3D6O: δ H 2.05/δ C 49.9; Pyridine-d 5: δ H 8.74, 7.58, 7.22/δ C 150.4, 135.9, and 123.9) on a Bruker 600 spectrometer (1H: 600 MHz) and a Bruker Avance III 500 spectrometer (1H: 500 MHz; 13C: 125 MHz) (Bruker, Rheinstetten, Germany).

UPLC-Q-TOF MS Analysis

AFB1 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 AFB1

All the degraded products and AFB1 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% CO2 incubator at 37°C. The cytotoxicity tests were performed using the MTS (Promega, United States) (Ahmed et al., 2019).

Results and Discussion

UPLC-Q-TOF-MS/MS Base Peak Intensity and the UPLC Chromatogram of Degraded Products

The UPLC-Q-TOF-MS/MS BPI of AFB1 and its degraded products in methanol–H2O and acetone–H2O solvents are shown in Figures 1 and 2. The retention time and molecular weight of AFB1 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.

FIGURE 1

UPLC-Q-TOF-MS/MS profiles of the degradation products of aflatoxin B1 in methanol solvent. (A) Base peak intensity (BPI) of aflatoxin B1. (B) Base peak intensity (BPI) of degradation products of aflatoxin B1.

FIGURE 2

UPLC-Q-TOF-MS/MS profiles of the degradation products of aflatoxin B1 in acetone solvent. (A) Base peak intensity (BPI) of aflatoxin B1. (B) Base peak intensity (BPI) of degradation products of aflatoxin B1.

TABLE 1

HR-ESI and MS/MS data of seventeen degraded products and aflatoxin B1.

Structure		Retention time (min)	Extract Mass (m/z)	Formula	Diff (ppm)	Loss mass	Loss formula
		3.48	347.0758	C17H15O8	−2.6
			329.0648	C17H13O7	−4.0	18.0110	[M + H]+-H2O
			319.0811	C16H15O7	−2.2	27.9947	[M + H]+-CO
			311.0538	C17H11O6	−5.8	36.0220	[M + H]+-H2O-H2O
			301.0697	C16H13O6	−5.0	46.0062	[M + H]+-CO-H2O
			283.0595	C16H11O5	−3.9	64.0163	[M + H]+-H2O-H2O-CO
			273.0747	C15H13O5	−5.9	74.0008	[M + H]+-CO-H2O-CO
			271.0595	C15H11O5	−4.1	76.0163	[M + H]+-CO-H2O-HCHO
		4.06	331.0801	C17H15O7	−5.4
		4.18	313.0697	C17H13O6	−4.8	18.0104	[M + H]+-H2O
			301.0699	C16H13O6	−4.3	30.0102	[M + H]+-HCHO
			285.0746	C16H13O5	−6.0	46.0055	[M + H]+-H2O-CO
			283.0598	C16H11O5	−2.8	48.0203	[M + H]+-CH2O-H2O
			273.0378	C14H9O6	−7.7	58.0423	[M + H]+- CH2O-CO
		4.47	361.0905	C18H17O8	−5.0
		4.82	343.0799	C18H15O7	−5.5	18.0106	[M + H]+-H2O
			329.0643	C17H13O7	−5.5	32.0262	[M + H]+-CH3OH
			315.0858	C17H15O6	−3.5	46.0047	[M + H]+-H2O-CO
			311.0542	C17H11O6	−4.5	50.0363	[M + H]+-H2O-CH3OH
			301.0698	C16H13O6	−4.7	60.0207	[M + H]+-CH3OH-CO
			283.0588	C16H11O5	−6.4	78.0317	[M + H]+-CH3OH-CO-H2O
			273.0753	C15H13O5	−3.7	88.0152	[M + H]+-CH3OH-CO-CO
			255.0646	C15H11O4	−4.3	106.0259	[M + H]+-CH3OH-CO-H2O-CO
		4.86	401.1224	C21H21O8	−3.0
			369.0967	C20H17O7	−1.9	32.0257	[M + H]+-CH3OH
			343.0804	C18H15O7	−4.1	58.0420	[M + H]+-CH3COCH3
			315.0851	C17H15O6	−5.7	86.0373	[M + H]+-CH3COCH3-CO
			313.0697	C17H13O6	−4.8	88.0527	[M + H]+-CH3COCH3-CH2O
		6.53					[M + H]+-CH3OH-C3H4O
			287.0538	C15H11O6	−6.3	114.0686	[M + H]+-CH3COCH3-CO-CO
			285.0747	C16H13O5	−5.6	116.0477	[M + H]+-CH3COCH3-CO-CH2O
			283.0601	C16H11O5	−1.8	118.0623	[M + H]+-CH3COCH3-CH2O-CH2O
							[M + H]+-CH3OH-C3H4O-CH2O
		4.345.63	371.1120	C20H19O7	−3.0
			313.0698	C17H13O6	−4.5	58.0422	[M + H]+-CH3COCH3
			285.0751	C16H13O5	−4.2	86.0369	[M + H]+-CH3COCH3-CO
			257.0796	C15H13O4	−7.0	114.0324	[M + H]+-CH3COCH3-CO-CO
		4.70	345.0971	C18H17O7	−0.9
		5.40	313.0721	C17H13O6	2.0	32.0250	[M + H]+-CH3OH
		5.84	285.0751	C16H13O5	−4.2	60.0220	[M + H]+-CH3OH-CO
		5.99	269.0802	C16H13O4	−4.5	76.0169	[M + H]+-CH3OH-CO2
			257.0794	C15H13O4	−7.8	88.0171	[M + H]+-CH3OH-CO-CO
			243.0647	C14H11O4	−4.1	102.0324	[M + H]+-CH3OH-CO-CO-CH2
			241.0846	C15H13O3	−7.9	104.0125	[M + H]+-CH3OH-CO2-CO
		6.09	313.0705	C17H13O6	−2.2	27.9955	[M + H]+-CO
		AFB1	285.0750	C16H13O5	−4.6	43.9900	[M + H]+-CO2
			269.0805	C16H13O4	−3.3	55.9912	[M + H]+-CO-CO
			257.0793	C15H13O4	−8.2	72.0211	[M + H]+-CO2-CO
			241.0494	C14H9O4	−2.9	83.9857	[M + H]+-CO-CO-CO
			229.0848	C14H13O3	−1.7
		6.41	391.1380	C20H23O8	−3.3
			359.1111	C19H19O7	−5.6	32.0269	[M + H]+-CH3OH
			345.0955	C18H17O7	−5.5	46.0425	[M + H]+-CH3OH-CH2
			327.0852	C18H15O6	−5.2	64.0528	[M + H]+-CH3OH-CH3OH
			313.0696	C17H13O6	−5.1	78.0684	[M + H]+-CH3OH-CH2-CH3OH
			297.0749	C17H13O5	−4.7	94.0631	[M + H]+-CH3OH-CH3OH-CH2O
			285.0754	C16H13O5	−3.2	106.0626	[M + H]+-CH3OH-CH2-CH3OH-CO
			283.0597	C16H11O5	−3.2	108.0783	[M + H]+-CH3OH-CH3OH-CH2O-CH2
			255.0653	C15H11O4	−1.6	136.0727	[M + H]+-CH3OH-CH2-CH3OH-CO
							[M + H]+-CH3OH-CH3OH-CH2O-CH2-CO
		4.93	359.1121	C19H19O7	−2.8
		7.30	345.0957	C18H17O7	−4.9	14.0164	[M + H]+-CH2
			327.0856	C18H15O6	−4.0	32.0265	[M + H]+-CH3OH
			313.0693	C17H13O6	−6.1	46.0428	[M + H]+-CH2-CH3OH
			301.0697	C16H13O6	−5.0	58.0424	[M + H]+-CH3OH-C2H2
			299.0904	C17H15O5	−5.0	60.0217	[M + H]+-CH3OH-CO
			287.0564	C15H11O6	2.8	72.0557	[M + H]+-CH3OH-C2H2-CH2
			273.0746	C15H13O5	−6.2	86.0375	[M + H]+-CH3OH-C2H2-CO
			259.0594	C14H11O5	−4.6	100.0527	[M + H]+-CH3COCH3-CO-CO

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 (OCH3 •) were produced when methanol–H2O and acetone–H2O solvents were irradiated under UV (White, 2001; Azrague et al., 2005). These free radicals could attack the AFB1 structure to form different degraded products. The double bond C8-C9 in AFB1 was broken easily by these free radicals via addition reactions. Ten and seven main degraded products in methanol–H2O and acetone–H2O 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 AFB1 (Figure 3 and Supplementary Figures S2–S9).

FIGURE 3

Fragmentation pathway of AFB1.

Four ions as (stereo) isomers (m/z 345, C18H16O7) appeared at t R = 4.70, 5.40, 5.84 and 5.99 min in methanol–H2O solvent with 32 Da (CH4O) more than that of AFB1 (Supplementary Figure S2). After a neutral loss of CH3OH from ion (m/z 345), the fragmentation pathways of these four ions were nearly the same as those of AFB1. It is suggested that these four degraded compounds might be addition products of CH3OH with AFB1 at C-8/C-9. The possible fragmentation pathways of these four (stereo) isomers are depicted in Supplementary Figure S2.

The molecular formula of the ion at m/z 361 ([M+1], t R = 4.47 and 4.82 min) was determined to be C18H16O8 based on HR-ESI-MS with 16 Da (O) more than that of degraded products (m/z 345) (Supplementary Figure S3), suggesting one more oxygen atom connected on C8 or C9. Both of them were suggested to be the addition products from free radical hydrogen atoms (OH•) and methoxy species (OCH3 •) with C8/C9 or C9/C8 of AFB1 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 C19H18O7 based on HR-ESI-MS. The neutral loss of -CO, -CH3OH, and -C2H2 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 C20H22O8 based on HR-ESI-MS (Supplementary Figure S5). Sequential losses of two -CH3OH (391→359→327), one -CH2O (327→297), and one -CH2 (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, C17H14O7] in acetone–H2O at 4.06 and 4.18 min) possessed 18 Da (H2O) more than that of AFB1, 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 AFB1 after the loss of a molecule of H2O, which implied that two degraded products were the adducts of H2O 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 C18H14O8 based on HR-ESI-MS with 16 Da (O) more than that of degraded products (m/z 331), indicating two hydroxyl groups connected on C8 and C9, 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 C20H18O7 based on HR-ESI-MS with 58 Da (CH3COCH3) more than that of AFB1 (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 C21H20O8 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 determined only based on UPLC-Q-TOF-MS/MS analysis. Thus, further purification and NMR experiments are needed to elucidate their structures and stereochemistry.

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 1H-NMR spectrum (Waiss and Wiley, 1969). In this study, the structures of these three degraded products were elucidated in detail by analyzing 1H, 13C, and 2D-NMR spectra (Figure 5). The 1H-NMR data of 1–3 and 13C-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 C18H17O8 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 1H–1H 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).

FIGURE 4

Structures of AFB1 and 1-7

FIGURE 5

Key 2D-NMR and NOESY correlations of 1 and 3-7

TABLE 2

1H NMR data of compounds 1-4 in acetone-d 6 at 600 MHz and 5-7 in pyridine-d 5 at 500 MHz.

Pos	1	2	3	4	5	6	7
	δ H (J in Hz)	δ H (J in Hz)	δ H (J in Hz)	δ H (J in Hz)	δ H (J in Hz)	δ H (J in Hz)	δ H (J in Hz)
2	2.51, t (5.4)	2.48, m	2.48, m	2.47, dd (6.0, 4.8)	2.53, ddd (7.0, 4.5, 2.5)	2.57, t (5.5)	2.57, dt (6.5, 5.0)
3	3.42, dt (5.4, 4.2)	3.38, m	3.38, m	3.38, ddd (5.4, 4.2, 3.0)	3.03, m	3.14, m	3.13, m
5	6.54, s	6.54, s	6.52, s	6.50, s	6.54, s	6.59, s	6.56, s
6a	6.60, d (6.0)	6.48, d (6.0)	6.57, d (5.4)	6.65, d (6.0)	6.79, d (6.0)	6.95, d (5.5)	6.74, d (5.5)
8	5.27, d (4.8)	5.15, t (4.8)	4.10, dd (10.8, 1.2)	5.02, s	6.06, d (5.0)	4.44, d (10.0)	4.04, m
			3.66, dd (10.8, 3.0)			4.06, dd (10.0, 3.0)
9	2.42, ddd (13.2, 9.6, 4.8)	2.31, m	4.12, d (3.0)	4.37, d (3.6)	2.68, d (13.0)	5.02, d (3.0)	3.13, m
	2.27, d (13.2)	2.23, m			2.36, m
9a	4.19, dd (9.6, 6.0)	4.24, t (6.0)	4.15, d (5.4)	3.95, d (6.0)	4.20, dd (9.5, 6.0)	4.37, d (5.5)	3.93, dd (5.5, 1.0)
4-OCH3	4.03, s	4.01, s	4.00, s	3.99, s	3.72, s	3.84, s	3.84, s
8-OCH3	3.16, s	3.37, s		3.11, s
9-OCH3			3.43, s
1′							2.72, d (7.5)
3′							2.12, s

TABLE 3

13C NMR data of compounds 1, 3, 4 in acetone-d 6 and 5-7 in pyridine-d 5 at 125 MHz.

Pos	1	3	4	5	6	7
1	200.9, C	200.8, C	200.8, C	200.8, C	200.8, C	200.8, C
2	35.5, CH2	35.5, CH2	35.5, CH2	35.8, CH2	35.8, CH2	36.0, CH2
3	29.5, CH2	29.6, CH2	29.8, CH2	29.5, CH2	29.6, CH2	29.1, CH2
3a	178.1, C	177.9, C	178.1, C	177.7, C	177.7, C	178.3, C
3b	103.8, C	104.2, C	104.1, C	104.4, C	104.3, C	103.2, C
4	162.7, C	163.0, C	162.9, C	162.4, C	162.6, C	163.1, C
5	91.2, CH	91.0, CH	91.2, CH	92.2, CH	90.7, CH	90.3, C
5a	167.0, C	167.8, C	167.0, C	167.6, C	168.6, C	167.4, C
6a	115.0, CH	114.7, CH	115.0, CH	115.4, CH	115.0, CH	114.3, C
8	107.6, CH	72.6, CH2	112.5, CH	101.7, CH	76.9, CH2	73.3, CH2
9	37.8, CH2	84.7, CH	78.2, CH	38.9, CH2	74.7, CH	41.1, CH
9a	43.1, CH	50.7, CH	52.8, CH	43.4, CH	55.2, CH	50.3, CH
9b	109.8, C	107.6, C	106.3, C	110.1, C	105.1, C	107.2, C
9c	153.5, C	154.5, C	153.8, C	154.5, C	154.5, C	154.2, C
11	155.3, C	156.0, C	154.8, C	155.6, C	155.6, C	156.5, C
11a	117.8, C	117.0, C	117.5, C	117.5, C	117.8, C	117.7, C
1′						47.5, CH2
2′						206.5, C
3′						30.4, CH3
4-OCH3	56.9, CH3	57.1, CH3	57.1, CH3	56.7, CH3	57.8, CH3	56.8, CH3
8-OCH3	55.0, CH3		54.8, CH3
9-OCH3		56.8, CH3

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 1H and 13C spectra data of 5 were reported, and 6 was a new degraded product reported for the first time (Cox and Cole, 1977; Liu, Jin, Tao, Shan, et al., 2010; Wang et al., 2011; Wang et al., 2012; Stanley et al., 2020). The molecular formula of 5 and 6 was determined to be C17H14O7 on the basis of HR-ESI-MS, with 18 more daltons than that of AFB1, implying that 5 and 6 might be transformed from AFB1 through an addition reaction with H2O 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 C20H19O7 based on HR-ESI-MS. In the 1H 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 AFB1, which indicated that one molecule of acetone might be connected on C-8 or C-9. The 1H–1H 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 AFB1 and degraded products (1–7), the possible photocatalytic reactions were suggested: 1) addition reactions happened between MeOH, H2O, or acetone with AFB1 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, H2O, or CH3COCH3 formed potential free radicals (H•, OH•, OMe•, or CH3COCH2 •) 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 CH3COCH2 • to shape degraded products 1–3 and 5–7 (Waiss and Wiley, 1969; Iyer et al., 1994; Jamil et al., 2017; Mao et al., 2019). In addition, O2 in the air under UV irradiation could form O2 •-, which could attack on the double bond C-8/C-9 to produce 8,9-epoxide-AFB1. Addition reactions then happened fast by the highly unstable intermediate 8,9-epoxide-AFB1 with OMe• to form the degraded products of 4 (Figure 6) (Waiss and Wiley, 1969; Iyer et al., 1994; Jamil et al., 2017).

FIGURE 6

Possibly photocatalytic mechanism of AFB1 in MeOH and acetone.

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 α- or β-configuration. This demonstrated that steric hindrance (from right part of AFB1 structure) might exist and prevent different groups (OH•, OMe•, or CH3COCH2 •) 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 AFB1 (Cheung and Sim, 1964; van Soest and Peerdeman, 1970) revealed that the right part of the AFB1 structure was indeed closer to C-9 than C-8 in space, which might preclude different groups to attack C-9 from the positive face (β-position) due to spatial hindrance. The photocatalytic reactions are depicted in Figure 7.

FIGURE 7

Possible catalytic reaction happened at C-8 and C-9

Toxic Evaluation of Degraded Products

The cytotoxicity of AFB1 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. AFB1 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.

TABLE 4

Cytotoxic activity of seven degraded products and aflatoxin B1.

Compounds	Cytotoxic activity (μM)
	LO-2	Hep-G2	MCF-7
AFB1	22.47 ± 3.10	29.08 ± 4.92	36.57 ± 4.43
1	>100	>100	>100
2	>100	>100	>100
3	>100	>100	>100
4	>100	>100	>100
5/6	>100	>100	>100
7	>100	>100	>100
cis-platinum	6.54 ± 0.72	11.36 ± 1.47	21.47 ± 2.18

Conclusion

In this work, the degraded products of AFB1 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 OCH3 • through addition reactions under UV irradiation. The cytotoxic evaluation revealed that the toxicity of AFB1-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 AFB1, which can also be used to evaluate other mycotoxin degradation processes.