Establishment of non-targeted screening database and confirmation method for 18 mycotoxins in grains using ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry

doi:10.3724/SP.J.1123.2022.05015

Abstract

Abstract:

A mass spectral library of 18 mycotoxins was developed based on ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF/MS), which was used to establish a non-targeted screening method for mycotoxins in rice and wheat matrices. Eighteen mycotoxin standards were separated on an HSS T3 column, and data were collected for both positive and negative ionization under the MS^E mode of the UPLC-Q-TOF/MS. Details including formulas, retention times, theoretical exact masses, measured exact masses of the adduct and fragment ions, and ion abundance ratios were recorded to establish the mass spectral library of the 18 mycotoxins in UNIFI Software. Analyte detection was based on a retention time deviation of 0.3 min, and the exact mass deviation of the adduct ions and fragment ions was set to 5×10^-6. The screening detection limit (SDL) was used as the main threshold for verifying the screening method. In the validation process, 18 mycotoxins were classified into two types: with maximum levels (MLs) and without MLs. The results showed that the mycotoxins with MLs could be accurately screened at their limited level, and the mycotoxins without MLs had a range of SDL concentration from 2 to 800 μg/kg. The matrix effect results showed that 14 mycotoxins in rice and 11 in wheat had moderate matrix effects. Finally, 25 batches of rice and wheat were purified using QuEChERS and HLB columns after acetonitrile extraction and screening were performed by employing the established method. The results revealed that aflatoxin G₁ (AFG₁), aflatoxin G₂ (AFG₂), fumonisins B₁ (FB₁), and sterigmatocystin (ST) were detected in one batch of rice, FB₁ and ST were detected in another batch of rice, FB₁ and ochratoxin A (OTA) were detected in two batches of wheat, and no other mycotoxins were detected. This method is characterized by high throughput, simplicity, rapidity, accuracy, and can be applied to accurately screen mycotoxins with concentrations higher than the SDLs and qualitatively screen various mycotoxins in rice and wheat without standards.

Key words: ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF/MS), mycotoxins, rice, wheat, database, screening and confirmation

CLC Number:

O658

ZHANG Luxing, HUANG Zhaohui, LUO Shuqing, CAO Lin, XIE Ying, QIAN Jiang. Establishment of non-targeted screening database and confirmation method for 18 mycotoxins in grains using ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry[J]. Chinese Journal of Chromatography, 2023, 41(1): 66-75.

Figures/Tables 12

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Compound	Abbreviation	Conc. of standard solution / (μg/mL)	Purity of standard substance/%	Conc. in mixed standard solution /(μg/L)	Source
Aflatoxin B₁ (黄曲霉素B₁)	AFB₁	10	-	5.0	Welch
Aflatoxin B₂ (黄曲霉素B₂)	AFB₂	10	-	5.0
Aflatoxin G₁ (黄曲霉素G₁)	AFG₁	10	-	5.0
Aflatoxin G₂ (黄曲霉素G₂)	AFG₂	10	-	5.0
Ochratoxin A (赭曲霉素)	OTA	50.4	-	15.1	Sigma
Sterigmatocystin (杂色曲霉毒素)	ST	50.1	-	10.0	Romer
HT-2 toxin (HT-2毒素)	HT-2	100.4	-	50.2
T-2 toxin (T-2)	T-2	100.4	-	10.0	Alta
Fumonisins B₁ (伏马毒素B₁)	FB₁	100.3	-	100.3
Fumonisins B₂ (伏马毒素B₂)	FB₂	99.9	-	99.9
Deoxynivalenol 3-glucoside	DON-3G	10.0	-	2000.0
(脱氧雪腐镰刀菌烯醇-3-葡萄糖苷)
Acetyl-deoxynivalenol (15-乙酰基脱氧雪腐镰刀菌烯醇)	15-ACDON	99.9	-	799.2
Patulin (展青霉素)	PAT	100.0	-	1000.0
Deoxynivalenol (脱氧雪腐镰刀菌烯醇)	DON	-	99.62	5000.0	Fermentek
Nivalenol (雪腐镰刀菌烯醇)	NIV	-	100	4000.0
Zearalenone (玉米赤霉烯酮)	ZEN	-	99.24	300.0
Citrinin (桔青霉素)	CIT	-	100	100.0

Compound	Abbreviation	Conc. of standard solution / (μg/mL)	Purity of standard substance/%	Conc. in mixed standard solution /(μg/L)	Source
Aflatoxin B₁ (黄曲霉素B₁)	AFB₁	10	-	5.0	Welch
Aflatoxin B₂ (黄曲霉素B₂)	AFB₂	10	-	5.0
Aflatoxin G₁ (黄曲霉素G₁)	AFG₁	10	-	5.0
Aflatoxin G₂ (黄曲霉素G₂)	AFG₂	10	-	5.0
Ochratoxin A (赭曲霉素)	OTA	50.4	-	15.1	Sigma
Sterigmatocystin (杂色曲霉毒素)	ST	50.1	-	10.0	Romer
HT-2 toxin (HT-2毒素)	HT-2	100.4	-	50.2
T-2 toxin (T-2)	T-2	100.4	-	10.0	Alta
Fumonisins B₁ (伏马毒素B₁)	FB₁	100.3	-	100.3
Fumonisins B₂ (伏马毒素B₂)	FB₂	99.9	-	99.9
Deoxynivalenol 3-glucoside	DON-3G	10.0	-	2000.0
(脱氧雪腐镰刀菌烯醇-3-葡萄糖苷)
Acetyl-deoxynivalenol (15-乙酰基脱氧雪腐镰刀菌烯醇)	15-ACDON	99.9	-	799.2
Patulin (展青霉素)	PAT	100.0	-	1000.0
Deoxynivalenol (脱氧雪腐镰刀菌烯醇)	DON	-	99.62	5000.0	Fermentek
Nivalenol (雪腐镰刀菌烯醇)	NIV	-	100	4000.0
Zearalenone (玉米赤霉烯酮)	ZEN	-	99.24	300.0
Citrinin (桔青霉素)	CIT	-	100	100.0

Mycotoxin	GB 2761-2017		EC No. 1881/2006
Mycotoxin	Rice	Wheat	Rice	Wheat
AFG₁	none	none	total: 4.0	total: 4.0
AFG₂	none	none
AFB₁	10	5.0
AFB₂	none	none
OTA	5.0	5.0	3.0	3.0
DON	none	1000	1250	1750
ZEN	none	60	100	100

Mycotoxin	GB 2761-2017		EC No. 1881/2006
Mycotoxin	Rice	Wheat	Rice	Wheat
AFG₁	none	none	total: 4.0	total: 4.0
AFG₂	none	none
AFB₁	10	5.0
AFB₂	none	none
OTA	5.0	5.0	3.0	3.0
DON	none	1000	1250	1750
ZEN	none	60	100	100

No.	Compound	Molecular formula	CAS No.	Ion mode	Theoretical precursor ion (m/z)	Experimental precursor ion (m/z)	Product ions (m/z)	t_R/ min	m/z error/ 10^-6
1	AFG₁	C₁₇H₁₂O₇	1165-39-5	[M+H]⁺	329.06558	329.0655	311.0525, 43.0659, 283.0544	13.91	-0.24
2	AFG₂	C₁₇H₁₄O₇	7241-98-7	[M+H]⁺	331.08123	331.0801	313.0673, 285.0701, 245.0842	13.55	-3.41
3	AFB₁	C₁₇H₁₂O₆	1162-65-8	[M+H]⁺	313.07066	313.0699	285.0716, 270.0477, 242.0516	14.55	-2.43
4	AFB₂	C₁₇H₁₄O₆	7220-81-7	[M+H]⁺	315.08631	315.0851	287.0900, 259.0551, 241.0777	14.27	-3.84
5	ST	C₁₈H₁₂O₆	163391-76-2	[M+H]⁺	325.07066	325.0706	310.0475, 281.0443	17.52	-0.18
6	OTA	C₂₀H₁₈ClNO₆	303-47-9	[M+H]⁺	404.08954	404.0883	358.0783, 239.0052, 166.1944	16.71	-3.07
7	T-2	C₂₄H₃₄O₉	21259-20-1	[M+NH₄]⁺	484.25411	484.2565	245.1135, 305.1354	16.22	4.94
8	FB₁	C₃₄H₅₉NO₁₅	116355-83-0	[M+H]⁺	722.39575	722.3990	334.3086, 352.3222	15.79	4.50
9	FB₂	C₃₄H₅₉NO₁₄	116355-84-1	[M+H]⁺	706.40083	706.4034	336.3198, 336.3263, 318.3171	17.52	3.64
10	CIT	C₁₃H₁₄O₅	518-75-2	[M+H]⁺	251.09140	251.0922	205.0828, 191.0692	14.55	3.19
11	15-ACDON	C₁₇H₂₂O₇	88337-96-6	[M+NH₄]⁺	356.17043	356.1701	321.1331, 279.0944	12.05	-0.92
12	DON	C₁₅H₂₀O₆	51481-10-8	[M+CH₃COO]^-	355.13985	355.1409	295.1158, 265.1033, 247.0968	4.95	2.96
13	NIV	C₁₅H₂₀O₇	23282-20-4	[M+CH₃COO]^-	371.13475	371.1353	311.1050, 281.0974	3.36	1.48
14	ZEN	C₁₈H₂₂O₅	17924-92-4	[M-H]^-	317.13945	317.1382	273.1464, 175.0364, 149.0561	17.08	-3.94
15	DON-3G	C₂₁H₃₀O₁₁	131180-21-7	[M+CH₃COO]^-	517.19265	517.1905	457.1694, 427.1625	5.10	-4.16
16	HT-2	C₂₂H₃₂O₈	26934-87-2	[M+CH₃COO]^-	483.22355	483.2249	59.0111, 141.0143	15.50	2.79
17	3-ACDON	C₁₇H₂₂O₇	50722-38-8	[M+CH₃COO]^-	397.15040	397.1516	337.1325, 307.1198, 173.0569	11.94	3.02
18	PAT	C₇H₆O₄	149-29-1	[M-H]^-	153.01932	153.0189	109.0329	2.87	-2.94