Development and validation of TOF/Q-TOF MS/MS, HPLC method and in vitro bio-strategy for aflatoxin mitigation

Figures & data

Figure 1. Experimental flow diagram for in-vitro biocontrol study of aflatoxin mitigation (micro-laboratory based) and metabolite determination using bio-analytical techniques. Undamaged maize kernels were surface sterilised and co-infected with a known concentration of atoxigenic isolate KSM012 and/or aflatoxigenic isolates KSM014 of A. flavus at varying proportions prior to metabolite extraction for TLC screening and LCMS/MS analysis. The isolates were cultured on inducing and non-inducing media (YES: yeast extract sucrose agar; YEP: yeast extract peptone agar; Tox: toxigenic and Atox: atoxigenic respectively; TLC: thin layer chromatography; LCMS/MS: liquid chromatography coupled to mass spectrometry)

Table 1. Percentage extraction yield at varying parameters (solvent proportions, bead beating, time frame and sonication)

Extraction solvent	Starting material (mg)	Sonication time	% yield without bead beating	% yield with bead beating
MeOH: DCM: EtOAc (1:2:3, v/v/v) and 1 % (v/v) FA	700	1 hr at 65 ° C	0.20 %	15 %~105 mg
MeOH: DCM: EtOAc (1:2:3, v/v/v)	700	1 hr at 65 ° C	0.00 %	<0.01 %
MeOH: DCM: EtOAc (1:2:3, v/v/v) and 1 % (v/v) FA	700	15 min at 30 ° C	1.29 %	23 %~161 mg
MeOH: DCM: EtOAc (1:2:3, v/v/v)	700	15 min at 30 ° C	0.00 %	<0.01 %
70 % MeOH in H2O	700	30 min at 40 ° C	0.11 %	12 %~83 mg
90 % ACN in H2O	700	30 min at 40 ° C	0.01 %	0.24 %~1.7 mg

N/B. Solvents; MeOH: methanol; DCM: dichloromethane; ACN: acetonitrile; FA: formic acid; EtOAc: ethyl acetate.

Figure 2. Standard curves of various aflatoxin standards used in the current research (AFG2, AFG1, AFB2 and AFB1) respectively showing linear regression and equation

Figure 3. HPLC chromatographic separations. Standard chromatogram achieved after the analysis of aflatoxins standard mix with certificated concentration of 1.70 µg/mL for AFG2 and AFB2 and 5.86 µg/mL for AFG1 and AFB1 at wavelength 320 nm

Figure 4. HPLC chromatographic separations for (a) isolate KSM012 (non-toxigenic strain) confirmed the possibility of its non-toxigenicity and (b) Aflatoxigenic isolate KSM014 (toxigenic strain) achieved at wavelength 365 nm respectively

Figure 5. MS/MS spectra and fragmentation pathway showing mycotoxin metabolites and their derivatives identified from eight different samples (KSM014, KSM015, KSM018, KSM019, HB023, MC033, MC037, MC038) isolated from four different climatic regions of Kenya based on electrospray ionisation mass-to-charge (m/z) ratio and targeted mass. The mycotoxins identified (AFG1: aflatoxin G1; AFG2: aflatoxin G2; AFB2: aflatoxin B2; AFM1: aflatoxin M1; AFM2: aflatoxin M2 and OTA: ochratoxin A). (MC: Makueni; HB: Homa Bay; KSM: Kisumu respectively)

Table 2. Metabolites identified by formula mass, possible compounds, monoisotopic masses and other chromatographic properties

Base peak (m/z)	Type of adduct	Neutral molecule (Da)	Suggested elemental composition	Possible compound	Mass error value (Da) * (detected m/z & theoretical m/z)	Base peak found in samples
183.1021	M + H	182.0943	C10H14O3	Barnol	−0,000006	NC04
233.1164	M + H	232.1086	C14H16O3	Sorbicillin	0,001344	NC05, NC08, KSM014, HB022, HB023, HB026
235.1312	M+ Na	212.1414	C12H20O3	Dihydrojasmonic acid	−0,000156	KSM018, HB021, MC037
235.1331	M + H	234.1253	C14H18O3	2ʹ,3ʹ-Dihydrosorbicillin	0,000294	NC010, KSM012, KSM013, KSM020, HB029, HB030, MC040
251.0920	M + H	250.0842	C13H14O5	Citrinin	−0,000076	HB029
251.1278	M + H	250.1200	C14H18O4	Vertinolide	0,000509	HB021
275.1612	M+ Na	252.1714	C15H24O3	Walleminone	0,001145	KSM012
277.1060	M + H	276.0982	C15H16O5	Ascochitine	0,001574	NC05
279.1250	M + K	240.1618	C16H20N2	Pyroclavine, costaclavine or secoclavine	0,000849	HB025

<1> (Nielsen & Smedsgaard, 2003). Ranges indicate the highest and lowest mass difference found within the different samples. * Absolute values reported. N/B: NC: Nandi county; KSM: Kisumu county; HB: Homa Bay and MC: Makueni county.

Table 3. Metabolites identified by targeted mass algorithm

Sample ID	Monoisotopic mass (Da)	Base peak (m/z)	Type of adduct	Retention time (min)	Algorithm	Suggested elemental composition	Possible compound
KSMO14	314.1845	315.2	M + H	15.18	Targeted MS/MS	C17H14O6	Aflatoxin B2
KSM015	330.1896	331.2	M + H	9.53	“	C17H14O7	Aflatoxin M2
KSM018	403.2463	404.2	(M + H)+	15.03	“	C20H18ClNO6	Ochratoxin A
KSM019	330.1991	331.2	M + H	16.76	“	‎ C17H14O7	Aflatoxin G2
HB023	330.2024	331.2	M + H	15.53	“	C17H14O7C17H12O6	Aflatoxin G2 ά
MC033	330.1895	331.2	M + H	9.06	“	C17H14O7	Aflatoxin M2
MC037	328.0927	329.1	M + H	14.64	“	C17H12O7	Aflatoxin G1
MC038	328.1138	329.1	M + H	1.12	“	C17H12O7	Aflatoxin M1

Figure 6. Images of thin-layer chromatography plates (a) showing aflatoxin suspects or presence from the isolates in comparison to the standards of aflatoxins (b) the staining effects on TLC plate with p-anisaldehyde. Lanes: (1) Blank; (2) Standard; (3) G100/0; (4) G75/25; (5) G50/50; (6) G25/75; (7) G0/100; (8) K100/0; (9) K75/25; (10) K50/50; (11) K25/75; (12) K0/100. (atoxigenic/toxigenic. (G1: aflatoxin AFG1; G2: aflatoxin AFG2; B1: aflatoxin AFB1; B2: aflatoxin AFB2)

Figure 7. Analysis of high-performance liquid chromatography highlighting biocontrol strategy exhibited when two maize lines (KDV1 and GAF4) were co-infected with non-toxin producing (KSM012) and toxin-producing (KSM014) strains of Aspergillus flavus. At co-infection ratio (a) indicated by red arrow: 50:50; aflatoxin AFG2, AFB1 and at 75:25; AFG2 were detected whereas AFG1, AFB1 and AFB2 were not detected for GAF4 maize line respectively); (b) at 50:50 and 75:25) there was significant decrease in presence of both aflatoxins or no signals detected by HPLC on KDV1 maize line, an indication of probable complete inhibition of the aflatoxigenic isolate by atoxigenic counterpart. KDV1 maize line appeared more susceptible to Aspergillus flavus colonisation compared to GAF4. (AFG1: aflatoxin G1; AFG2: aflatoxin G2; AFB1: aflatoxin B1; AFB2: aflatoxin B2)

Nielsen KF, Smedsgaard J. 2003. Fungal metabolite screening: database of 474 mycotoxins and fungal metabolites for dereplication by standardised liquid chromatography-UV-mass spectrometry methodology. J Chromatogr A. 1002(1–2):111–136. doi:10.1016/S0021-9673(03)00490-4.

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