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Alternaria Toxins, Ergot Alkaloid Epimers, and Other Major Mycotoxins in Fortified Flour Sample on Raptor Biphenyl by LC-MS/MS

LC_FS0550
PeakstR (min)Precursor IonProduct Ion 1Product Ion 2
1.Nivalenol 0.92295.1137.191.0
2.Patulin1.03155.099.081.0
3.Ergometrine1.27326.2223.2208.1
4.Deoxynivalenol1.30297.2231.0249.0
5.Ergometrinine1.83326.2223.2208.1
6.Fusarenon-X1.98355.1137.1247.1
7.15-Acetyldeoxynivalenol3.14339.2137.1321.2
8.3-Acetyldeoxynivalenol3.21339.2213.1231.1
9.Tenuazonic acid4.22198.1125.0153.1
10.Altenuene4.70293.2257.1275.2
11.Alternariol5.30259.0185.1130.0
12.Citrinin5.43251.2233.1205.1
13.Ergosine5.47548.4208.1223.2
14.Fumonisin B15.63722.5352.3334.2
15.Ergosinine5.67548.4208.1223.2
16.Diacetoxyscirpenol5.73384.2247.1307.2
17.Ergotamine5.90582.4223.2268.2
18.Ergocornine6.03562.4268.2223.2
PeakstR (min)Precursor IonProduct Ion 1Product Ion 2
19.Ergotaminine6.13582.4223.2268.2
20.HT-26.20447.2345.1285.1
21.Fumonisin B36.32706.4336.2318.3
22.Ergocryptine6.32576.4268.2223.2
23.Ergocristine6.56610.4223.2592.4
24.Fumonisin B26.68706.4336.2318.3
25.Tentoxin6.70415.2312.2302.2
26.α-Zearalenol6.96303.1285.1175.0
27.Ergocorninine7.07562.4268.2223.2
28.T-27.14489.2387.1245.1
29.Aflatoxin G27.15331.2189.0313.0
30.Ergocryptinine7.31576.4268.2223.2
31.Ergocristinine7.53610.4223.2592.4
32.Aflatoxin G17.62329.1199.7243.0
33.Zearalenone7.65319.2283.1187.0
34.Alternariol monomethylether7.69273.0199.1128.0
35.Aflatoxin B27.81315.1287.0259.0
36.Aflatoxin B18.20313.2241.1284.9
37.Ochratoxin A8.31404.1239.0358.0
ColumnRaptor Biphenyl (cat.# 9309A12)
Dimensions:100 mm x 2.1 mm ID
Particle Size:2.7 µm
Pore Size:90 Å
Guard Column:Raptor Biphenyl EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 µm (cat.# 9309A0252)
Temp.:60 °C
Standard/SampleAflatoxins standard (cat.# 34121)
Ochratoxin A standard (cat.# 34122)
Diluent:50:50 Water:methanol
Conc.:6.25 ng/mL final concentration after sample preparation
Inj. Vol.:5 µL
Mobile Phase
A:Water, 0.05% formic acid
B:Methanol, 0.05% formic acid
Time (min)Flow (mL/min)%A%B
0.000.47525
5.000.45050
9.000.40100
9.010.47525
11.00.47525
DetectorMS/MS
Ion Mode:ESI+
Mode:MRM
InstrumentUHPLC
Sample PreparationA blended flour was prepared by mixing white rice flour (75%); brown rice flour (5%); millet flour (5%); oat flour (5%); all-purpose wheat flour (5%); and all-purpose, gluten-free flour (5%). Two grams of the flour sample were weighed into a 50-mL polypropylene centrifuge tube (cat.# 25846) and fortified at 50 µg/kg for all analytes with a stock standard solution. After sitting at room temperature for 10 minutes, 16 mL of extraction solution (80:20 acetonitrile:water) containing 0.5% formic acid were added, and the tube was stirred to create a homogenous suspension. The extraction was carried out by shaking horizontally on a digital pulse mixer (Glas-Col LLC, Terre Haute, IN) at 800 rpm for 20 minutes. After centrifuging for 5 minutes at 4000 rpm, 1 mL of extract was evaporated to dryness at 45 °C under a gentle stream of nitrogen. The dried extract was reconstituted with 1 mL of 50:50 water:methanol solution, and a 0.4 mL aliquot was transferred to and filtered using a Thomson SINGLE StEP filter vial with a 0.2 µm PTFE filter (cat.# 25874). Five µL of the filtered solution was injected for the LC-MS/MS analysis.

NotesThe chromatogram shows peaks with the MS transition of product ion 1.

Note that method development work demonstrated that whenever a new column is installed it must be rinsed and maintained under mobile phase overnight to ensure an acceptable peak shape for tenuazonic acid.