Analysis of Ergot Alkaloid Mycotoxins in Blended Flour by LC-MS/MS Under Acidic Conditions

• Baseline separation of six critical ergot alkaloids and their epimers allows definitive quantification.
• Fast, 11-min total cycle time supports high-throughput testing.
• Acidic conditions suitable for the simultaneous analysis of ergot alkaloids, Alternaria toxins, and major regulated mycotoxins.

Ergot alkaloids are mycotoxin contaminants of cereal plants that are of increasing regulatory interest due to their widespread presence and the dietary importance of cereals. Of more than 40 known ergot alkaloids, six compounds including ergocornine, ergocristine, ergocryptine, ergometrine, ergosine, and ergotamine, together with their isomeric -inine epimers, are currently regulated by the EU. When these ergot alkaloids are analyzed on a C18 column, high pH conditions are needed to separate all 12 epimers. In contrast, when a Raptor Biphenyl column is used, baseline separation can be achieved under regular column- and MS-friendly, acidic conditions. As shown here, complete resolution of all 12 epimers, including the challenging ergotamine/ergotaminine and ergosine/ergosinine epimer pairs, was obtained in 11 minutes of total cycle time.

The use of a Raptor Biphenyl column and acidic conditions also allows concurrent analysis of ergot alkaloids with other mycotoxins of interest. A separate study established that these conditions are suitable for quantitative analysis of ergot alkaloids in the same run as Alternaria toxins and priority regulated mycotoxins in a variety of foods [1]. Quantitation in that study was performed in wheat baby cereal, peanut, tomato puree, and blended flour using matrix-matched standards because isotopically labeled standards were not available for these mycotoxins. Suitability of this method for multiresidue mycotoxin testing provides food safety labs with a more efficient workflow and the time-saving benefit of combined analysis.

	Peaks	tR (min)	Precursor Ion	Product Ion 1	Product Ion 2
1.	Ergometrine	1.27	326.2	223.2	208.1
2.	Ergometrinine	1.83	326.2	223.2	208.1
3.	Ergosine	5.47	548.4	208.1	223.2
4.	Ergosinine	5.67	548.4	208.1	223.2
5.	Ergotamine	5.90	582.4	223.2	268.2
6.	Ergocornine	6.03	562.4	268.2	223.2

	Peaks	tR (min)	Precursor Ion	Product Ion 1	Product Ion 2
7.	Ergotaminine	6.13	582.4	223.2	268.2
8.	Ergocryptine	6.32	576.4	268.2	223.2
9.	Ergocristine	6.56	610.4	223.2	592.4
10.	Ergocorninine	7.07	562.4	268.2	223.2
11.	Ergocryptinine	7.31	576.4	268.2	223.2
12.	Ergocristinine	7.53	610.4	223.2	592.4

Column	Raptor 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/Sample
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 0.00 0.4 75 25 5.00 0.4 50 50 9.00 0.4 0 100 9.01 0.4 75 25 11.0 0.4 75 25

Detector	MS/MS
Ion Mode:	ESI+
Mode:	MRM
Instrument	UHPLC
Sample Preparation	A 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.
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References

1. S.H. Liang, J. York, J. Konschnik, H. Majer, J. Steimling, Comprehensive mycotoxin analysis: simultaneous determination of Alternaria toxins, ergot alkaloid epimers, and other major mycotoxins in various food matrices by LC-MS/MS, Restek Corporation, 2022.

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