Detection of Regulated Ergot Alkaloids in Food Matrices by Liquid Chromatography-Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometry

Summary

This protocol presents a validated liquid chromatography-ion mobility-high resolution mass spectrometry method to determine the presence of ergot alkaloids in food in compliance with the recently released Commission Regulation (EU) 2023/915.

Abstract

Ion mobility mass spectrometry (IMS) acts as an additional separation dimension when integrated into liquid chromatography-mass spectrometry (LC-MS) workflows. LC-IMS-MS methods provide higher peak resolution, enhanced separation of isobaric and isomeric compounds, and improved signal-to-noise ratio (S/N) compared to traditional LC-MS methods. IMS provides another molecular characteristic for the identification of analytes, namely the collision cross section (CCS) parameter, reducing false positive results. Therefore, LC-IMS-MS methods address important analytical challenges in the field of food safety (i.e., detection of compounds at trace levels in complex food matrices and unambiguous identification of isobaric and isomeric molecules).

Ergot alkaloids (EAs) are a family of mycotoxins produced by fungi that attack a wide variety of grass species, including small grains such as rye, triticale, wheat, barley, millet, and oats. Maximum levels (MLs) of these mycotoxins have been established in several foodstuffs, as detailed in the Commission Regulation EC/2023/915. This new legislation includes six main EAs and their corresponding epimers, so an efficient methodology is required to properly distinguish these isomeric molecules considering their co-occurrence.

Therefore, the goal of this protocol is to show how the integration of IMS in LC-MS workflows contributes to the separation of isomeric EAs, enhancing the selectivity of the analytical method. Additionally, it illustrates how the generation of CCS libraries through the characterization of analytical standards provides higher confidence for the identification of mycotoxins. This protocol is designed to clearly explain the benefits of implementing IMS in food safety, taking as an example the determination of EAs in cereals. A QuEChERS-based extraction followed by an LC-trapped ion mobility spectrometry (TIMS)-MS analysis provided limits of quantification ranging from 0.65 to 2.6 ng/g with acceptable accuracy (although low recovery for ergotaminine) at 1.5x, 1x, and 0.5x the ML and exhibited a negligible matrix effect.

Introduction

Ion mobility mass spectrometry (IMS) is becoming a growingly used analytical technique, often presented as an additional separation dimension integrated into traditional liquid/gas chromatography (LC/GC) coupled to MS workflows. IMS consists of the separation of molecules along a mobility cell, filled with a buffer gas, under an electric field and at atmospheric pressure¹. Depending on the mass-to-charge ratio (m/z) and the geometrical conformation, an ionized molecule will interact with the buffer gas as it moves across the mobility cell, which is reflected in the ion mobility (K) parameter² and calculated thro....

Protocol

1. Preparation of stock, intermediate, and working standard solutions

NOTE: Use nitrile gloves, laboratory coat, and safety glasses.

1. Prepare individual stock solutions of the 12 EAs (see Table of Materials) at 10,000 ng/mL in 4 mL amber glass vials using acetonitrile. The R-forms (-ine) were previously aliquoted in 25,000 ng portions, whereas the S-forms (-inine) were distributed in 10,000 ng portions. This study began with t.......

Discussion

The successful use of this protocol is based on the optimization of the extraction procedure, previously carried out by Carbonell-Rozas et al.¹⁷, who implemented the use of an extraction solvent effective enough to extract EAs from complex food matrices such as barley and wheat, and a clean-up that provided relatively low SSE values. The choice of extraction solvent represents a critical step considering the chemical characteristics of the analytes and the lability of EAs to decomposition and epim.......

Materials

Name	Company	Catalog Number	Comments
Acetonitrile	VWR	83640.32
Amber glass tubes 4 mL	VWR	548-0052
Amber glass tubes 12 mL	VWR	548-0903
Amber vials 1.5 mL	Agilent	5190-9063
Ammonium carbonate	Fluka	9716
Analytical balance BAS 31	Boeco	4400519
Balance CP 323 S	Sartorius	23-84182
C18	Supelco	52604-U
Centrifuge tubes, 15 mL	VWR	525-1082
Centrifuge tubes, 50 mL	VWR	525-0155
Centrifuge Universal 320 R	Hettich	1406
Compass HyStar	Bruker		Acquisition software
DataAnalysis	Bruker		Qualitative software
Elute PLUS UHPLC	Bruker
EVA EC-S evaporator	VLM	V830.012.12
Formic acid GR for analysis ACS, Reag. Ph Eur	Merck	100264
Grinder TitanMill300	Cecotec	1559
Methanol	VWR	83638.32
Milli-Q water purification system (18.2 MΩ cm)	Millipore	ZD5211584
Pipette tips 1- 5 mL	Labortecnic	162005
Pipette tips 100 - 1000 µL	Labortecnic	1622222
Pipette tips 5 - 200 µL	Labortecnic	162001
Pippette Transferpette S variable, DE-M 10 - 100 µL	BRAND	704774
Pippette Transferpette S variable, DE-M 100 - 1000 µL	BRAND	704780
Pippette Transferpette S variable, DE-M 500 - 5000 µL	BRAND	704782
Syringe 2 mL	VWR	613-2003
Syringe Filter 13 mm, 0.22µm	Phenomenex	AF-8-7707-12
TASQ	Bruker		Quantitative software
timsTOFPro2 IM-HRMS	Bruker
Vortex Genie 2	Scientific Industries	15547335
Zorbax Eclipse Plus RRHD C18 column (50 x 2.1 mm, 1.8 µm particle size)	Agilent	959757-902
Z-Sep+	Supelco	55299-U	Zirconia-based sorbent
Ergot alkaloids			CAS registry sorbent
Ergocornine (Eco)	Techno Spec	E178	564-36-3
Ergocorninine (Econ)	Techno Spec	E130	564-37-4
Ergocristine (Ecr)	Techno Spec	E180	511-08-0
Ergocristinine (Ecrn)	Techno Spec	E188	511-07-9
Ergokryptine (Ekr)	Techno Spec	E198	511-09-1
Ergopkryptinine (Ekrn)	Techno Spec	E190	511-10-4
Ergometrine (Em)	Romer Labs	"002067"	60-79-7
Ergometrinine (Emn)	Romer Labs	LMY-090-5ML	479-00-5
Ergosine (Es)	Techno Spec	E184	561-94-4
Ergosinine (Esn)	Techno Spec	E194	596-88-3
Ergotamine (Et)	Romer Labs	"002069"	113-15-5
Ergotaminine (Etn)	Romer Labs	"002075"	639-81-6