Multimycotoxin Screening and Quantitation in Beer Samples Using UHPLC and a High-Resolution and Accurate Mass Approach

Mycotoxins are toxic secondary metabolites produced by many species of microscopic filamentary fungi occurring on field cereals including barley, which is used in the production of beer. The most abundant fungal genera affecting malting barley are Alternaria, Aspergillus, Penicillium, and Fusarium, which have all shown high producing potential for a wide range of mycotoxins.1 In addition to these relatively common micromycetes, the Claviceps purpurea pathogen, which causes ergot disease, is also commonly found in barley.

There is a significant trend in analytical laboratories toward the simplification of sample preparation strategies. With respect to the determination step, full spectral data acquisition techniques are gaining popularity because of their ease of use and the possibility of retrospective archived data mining. Until recently, the most common full spectral mass spectrometric approach for food analysis has been time-of-flight technology (TOF-MS), with a typical resolving power of approximately 12,500 FWHM (full width at half maximum). However, in complex food matrices such as beer, this limited mass resolving power leads to the risk of inaccurate mass measurements caused by unresolved background matrix interferences.2,3 Systems based on Orbitrap™ technology (Thermo Fisher Scientific, Bremen, Germany) allow scientists to routinely achieve mass resolving power of up to 100,000 FWHM and maintain very high mass accuracy of <5 ppm without the use of internal mass correction.4

Mycotoxin analysis

Although the carryover of aflatoxins, ochratoxin A, zearalenone, fumonisins, and ergot alkaloids from malted grains into beer has been documented,5,6 the main research in this area has been focused on deoxynivalenol, the most frequently found Fusarium mycotoxin. In recent years, the presence of its primary metabolite, deoxynivalenol-3-glucoside, in malt and beer has been reported at relatively high levels (the deoxynivalenol-3-glucoside/deoxynivalenol molar ratio reported as ≥1.7 This has been confirmed in a followup study in which both deoxynivalenol and its glucoside were determined as the main contaminants of beers retailed on the European market.8 Since beer is commonly consumed worldwide, the control of mycotoxin presence is essential. For this purpose, reliable analytical methods for fast and effective monitoring of mycotoxins during beer production are needed.

The aim of this study was to introduce a fast multimycotoxin method for the analysis of 32 mycotoxins in beer based on very simple sample preparation and ultrahigh-performance liquid chromatography (UHPLC) coupled with full spectral orbital trapping MS detection, and to explore the potential of the system for routine work.

Experimental/methods

Reagents and chemicals

Mycotoxin standards were obtained from Sigma Aldrich (Taufkirchen, Germany), Biopure (Tulln, Austria), and The Czech Agricultural and Food Inspection Authority (Prague, Czech Republic). These included:

  • Fusarium toxins, major conjugates, and other products of transformation—nivalenol, deoxynivalenol, deoxynivalenol-3-glucoside, deepoxydeoxynivalenol, fusarenon-X, neosolaniol, 3-acetyldeoxynivalenol, diacetoxyscirpenol, HT-2 toxin, T-2 toxin, verrucarol, zearalenone, α-zearalenole, and ß-zearalenole (Sigma Aldrich)
  • Aflatoxins—aflatoxin G1, aflatoxin G2, aflatoxin B1, and aflatoxin B2 (Biopure)
  • Sterigmatocystin (Biopure)
  • Ochratoxins—ochratoxin A and ochratoxin α (Biopure)
  • Alternaria toxins—altenuene, alternariol, and alternariol-methylether (Biopure)
  • Ergot alkaloids—ergosine, ergocornine, ergocryptine, and ergocristine (The Czech Agricultural and Food Inspection Authority).

The purity of standards was declared in the range 96–98.9%.

Solid standards of nivalenol, deoxynivalenol, fusarenon-X, neosolaniol, 3-acetyldeoxynivalenol, T-2 toxin, verrucarol, zearalenone, α-zearalenole, β-zearalenole, sterigmatocystin, ochratoxin A, altenuene, alternariol, and alternariolmethylether were dissolved in acetonitrile. Liquid standards of deepoxydeoxynivalenol, diacetoxyscirpenol, HT-2 toxin, α-zearalenole, ß-zearalenole, ochratoxin α, and ergot alkaloids were supplied in acetonitrile, and deoxynivalenol-3-glucoside was delivered in acetonitrile:water (1:1, vol/vol) solution. All of the standards were stored at –20 ºC. For spiking experiments and calibration purposes, a composite working standard solution in acetonitrile (1000 µg L–1) was prepared. All of the standards were brought to room temperature before use.

Sample preparation

The aliquot of 4-mL beer sample in a PTFE cuvette was degassed in an ultrasonic bath. Acetonitrile (16 mL) was added and the content was shaken vigorously for approximately 1 min. The dark-colored matrix precipitated under these conditions was then separated by centrifugation (10 min at 11,000 rpm). The 5-mL aliquot of the supernatant was evaporated to dryness and reconstituted in 1 mL of methanol:water (50:50, vol/vol). To avoid obstruction of the UHPLC system, microfiltration was performed prior to injection (centrifugation through a 0.2-µm PVDF Zentrifugenfilter microfilter [Alltech® Grace Davison Discovery Sciences, Deerfield, IL]).

To control potential losses, for example, due to a partition between the precipitate and aqueous phases, an aliquot of 13C-labeled deoxynivalenol and 13C-labeled zearalenone standard solution was added as a surrogate to the beer at a contamination level of 20 µg L–1 prior to processing (13C-deoxynivalenol and 13C-zearalenone for correction of more and less polar analytes, respectively).