An integrated targeted and untargeted approach for the analysis of ergot alkaloids in cereals using UHPLC - hybrid quadrupole time-of-flight mass spectrometry

An ultra-high performance liquid chromatography hybrid quadrupole time of flight (Q-TOF) mass spectrometry (MS) method is described for the simultaneous quantitative determination of common ergot alkaloids and the screening, detection and identification of unexpected (less studied or novel) members of this class of toxic fungal secondary metabolites. The employed analytical strategy involves an untargeted data acquisition (consisting of full scan TOF MS survey and information dependent acquisition MS/MS scans) and the processing of data using both targeted and untargeted approaches. Method performance characteristics for the quantitative analysis of 6 common ergot alkaloids i.e. ergometrine, ergosine, ergotamine, ergocornine, ergocristine, ergokryptine and their corresponding epimers in rye were comparable to those previously reported for triple-quadrupole (QqQ) MS/MS. The method limits of quantification (LOQ) were in the range from 3 to 19 mu g/kg, and good linearity was observed for the different ergot alkaloids in the range from LOQ to 1000 mu g/kg. Furthermore, the method demonstrated good precision (relative standard deviations at 50 mu g/kg not higher than 14.6 and 16.2% for the intra-day and inter-day precision, respectively), and the trueness values at different concentration levels were all between 89 and 115%. The method was applied for the analysis of a set of 17 rye samples and demonstrated the presence of these ergot alkaloids in the range from <LOQ to 2,811 mu g/kg. Further mining of the same data based on a 'non-targeted peak finding' algorithm and the use of full MS and MS/MS accurate mass data allowed the detection and identification of 19 ergot alkaloids that are commonly not included in most analytical methods using QqQ instruments. Some of these alkaloids are reported for the first time in naturally contaminated samples

Microbial detoxification of deoxynivalenol (DON), assessed via a Lemna minor L. bioassay, through biotransformation to 3-epi-DON and 3-epi-DOM-1

Mycotoxins are toxic metabolites produced by fungi. To mitigate mycotoxins in food or feed, biotransformation is an emerging technology in which microorganisms degrade toxins into non-toxic metabolites. To monitor deoxynivalenol (DON) biotransformation, analytical tools such as ELISA and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) are typically used. However, these techniques do not give a decisive answer about the remaining toxicity of possible biotransformation products. Hence, a bioassay using Lemna minor L. was developed. A dose-response analysis revealed significant inhibition in the growth of L. minor exposed to DON concentrations of 0.25 mg/L and higher. Concentrations above 1 mg/L were lethal for the plant. This bioassay is far more sensitive than previously described systems. The bioassay was implemented to screen microbial enrichment cultures, originating from rumen fluid, soil, digestate and activated sludge, on their biotransformation and detoxification capability of DON. The enrichment cultures originating from soil and activated sludge were capable of detoxifying and degrading 5 and 50 mg/L DON. In addition, the metabolites 3-epi-DON and the epimer of de-epoxy-DON (3-epi-DOM-1) were found as biotransformation products of both consortia. Our work provides a new valuable tool to screen microbial cultures for their detoxification capacity

Validated UPLC-MS/MS methods to quantitate free and conjugated Alternaria toxins in commercially available tomato products and fruit and vegetable juices in Belgium

Ultraperformance liquid chromatography tandem mass spectrometry and Quick, Easy, Cheap, Effective, Rugged, and Safe based analytical methodologies to quantitate both free (alternariol (1), alternariol monomethyl ether (2), tenuazonic acid (3), tentoxin (4), altenuene (5), altertoxin-I (6)) and conjugated (sulfates and glucosides of 1 and 2) Alternaria toxins in fruit and vegetable juices and tomato products were developed and validated. Acceptable limits of quantitation (0.7-5.7 mu g/kg), repeatability (RSDr < 15.7%), reproducibility (RSDR < 17.9%), and apparent recovery (87.0-110.6%) were obtained for all analytes in all matrices investigated. 129 commercial foodstuffs were analyzed, and 3 was detected in 100% of tomato product samples (<LOQ to 333 mu g/kg), while 1, 2, 4, and 5 were also frequently detected (21-86%, <LOQ to 62 mu g/kg). Moreover, low levels (<LOQ to 9.9 mu g/kg) of modified Alternaria toxins (sulfates of 1 and 2) were repeatedly detected. A deterministic dietary exposure assessment revealed the possible risk for human health related to the presence of 1 and 2 in tomato based foodstuffs, whereas 3 is unlikely to be of human health concern

Detached leaf in vitro model for masked mycotoxin biosynthesis and subsequent analysis of unknown conjugates

The manuscript details the development of an in vitro model plant system using detached leaves because there is a need for biosynthetic methods for the production and isolation of masked mycotoxins. This detached leaf in vitro model was firstly applied to deoxynivalenol with satisfying results. The biosynthesis of deoxynivalenol-3-glucoside was confirmed using its respective commercially available reference standard. Secondly, the detached leaf in vitro model was applied to T-2 toxin. Mono- and tri-glucoside derivatives of T-2 toxin and HT-2 toxin, T-2-(3)-glucoside, T-2-(3)-triglucoside and HT-2-(3)-glucoside were identified and characterised using Orbitrap high-resolution mass spectrometry. This is the first report on a triglucoside of T-2 toxin. The discovery of new masked forms implies the importance of the development of analytical methods for their detection, the constitution of toxicity studies, and proving the relevance of their presence in the food and feed chain

New insights into electrospray ionization of patulin

Patulin is a mycotoxin produced by several fungal species, mainly by Penicillium spp. and Aspergillus spp. Since patulin-producing fungi are widely spread, this toxin has been detected in food (fruit- and vegetable-based products, cereal products, cheese), feed and even in mouldy water-damaged dwellings. Co-occurrence of patulin with other mycotoxins has also been reported [1]. Patulin is commonly analyzed by liquid chromatography with UV detection. Liquid chromatography coupled to mass spectrometry (LC-MS/MS) is considered as a more specific tool for mycotoxin detection and confirmation. However, the implementation of this technique for the determination of patulin, especially in the context of multi-mycotoxin analysis, is limited due to ionization problems. In this study, the effect of different solvents, mobile phase additives and pH on the ionization and fragmentation pattern of patulin was investigated. The preliminary results showed that under alkaline conditions and using methanol as organic modifier, an intense and stable signal for the methanol-adduct of patulin was obtained in the positive electrospray ionization mode. The fragmentation of this protonated methanol-adduct gave a strong and stable product ion signal. The production spectra were overall more useful than those obtained with the protonated or the deprotonated molecule. These findings indicate the possibility of using the protonated methanol-adduct of patulin for its identification and quantification by LC-MS/MS. Further results that will be presented include the optimization, by means of experimental design, of the parameters that have an influence on the formation of the protonated methanol-adduct of patulin and on its fragmentation behaviour, as well as the inclusion of this toxin in a multi-mycotoxin LC-MS/MS method. This study is the first report of the LC-MS/MS determination of patulin using its protonated methanol-adduct. References: [1] Nielsen KF (2003) Mycotoxin production by indoor molds. Fungal Genetics and Biology. 39: 103-117