Immunomagnetic microbeads for screening with flow cytometry and identification with nano-liquid chromatography mass spectrometry of ochratoxins in wheat and cereal

Abstract

Multi-analyte binding assays for rapid screening of food contaminants require mass spectrometric identification of compound(s) in suspect samples. An optimal combination is obtained when the same bioreagents are used in both methods; moreover, miniaturisation is important because of the high costs of bioreagents. A concept is demonstrated using superparamagnetic microbeads coated with monoclonal antibodies (Mabs) in a novel direct inhibition flow cytometric immunoassay (FCIA) plus immunoaffinity isolation prior to identification by nano-liquid chromatography–quadrupole time-of-flight-mass spectrometry (nano-LC-Q-ToF-MS). As a model system, the mycotoxin ochratoxin A (OTA) and cross-reacting mycotoxin analogues were analysed in wheat and cereal samples, after a simple extraction, using the FCIA with anti-OTA Mabs. The limit of detection for OTA was 0.15 ng/g, which is far below the lowest maximum level of 3 ng/g established by the European Union. In the immunomagnetic isolation method, a 350-times-higher amount of beads was used to trap ochratoxins from sample extracts. Following a wash step, bound ochratoxins were dissociated from the Mabs using a small volume of acidified acetonitrile/water (2/8 v/v) prior to separation plus identification with nano-LC-Q-ToF-MS. In screened suspect naturally contaminated samples, OTA and its non-chlorinated analogue ochratoxin B were successfully identified by full scan accurate mass spectrometry as a proof of concept for identification of unknown but cross-reacting emerging mycotoxins. Due to the miniaturisation and bioaffinity isolation, this concept might be applicable for the use of other and more expensive bioreagents such as transport proteins and receptors for screening and identification of known and unknown (or masked) emerging food contaminants.FigureMicrobead coated with antibody

Colour-encoded paramagnetic microbead-based direct inhibition triplex flow cytometric immunoassay for ochratoxin A, fumonisins and zearalenone in cereals and cereal-based feed

“…Only OTA at a very high concentration (1 μg/ml, corresponding with 32 mg/kg in a cereal product) showed an impact on the FB 1 assay causing 40 % inhibition. The OTA antibody showed high cross-reactivity for OTB (43 % in maize), which is less desired since OTB is not as hazardous as OTA [35]. However, OTB occurs in much lower concentrations than OTA and therefore will not cause a significant problem [38].…”

“…The OTA-RPE conjugation procedure was identical to the one described previously by Aqai et al [35]. The conjugation of FB 1 to RPE was based on the method of Szurdoki et al [31] with slight modifications: A glutaraldehyde buffer was prepared just before use by adding 400 μl of a 25 % glutaraldehyde solution, 0.4 g of NaCl and 5 ml of a 0.1 M sodium phosphate solution (pH 7.5) to a 50-ml tube, and the volume was adjusted to 50 ml using fresh double distilled water.…”

Application of EU guidelines for the validation of screening methods for veterinary drugs

“…[7] By varying the ratio of the two fluorophores, up to 100 different colour-coded microsphere sets can be distinguished, and each microsphere set can be coupled with a different biological probe. [8,9] Within the EU project, CONffIDENCE, this approach was applied to eggs and feed for the detection of the following coccidiostats: narasin, salinomycin, diclazuril, lasalocid, monensin, and nicarbazin in feed and egg extract. For validation purposes all coccidiostats had to be validated separately due to possible cross reactivity of the coccidiostat with the antibody of a second coccidiostats coated an a different microsphere.…”

Theory and Practice of UHPLC and UHPLC–MS

“…Food products reach the consumer through human handling and action, which can introduce the presence of exogenous molecules that could endanger human health or impair the food quality. The second part of Table 9.4 describes selected recent research works in nano-LC and CLC regarding the determination of harmful compounds in food matrices such as antibiotics (LombardoAgüí et al, 2011;Ruiz-Viceo et al, 2012;D'Orazio et al, 2012a;Quesada-Molina et al, 2013;Liu et al, 2016), drugs (Berlioz-Barbier et al, 2015), pesticides (Mirabelli et al, 2016;Kruve et al, 2011;Moreno-González et al, 2011;Gure et al, 2014Gure et al, , 2015, mycotoxins (Arroyo-Manzanares et al, 2011Aqai et al, 2011;Liu et al, 2013), and phthalates (Muñoz-Ortuño et al, 2012).…”