Development and Validation of a Liquid Chromatography High-Resolution Mass Spectrometry Method for the Simultaneous Determination of Mycotoxins and Phytoestrogens in Plant-Based Fish Feed and Exposed Fish

Abstract

New protein sources in fish feed require the assessment of the carry-over potential of contaminants and anti-nutrients from feed ingredients into the fish, and the assessment of possible health risks for consumers. Presently, plant materials including wheat and legumes make up the largest part of aquafeeds, so evaluation of the transfer capabilities of typical toxic metabolites from plant-infesting fungi and of vegetable phytoestrogens into fish products is of great importance. With the aim of facilitating surveillance of relevant mycotoxins and isoflavones, we have developed and validated a multi-analyte LC-HRMS/MS method that can be used to ensure compliance to set maximum levels in feed and fish. The method performance characteristics were determined, showing high specificity for all 25 targeted analytes, which included 19 mycotoxins and three isoflavones and their corresponding aglycons with sufficient to excellent sensitivities and uniform analytical linearity in different matrices. Depending on the availability of matching stable isotope-labelled derivates or similar-structure homologues, calibration curves were generated either by using internal standards or by matrix-matched external standards. Precision and recovery data were in the accepted range, although they varied between the different analytes. This new method was considered as fit-for-purpose and applied for the analysis of customised fish feed containing wheat gluten, soy, or pea protein concentrate as well as salmon and zebrafish fed on diets with these ingredients for a period of up to eight weeks. Only mycotoxin enniatin B, at a level near the limit of detection, and low levels of isoflavones were detected in the feed, demonstrating the effectiveness of maximum level recommendations and modern feed processing technologies in the Norwegian aquaculture industry. Consequently, carry-over into fish muscle was not observed, confirming that fillets from plant-fed salmon were safe for human consumption.

Co-Occurrence and Levels of Mycotoxins in Fish Feeds in Kenya

“…One advantage of the method used in this study, besides the ability to detect several mycotoxins simultaneously, was that it was not necessary to carry out a clean-up step after extraction. Additionally, it has been validated and has been used several times in previous studies [ 56 , 57 , 58 ]. However, there are drawbacks with multi-analyte methods that employ such an extract-and-shoot approach.…”

“…Acetonitrile (Romil Pure Chemistry Acetonitrile 190) (MeCN), deoxynivalenol (DON), 3-OAc-deoxynivalenol (3ADON), 15-OAc-deoxynivalenol (15ADON), deoxynivalenol-3-glucoside (DON3G), zearalenone (ZEN), α-zearalenol (αZEL), β-zearalenol (βZEL), ochratoxin A (OTA), T-2 toxin (T2), T-2 toxin triol, T-2 toxin tetraol, HT-2 toxin (HT2), nivalenol (NIV), neosolaniol (NEO), ergocristine (ECR), ergosine (ESN), α-ergocryptine (αECP), moniliformin (MON), fusarenon-X (FUS-X), diacetoxyscirpenol (DAS), alternariol (AOH), alternariol methyl ether (AME), sterigmatocystin (STC), beauvericin (BEA), 15-hydroxyculmorin (CUL), 5-acetamido-butenolide (BUT), 2-amino-14,16-dimethyloctadecan-3-ol (AOD), aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), fumonisin B1 (FUMB1), fumonisin B2 (FUMB2) as well as the stable isotope-labelled analogues U-[ 13 C-15]-NIV, U-[ 13 C-15]-DON, U-[ 13 C-21]-DON3G, U-[ 13 C-17]-3ADON, U-[ 13 C-17]-15ADON, U-[ 13 C-22]-HT2, U-[ 13 C-24]-T2, U-[ 13 C-20]-OTA, U-[ 13 C-18]-ZEN, U-[ 13 C17]-AFB1, U-[ 13 C17]-AFB2, U-[ 13 C17]-AFG1 and U-[ 13 C17]-AFG2 were provided by Romer labs (Tulln, Austria) [ 56 , 58 ]. Formic acid (>98%) (HCOOH), acetic acid (>99.8%) (CH 3 COOH) and ammonium acetate (>98%) (CH 3 COONH 4 ) were provided by Merck KGaA (Darmstadt, Germany).…”

“…LC-MS grade MeCN (Optima), LC-MS grade MeOH (Optima) and UHPLC-UV grade water (W6-212 water) were provided by Fisher Scientific (Loughborough, Leics., UK). The enniatins A (ENNA), A1 (ENNA1), B (ENNB), and B1 (ENNB1) and the ergot alkaloids, ergonovine (ENV), ergotamine (ETA), ergocornine (ECO), methysergide maleate (MSM) and bromocriptine mesylate (BCM) were provided as solids by Sigma-Aldrich (St. Louis, MO, USA) [ 56 , 58 ].…”

“…Stock solutions of the ergot alkaloids, ENV, ETA, ECO, MSM and BCM were prepared by dissolving the respective solids in MeCN or MeOH to concentrations of 100–500 mg/L. Similarly, stock solutions of the enniatins, ENNA, ENNA1, ENNB and ENNB1 were prepared by dissolving the respective solids in MeOH to 200 mg/L [ 58 ]. This was prepared fresh prior to extraction.…”

Rapid and sensitive detection of mycotoxins by advanced and emerging analytical methods: A review

“…Methods like LC‐MS/MS are developed to detect the both DON and DON‐3G in the bakery products (Generotti et al, ). Similarly, Johny et al () have developed high‐resolution LC‐MS method to detect DON‐3G exposed fish and in plant‐based fish feed. The LOD was obtained 176 µg/kg for DON‐3G in salmon, zebrafish, and fish feed.…”