Optimization and validation of a quantitative liquid chromatography–tandem mass spectrometric method covering 295 bacterial and fungal metabolites including all regulated mycotoxins in four model food matrices

Abstract

An LC-MS/MS "dilute and shoot" method for the determination of 295 fungal and bacterial metabolites was optimized and validated according to the guidelines established in the Directorate General for Health and Consumer Affairs of the European Commission (SANCO) document No. 12495/2011. Four different types of food matrices were chosen for validation: apple puree for infants (high water content), hazelnuts (high fat content), maize (high starch and low fat content) and green pepper (difficult or unique matrix). Method accuracy and precision was evaluated using spiked samples in five replicates at two concentration levels. Method trueness was demonstrated through participation in various proficiency tests. Although the method covers a total number of 331 analytes, validation data were acquired only for 295 analytes, either due to the non-availability of analytical standards or due other reasons described in this paper. Concerning the apparent recovery, the percentage of 295 analytes matching the acceptable recovery range of 70-120% lied down by SANCO varied from 21% in green pepper to 74% in apple puree at the highest spiking level. At the levels close to limit of quantification only 20-58% of the analytes fulfilled this criterion. The extent of matrix effects was strongly dependent on the analyte/matrix combination. In general, the lowest matrix effects were observed in apple puree (59% of analytes were not influenced by enhancement/suppression at all at the highest validation level). The highest matrix effects were observed in green pepper, where only 10% of analytes did not suffer from signal suppression/enhancement. The repeatability of the method was acceptable (RSD≤20) for 97% of all analytes in apple puree and hazelnuts, for 95% in maize and for 89% in green pepper. Concerning the trueness of the method, Z-scores were generally between -2 and 2, despite a broad variety of different matrices. Based on these results it can be concluded that quantitative determination of mycotoxins by LC-MS/MS based on a "dilute and shoot" approach is also feasible in case of complex matrices.

Advanced LC–MS-based methods to study the co-occurrence and metabolization of multiple mycotoxins in cereals and cereal-based food

“…The z scores obtained are interpreted as follows: |z| ≤ 2 is an acceptable result, 2 < |z| ≤ 3 is considered a questionable result, and |z| > 3 is an unacceptable result. An example of the z scores obtained by the Bdilute and shoot^multimycotoxin LC-MS/MS method [36] in routine proficiency testing organized by the Bureau Interprofessionnel des Études Analytique (BIPEA) is displayed in Fig. 2.…”

“…Faster instrument electronics and improved design of the collision cell significantly shorten the minimum dwell times that need to be used for each precursor ion-product ion pair monitored. The rapid multimethods that fully exploit the potential of the stateof-the-art QqQ/QLIT instruments are capable of the simultaneous analysis of up to 300 mycotoxins, their metabolites, or other related food contaminants depending on the length of the chromatographic run [36,49,55]. Such multimycotoxin methods are considered to be semiquantitative methods, since no pure analytical standards are available on the market and only inhouse purified standards are used for quantification.…”

“…The method performance characteristics for some analytes are given in the electronic supplementary material. The method has been continuously extended to include 331 bacterial, plant, and fungal metabolites, and has been fully validated for 295 analytes in maize and three other matrices [36]. To successfully acquire as many MRM transitions with acceptable sensitivity and repeatability in a reasonable time, the method was transferred onto the next generation of a QTRAP instrument (QTRAP 5500).…”

“…However, despite the use of highly sensitive LC-MS instrumentation, achieving trace detection levels of some analytes in more than 200 multidetection methods is impossible when compromises have to be made with regard to both sample preparation and LC-MS/MS conditions. These methods rely on the injection of raw extracts [43,64], Bdilute and shoot^approaches [36,47], or different modifications of the Bquick, easy, cheap, effective, rugged, and safe^(QuEChERS) approach [48,62,64,65].…”

Fungal secondary metabolite analysis applied to Cultural Heritage: the case of a contaminated library in Venice

“…were punched out using a cork borer and treated with 4 mL of extraction solvent (acetonitrile/water/acetic acid 79:20:1, v/v/v). The extraction was performed for 1.5 h using a Universal Table Shaker Malachová et al (2014), which includes 320 analytes. As mass-spectrometer, a QTrap 5500 LC-MS/MS System (Applied Biosystems, Foster City, CA) equipped with TurbolonSpray electronspray ionization (ESI) source was used connected with a 1290 Series HPLC System (Agilent, Waldbronn, Germany).…”