Development and validation of a confirmative LC-MS/MS method for the determination of ß-exotoxin thuringiensin in plant protection products and selected greenhouse crops

Abstract

Bacterial products based on Bacillus thuringiensis are registered in many countries as plant protection products (PPPs) and are widely used as insecticides and nematocides. However, certain B. thuringiensis strains produce harmful toxins and are therefore not allowed to be used as PPPs. The serotype B. thuringiensis thuringiensis produces the beta-exotoxin thuringiensin (ßeT) which is considered to be toxic for almost all forms of life including humans (WHO 1999). The use of a non-registered PPP based on B. thuringiensis thuringiensis called bitoxybacillin was established through the determination of ßeT. First, an analytical reference standard of ßeT was characterized by nuclear magnetic resonance, liquid chromatography-high-resolution mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Then, a confirmatory quantitative method for the determination of ßeT in PPPs and selected greenhouse crops based on LC-MS/MS was developed and validated. A limit of quantitation of 0.028 mg/kg was established, and average recoveries ranged from 85.6 % to 104.8 % with repeatability (RSDr) of 1.5-7.7 % and within-lab reproducibility (RSD(WLR)) of 17 %. The method was used for analysis of >100 samples. ßeT was found in leaves of ornamentals, but no evidence was found for use in edible crops.

Thuringiensin: A Thermostable Secondary Metabolite from Bacillus thuringiensis with Insecticidal Activity against a Wide Range of Insects

“…In addition, other methods have been developed, including bioassay, ion-exchange chromatography, spectrophotometry, reverse phase liquid chromatography, micellar electrokinetic capillary chromatography, and enzyme-linked immunosorbent assay [34,35,36,37]. De Rijk et al (2013) established a confirmatory quantitative method for the determination of Thu in plant protection products and selected greenhouse crops based on liquid chromatography tandem mass spectrometry; the limit of quantitation was 0.028 mg/kg [38]. …”

Characterization of Bacillus thuringiensis isolates by their insecticidal activity and their production of Cry and Vip3 proteins

“…kurstaki strain HD1 were used as positive and negative control of β-exotoxin, respectively. β-exotoxin was detected by looking for the transitions 702.1–136.2 and 702.1–412.0 in the chromatogram peaks [ 17 ].…”

Quality assurance and control of methods to examine visually recognisable substances in feed and food : In cooperation with International Association for Feedingstuff Analysis, Section Feedingstuff Microscopy

“…The upper confidence limit as defined by Pocklington and AOC is based on the equation 𝑅𝑆𝐷 𝑅 = 2 • 𝐶 −0.15 , whereas Codex Alimentarius used a factor 4 (𝑅𝑆𝐷 𝑅 = 4 • 𝐶 −0.15 ). Chemical studies show levels of measurement uncertainty which are lower than the accepted maximum values according to the standard models (De Rijk et al, 2013;Ciasca et al, 2018;González Medina et al, 2018). The models and values from practice are shown in Figure A1.…”

“…The level of inhomogeneity for samples containing units in the mm size range remains much larger than the acceptable limits for relative standard deviations in analytical chemistry for comparable analyte levels (AOAC, 2002b, page 25;Codex Alimentarius, 2004, page 5;Gustavo Gonzales and Angeles Herrador, 2007), and larger than uncertainty ranges found in chemical studies in practice (De Rijk et al, 2013;Ciasca et al, 2018;Medina et al, 2018). Fluid matrices of a certain viscosity can be heated or diluted to decrease the viscosity and an acceptable homogeneity could be achieved (oil: inclusions; candy syrup: packaging material; honey: pollen).…”