Study of co-occurrence of mycotoxins in cocoa beans in Brazil by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

In this study, 135 samples of cocoa beans collected in the Amazon and Atlantic Forest regions of Brazil were analysed to evaluate the possible co-occurrence of 34 mycotoxins. The results indicate that 42% of the cocoa samples exhibited quantifiable levels for 11 mycotoxins: aflatoxins (AFs) B1, B2 and G1; ochratoxin A; citrinin; cyclopiazonic acid; tenuazonic acid; paxilline; sterigmatocystin; zearalenone and fumonisin B2. Of the samples, 18% exhibited the co-occurrence of up to six mycotoxins. No toxins belonging to the groups of trichothecenes or ergot alkaloids were detected. Contingency analysis of the incidence of mycotoxins did not show significant differences between the two regions evaluated. Seven samples were contaminated with AFs, while only one contained ochratoxin A above 10 µg kg-1. The accuracy of the method was evaluated by proficiency testing for ochratoxin A, where satisfactory Z-scores were obtained.

Validation and estimation of uncertainty of an LC-MS/MS method for the simultaneous determination of 34 mycotoxins in cocoa beans.

The aim of this manuscript was to validate and apply an analytical methodology for the simultaneous determination of 34 mycotoxins in cocoa. The extraction method used in the tests was a liquid-liquid partition by NaCl addition with a freezing step followed by quantification using LC-MS/MS. The results were discussed based on national and international directives for food contaminants. The recoveries and precision were adequate, except for the mycotoxins ionized with the ammonium adduct (NH4+), E-cristinine and ß-ZOL. This result directly influenced the measurement uncertainty of these mycotoxins, because the precision and the correction factor of the recovery were the factors with the greatest impact on the uncertainty of the method. The evaluation of the matrix effect showed considerable signal suppression for 53 % of the evaluated mycotoxins. Nevertheless, the mycotoxins exhibited relatively low quantification limits, with values between 1 and 75 µg kg-1. The validated methodology was applied to 15 cocoa samples collected in warehouses in Brazil. Positive results were found for all the evaluated samples, in which nine toxins were detected out of the 34 investigated.

Methodology development based on "dilute and shoot" and QuEChERS for determination of multiple mycotoxins in cocoa by LC-MS/MS.

This work proposes an extraction method based on the "dilute and shoot" approach and QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) for the simultaneous determination of 42 mycotoxins (34 quantified and 8 qualitatively studied) in dried cocoa bean samples. The purpose of the developed methodology was the reduction of co-extractives from the matrix and an efficient extraction without a cleanup step, and subsequent analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In order to obtain the best extraction conditions, gravimetric tests were performed and parameters that influenced the extraction efficiency were evaluated, such as the proportion of extraction phases, amount of salt, acidification, and extraction time. The performance of the developed method was evaluated to ensure its reliability. Considering the recovery range of 70-120% as an accuracy parameter, four of the mycotoxins under study (acetyl T-2, tenuazonic acid, wortmannin, and zearalenone) showed undesirable values at one of the levels evaluated. The repeatability of the method was assessed for 34 mycotoxins by the relative standard deviation (RSD%) of the responses, and all presented satisfactory values. The quantification limits ranged from 1.0 to 33.0 µg kg-1. Modification of the extraction methods made it possible to simultaneously analyze multiple mycotoxins, eliminating the need for the cleanup step, which led to analyte losses. The proposed methodology has a low cost, which makes it advantageous in routine analysis. It also has the potential for scope extension to cocoa-based foods, which are naturally exposed to a greater variety of mycotoxins. Graphical abstract.

Development and comparative analysis of single-drop and solid-phase microextraction techniques in the residual determination of 2-phenoxyethanol in fish.

Solid-phase microextraction (SPME) and single-drop microextraction (SDME) in headspace mode, were used in the residual determination of the anesthetic 2-phenoxyethanol in fish fillets, to ensure food safety. For the optimization of the methodologies the experimental central composite design (CCD) was used, resulting in accurate evaluations with less amount of analysis. The developed methodologies presented good precision in the evaluated range, o limits of detection (LD) and quantification (LQ) for SDME were 0.2 and 0.62 µg mL-1 and for SPME were 0.18 and 0.56 µg mL-1, respectively. In the analyzed samples the determined elimination time of post-anesthesia 2-phenoxyethanol was 12 h for the SDME and 24 h for the SPME, at the anesthesia concentrations evaluated (450-1050 µg mL-1). The two techniques presented viability of application for the residual determination of 2-phenoxyethanol in fish, SPME being more sensitive and automated and SDME with lower operation cost.

Residual determination of anesthetic menthol in fishes by SDME/GC-MS.

The SDME-GC/MS method was applied to residual determination of anesthetic menthol in fish. The extractions took place from the headspace of the sample using 1.8µL of octane as the extraction solvent. To obtain the ideal extraction condition, was used Response Surface Methodology, defining: extraction time 15min, temperature 30°C and salt 3g. The method showed LOD and LOQ of 0.021 and 1.56µgL-1 respectively, recovery of 94% and R2 of 0.9997. The analyzes were performed on tilapia fillets anesthetized in five concentrations between 5 and 15×104µgL-1 and with times of slaughter after anesthesia of 0, 12, 24 and 48h. It was determined that 48h is the required residual period for total metabolization of menthol in the fishes' organisms. This methodology becomes promising regarding the establishment of protocols to regulatory the use of menthol as an anesthetic in aquaculture.