Challenges on Production of a Certified Reference Material of Ochratoxin A in Roasted Coffee: A Brazilian Experience.

Background: Coffee is an important agricultural commodity with technical barriers for exportation because of possible contamination with ochratoxin A (OTA), a mycotoxin nephrotoxic and carcinogenic. The maximum limit for OTA in roasted coffee is 5.0 µg/kg in the European Union and 10 µg/kg in Brazil, and the use of certified reference materials (CRM) is required for reliable measurements. Objective: This paper describes the development of a candidate CRM of OTA in roasted coffee following the requirements of ISO 17034 and ISO Guide 35. Methods: A primary method of isotope dilution MS was developed and validated using (13C20)-OTA as internal standard. The sample preparation was based on AOAC Official Methods of AnalysisSM using immunoaffinity column. Results: The linear working range is 2.0-15.0 µg/kg, with recoveries of 92.2-110.8% and relative SDs lower than 12.4%. The method was successfully applied to the feasibility study, which defined the procedure for preparation of a large batch around 5 µg/kg. It was produced by spiking blank roasted coffee with OTA standard, mixing and filling in amber flasks with 50 g of coffee, and storing at -80°C. The homogeneity study showed an acceptable degree of heterogeneity of 1.44%, and the short-term-stability study defined the conditions for transportation as maximum temperature of 50°C up to 28 days. Conclusions: These results show that certification is possible. Highlights: The long-term stability study at -20°C is in progress, and the characterization will be conduzed by a interlaboratory comparison. This material will be an important tool for QC in laboratories.

Multi-omic and physiologic approach to understand Lotus japonicus response upon exposure to 3,4 dimethylpyrazole phosphate nitrification inhibitor.

Nitrogen fertilization is a major force in global greenhouse gases emissions and causes environmental contamination through nitrate leaching. The use of nitrification inhibitors has been proven successful to mitigate these effects. However, there is an increasing concern about the undesired effects that their potential persistence in the soil or accumulation in plants may provoke. In this study, we first exposed Lotus japonicus plants to high amounts of 3,4 dimethylpyrazole phosphate (DMPP) and 2-(N-3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture (DMPSA) nitrification inhibitors. Exposure to doses higher than 1 mg·L-1 provoked DMPP accumulation mostly in the aerial part, while DMPSA was only detected from 10 mg·L-1 and nearly no translocation. To evaluate the effect that DMPP accumulation in leaves may provoke on plant performance we combined a transcriptome, proteome, and physiological analysis in plants treated with 10 mg/ L of DMPP. This treatment provoked changes in the expression of 229 genes and 59 proteins. Overall, we evidence that when DMPP accumulates in leaves it induces stress responses, notably provoking changes in cell redox balance, hormone signaling, protein synthesis and turnover and carbon and nitrogen metabolism.

3,4-Dimethylpyrazole phosphate and 2-(N-3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture nitrification inhibitors: Quantification in plant tissues and toxicity assays.

Nitrification inhibitors are used to maintain ammonium available in the soil for longer periods while reducing nitrate leaching and N2O emission. In this work we evaluated the potential toxicity effects of 3,4-dimethylpyrazole phosphate (DMPP) and 2-(N-3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture (DMPSA) nitrification inhibitors. In order to determine the potential plant capacity to take up and translocate these inhibitors, we grew clover plants in hydroponic conditions and we developed a novel methodology for extracting DMPP and DMPSA that we quantified by HPLC. In addition, we also did toxicity bioassays: seed germination and Vibrio fischeri test. When clover was exposed to high amounts of nitrification inhibitors, plants accumulated DMPP, predominantly in leaves, and also DMPSA that preferentially accumulated in roots. These inhibitors did not provoke phytotoxicity at the equivalent of the maximum amount estimated in agriculture (0.5mg/kg soil). DMPP only provoked detrimental effects in plants at very high dose (100mg/kg soil). Interestingly, DMPSA was innocuous.

Evaluation of a fungal collection as certified reference material producer and as a biological resource center

Abstract Considering the absence of standards for culture collections and more specifically for biological resource centers in the world, in addition to the absence of certified biological material in Brazil, this study aimed to evaluate a Fungal Collection from Fiocruz, as a producer of certified reference material and as Biological Resource Center (BRC). For this evaluation, a checklist based on the requirements of ABNT ISO GUIA34:2012 correlated with the ABNT NBR ISO/IEC17025:2005, was designed and applied. Complementing the implementation of the checklist, an internal audit was performed. An evaluation of this Collection as a BRC was also conducted following the requirements of the NIT-DICLA-061, the Brazilian internal standard from Inmetro, based on ABNT NBR ISO/IEC 17025:2005, ABNT ISO GUIA 34:2012 and OECD Best Practice Guidelines for BRCs. This was the first time that the NIT DICLA-061 was applied in a culture collection during an internal audit. The assessments enabled the proposal for the adequacy of this Collection to assure the implementation of the management system for their future accreditation by Inmetro as a certified reference material producer as well as its future accreditation as a Biological Resource Center according to the NIT-DICLA-061.

Evaluation of a fungal collection as certified reference material producer and as a biological resource center.

Considering the absence of standards for culture collections and more specifically for biological resource centers in the world, in addition to the absence of certified biological material in Brazil, this study aimed to evaluate a Fungal Collection from Fiocruz, as a producer of certified reference material and as Biological Resource Center (BRC). For this evaluation, a checklist based on the requirements of ABNT ISO GUIA34:2012 correlated with the ABNT NBR ISO/IEC17025:2005, was designed and applied. Complementing the implementation of the checklist, an internal audit was performed. An evaluation of this Collection as a BRC was also conducted following the requirements of the NIT-DICLA-061, the Brazilian internal standard from Inmetro, based on ABNT NBR ISO/IEC 17025:2005, ABNT ISO GUIA 34:2012 and OECD Best Practice Guidelines for BRCs. This was the first time that the NIT DICLA-061 was applied in a culture collection during an internal audit. The assessments enabled the proposal for the adequacy of this Collection to assure the implementation of the management system for their future accreditation by Inmetro as a certified reference material producer as well as its future accreditation as a Biological Resource Center according to the NIT-DICLA-061.

The influence of different referencing methods on the accuracy of δ(13) C value measurement of ethanol fuel by gas chromatography/combustion/isotope ratio mass spectrometry.

RATIONALE: Brazil is the largest producer of sugar cane bioethanol in the world. Isotope ratio mass spectrometry (IRMS) is the technique of choice to certify the origin/raw materials for ethanol production, but the lack of certified reference materials (CRMs) for accurate measurements of δ(13) C values traceable to Vienna Pee Dee Belemnite (VPDB), the international zero point for (13) C/(12) C measurements, certified and compatible with gas chromatography (GC)/IRMS instruments may compromise the accuracy of δ(13) C determinations. METHODS: We evaluated the influence of methods for the calibration and normalization of raw δ(13) C values of ethanol samples. Samples were analyzed by GC/C/IRMS using two different GC columns. Different substances were used as isotopic standards for the working gas calibration. The δ(13) C values obtained with the three methods of normalization were statistically compared with those obtained with elemental analyzer (EA)/IRMS, since the δ(13) C results obtained using EA are traceable to VPDB via the NBS 22 reference material. RESULTS: It was observed that both the isotopic reference material for CO2 calibration and the GC column have a major effect on the δ(13) C measurements, leading to a bias of almost 2-3 ‰ in the δ(13) C values. All three methods of normalization were equivalent in performance, enabling an improvement in the GC/C/IRMS accuracy, compared with the EA/IRMS reference values for the samples. CONCLUSIONS: All the methods of CO2 calibration, chromatography and normalization presented in this work demonstrated several sources of traceability and accuracy loss for the determination of δ(13) C values in ethanol fuel samples by GC/C/IRMS. This work has also shown the importance of using proper CRMs traceable to VPBD that should be compatible and certified using GC/C/IRMS, ideally in a wide range of δ(13) C values. This is important not only for bioethanol fuel samples, but also for many analytes commonly analyzed by IRMS.

Determination of PAHs: a practical example of validation and uncertainty assessment.

The objective of this study was to present a reliable and practical example of method validation and uncertainty assessment with an analytical method for the determination of polycyclic aromatic hydrocarbons (PAHs) in urban dust. The method was gas chromatography-mass spectrometry in combination with isotope dilution principle to achieve better accuracy for the results. The method performance parameters for five PAHs were determined (phenanthrene, fluoranthene, benzo[a]anthracene, benzo[a]pyrene and benzo[ghi]perylene); this method was used in the key comparison of CCQM-K50b for PAHs in particulate matter. The limits of detection and quantification were lower than 0.075 and 0.250 µg/g, respectively. The linear correlation coefficients were greater than 0.99. The major uncertainty contributions resulted from the accuracy of each analyzed PAH and the repeatability of the process. Certified reference material (National Institute of Standards and Technology SRM 1649a, urban dust) was used to determine the accuracy and precision of the method. The obtained results were satisfactory and agreed with all evaluated performance parameters.