Metabolic Pathway Reconstruction Indicates the Presence of Important Medicinal Compounds in Coffea Such as L-DOPA.

The use of transcriptomic data to make inferences about plant metabolomes is a useful tool to help the discovery of important compounds in the available biodiversity. To unveil previously undiscovered metabolites of Coffea, of phytotherapeutic and economic value, we employed 24 RNAseq libraries. These libraries were sequenced from leaves exposed to a diverse range of environmental conditions. Subsequently, the data were meticulously processed to create models of putative metabolic networks, which shed light on the production of potential natural compounds of significant interest. Then, we selected one of the predicted compounds, the L-3,4-dihydroxyphenylalanine (L-DOPA), to be analyzed by LC-MS/MS using three biological replicates of flowers, leaves, and fruits from Coffea arabica and Coffea canephora. We were able to identify metabolic pathways responsible for producing several compounds of economic importance. One of the identified pathways involved in isoquinoline alkaloid biosynthesis was found to be active and producing L-DOPA, which is a common product of POLYPHENOL OXIDASES (PPOs, EC 1.14.18.1 and EC 1.10.3.1). We show that coffee plants are a natural source of L-DOPA, a widely used medicine for treatment of the human neurodegenerative condition called Parkinson's disease. In addition, dozens of other compounds with medicinal significance were predicted as potential natural coffee products. By further refining analytical chemistry techniques, it will be possible to enhance the characterization of coffee metabolites, enabling a deeper understanding of their properties and potential applications in medicine.

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.

Contamination of cachaça by PAHs from storage containers.

Cachaça is a distiled beverage obtained from the fermentation of sugar cane syrup that, depending on the production procedures, may be susceptible to contamination by polycyclic aromatic hydrocarbons (PAHs). These compounds present carcinogenic and/or mutagenic properties and offer a risk to human health. Sixteen PAHs were determined in cachaças that had been stored in glass bottles and in polyethylene tank by gas chromatography coupled with mass spectrometry. The quantification of the PAHs utilised an internal standard. The limits of detection and quantification varied from 0.05 to 0.10µgL(-)(1) and 0.20 to 0.30µgL(-)(1), respectively. A total PAH concentration of 51.57µgL(-)(1) was found in the beverages that were stored in the tank, while the concentration in the cachaça stored in glass jugs was 6.07µgL(-)(1). These results indicate that the polyethylene tank is a source for PAHs in cachaça.

Determination of ethyl carbamate in cachaça produced from copper stills by HPLC.

Ethyl carbamate (EC) is a common substance in fermented foods and drinks, and its quantification is important because of its carcinogenic nature and its usually presence in alcoholic beverages. The present work involved the development and validation of an analytical method for the evaluation of EC in cachaça by HPLC-FLD after previous derivatization with xanthydrol. The method presented a mean recovery of 94.88%, an intra-day precision of 4.19% (30.0 µgL(-1)) and 3.32% (75.0 µgL(-1)), a coefficient of determination (r(2)) equal to 0.9985, and limits of detection and quantification equal to 6.39 and 21.32 µgL(-1), respectively. The results show that the analytical method is accurate, reproducible and linear over the concentration range from 5.0 to 160 µg of EC per litre. The method was applied to the analysis of EC in cachaça, the analyses being rapid and efficient.

Voltammetric determination of Δ9-THC in glassy carbon electrode: An important contribution to forensic electroanalysis.

A new voltammetric method for the determination of Δ(9)-tetrahydrocannabinol (Δ(9)-THC) is described. The voltammetric experiments were accomplished in N-N dimethylformamide/water (9:1, v/v), using tetrabutylammonium tetrafluoroborate (TBATFB) 0.1mol/L as supporting electrolyte and a glassy carbon disk electrode as the working electrode. The anodic peak current was observed at 0.0V (vs. Ag/AgCl) after a 30s pre-concentration step under an applied potential of -1.2V (vs. Ag/AgCl). A linear dependence of Δ(9)-THC detection was obtained in the concentration range 2.4-11.3ng/mL, with a linear correlation coefficient of 0.999 and a detection limit of 0.34ng/mL. The voltammetric method was used to measure the content of Δ(9)-THC in samples (hemp and hashish) confiscated by the police. The elimination of chemical interferences from the samples was promptly achieved through prior purification using the TLC technique, by employing methanol/water (4:1, v/v) as the mobile phase. The results showed excellent correlation with results attained by HPLC.

Rapid and sensitive method for the determination of acetaldehyde in fuel ethanol by high-performance liquid chromatography with UV-Vis detection.

A high-performance liquid chromatography (HPLC) method for the determination of acetaldehyde in fuel ethanol was developed. Acetaldehyde was derivatized with 0.900 mL 2,4-dinitrophenylhydrazine (DNPHi) reagent and 50 microL phosphoric acid 1 mol L(-1) at a controlled room temperature of 15 degrees C for 20 min. The separation of acetaldehyde-DNPH (ADNPH) was carried out on a Shimadzu Shim-pack C18 column, using methanol/LiCl((aq)) 1.0 mM (80/20, v/v) as a mobile phase under isocratic elution and UV-Vis detection at 365 nm. The standard curve of ADNPH was linear in the range 3-300 mg L(-1) per injection (20 microL) and the limit of detection (LOD) for acetaldehyde was 2.03 microg L(-1), with a correlation coefficient greater than 0.999 and a precision (relative standard deviation, RSD) of 5.6% (n = 5). Recovery studies were performed by fortifying fuel samples with acetaldehyde at various concentrations and the results were in the range 98.7-102%, with a coefficient of variation (CV) from 0.2% to 7.2%. Several fuel samples collected from various gas stations were analyzed and the method was successfully applied to the analysis of acetaldehyde in fuel ethanol samples.

Simultaneous determination of zinc, copper, lead, and cadmium in fuel ethanol by anodic stripping voltammetry using a glassy carbon-mercury-film electrode.

A new, versatile, and simple method for quantitative analysis of zinc, copper, lead, and cadmium in fuel ethanol by anodic stripping voltammetry is described. These metals can be quantified by direct dissolution of fuel ethanol in water and subsequent voltammetric measurement after the accumulation step. A maximum limit of 20% ( v/ v) ethanol in water solution was obtained for voltammetric measurements without loss of sensitivity for metal species. Chemical and operational optimum conditions were analyzed in this study; the values obtained were pH 2.9, a 4.7-microm thickness mercury film, a 1,000-rpm rotation frequency of the working electrode, and a 600-s pre-concentration time. Voltammetric measurements were obtained using linear scan (LSV), differential pulse (DPV), and square wave (SWV) modes and detection limits were in the range 10(-9)-10(-8) mol L(-1) for these metal species. The proposed method was compared with a traditional analytical technique, flame atomic absorption spectrometry (FAAS), for quantification of these metal species in commercial fuel ethanol samples.