A new adsorptive 3D-printed sampling device for simultaneous determination of 63 urinary organic acids by LC-MS/MS.

BACKGROUND: The detection and quantification of urinary metabolites play an important role in disease diagnosis. In most cases, urinary analyses are done with liquid urine samples, which must be quickly transported to the laboratory to avoid metabolites degradation that is associated with temperature fluctuations. Consequently, dried sampling devices have emerged to minimize analyte degradation. However, most commercial dried sampling devices are expensive, aggregate low volumes, and need better analytical sensitivity. Therefore, a new dry urine sampling device that is inexpensive, suitable for domestic sampling operation, and efficient for quantifying metabolites without requiring high-resolution instruments is proposed in the present study. RESULTS: The newly designed dry urine sampling device was produced by 3D printing that efficiently determines 63 urinary organic acids using liquid chromatography coupled with mass spectrometry (LC-MS/MS). The system's efficiency was demonstrated with analytical figures of merit, such as precision, accuracy, and stability of analytes after the sampling and storing of ordinary urine samples. The limits of quantification ranged from 0.01 to 0.42 ng mL-1. Precision and accuracy tests showed relative standard deviations of less than 15 %. The urine stability in the sampling device was high within seven days without any significant degradation of the metabolites. The method was applied to the analysis of 10 human urine samples and compared to a conventional method without the use of the sampling device. The results showed no statistically significant differences, demonstrating the method's efficiency. SIGNIFICANCE: The proposed 3-D printing device was developed with fast, low-cost manufacturing features and can be manufactured with different volumetric capacities, adaptable to the needs of each user. Furthermore, it is innovative because this is the first sampling device that is effective for the simultaneous storage and preservation of several important urinary metabolites. Thus, it is anticipated that its application would contribute significantly to the identification of metabolic disorders.

Effect of sweetened beverages intake on salivary aspartame, insulin and alpha-amylase levels: A single-blind study.

The objective was to assess aspartame excretion in saliva and the salivary insulin, total protein (TP), and alpha-amylase (AMI) levels in response to the ingestion of sweetened beverages (sodium cyclamate, aspartame, acesulfame, and sucrose). Fifteen healthy participants were included in a single-blinded trial with the intake of Diet soft drink, Regular soft drink, Water + sweeteners, Low sucrose content (3.5 g), and Water (blank) in 5 different days. In each day, saliva was collected at T0 (fasting), T1 (15 min after test-drink intake), T2 (30 min), T3 (60 min), and T4 (120 min) for the measurement of salivary aspartame (HPLC), TP, AMI (ELISA assays) and insulin levels (chemiluminescence). Chi-square, Friedman, ANOVA and Spearman correlation tests were applied. The late-perceived sweet/sour residual flavor was reported at a frequency of 80%, 60% and 20% after ingestion of artificially sweetened drinks, beverages with sucrose, and plain water, respectively (p < 0.05). Aspartame was detected in saliva after artificially sweetened drinks intake, with highest area under the peak for the Diet soft drink (p = 0.014). No change was observed for TP and AMI levels during the 120 min. Insulin levels increased 1 h after soft-drinks ingestion (regular and diet), while the levels did not change for Low sucrose content and Water + sweeteners test-drinks. Salivary aspartame correlated with insulin levels only after Diet soft drink intake (rho ≥ 0.7; p < 0.05). As aspartame can be detected in saliva and swallowed again until completely excreted, these results contribute to the knowledge of the biological fate of artificial sweeteners and the study of health outcomes.

Biomonitoring of polycyclic aromatic hydrocarbons in Brazilian pregnant women: Urinary levels and health risk assessment.

Over the years, humans have been continuously exposed to several compounds directly generated by industrial processes and/or present in consumed products. Polycyclic aromatic hydrocarbons (PAHs) are legacy pollutants ubiquitous in the environment and represent the main chemical pollutants in urban areas. Worldwide, studies that aim to understand the impacts of exposure to these chemicals have gained increasing prominence due to their potential toxicity profile, mainly concerning genotoxicity and carcinogenicity. Human biomonitoring (HB) is an analytical approach to monitoring population exposure to chemicals; however, these studies are still limited in Brazil. Thus, this work aimed to evaluate the exposure of Brazilian pregnant women to PAHs through HB studies. Besides, the risk characterization of this exposure was performed. For this purpose, urine samples from 358 Brazilian pregnant women were used to evaluate 11 hydroxylated metabolites of PAHs employing gas chromatography coupled to mass spectrometry. The 1OH-naphthol and 2OH-naphthol were detected in 100% of the samples and showed high levels, corresponding to 16.99 and 3.62 µg/g of creatinine, respectively. 2OH-fluorene (8.12 µg/g of creatinine) and 9OH-fluorene (1.26 µg/g of creatinine) were detected in 91% and 66% of the samples, respectively. Benzo(a)pyrene (BaP) metabolites were detected in more than 50% of the samples (0.58-1.26 µg/g of creatinine). A hazard index of 1.4 and a carcinogenic risk above 10-4 were found for BaP metabolites in the risk characterization. Therefore, our findings may indicate that exposure to PAHs poses a potential risk to pregnant women's health and a high probability of carcinogenic risk due to their exposure to BaP. Finally, this work shows the need for more in-depth studies to determine the sources of exposure and the implementation of health protection measures regarding the exposure of the Brazilian population to PAHs.

Potential Health Risk to Brazilian Infants by Polybrominated Diphenyl Ethers Exposure via Breast Milk Intake.

Polybrominated diphenyl ethers (PBDEs) are ubiquitous flame retardants and are environmentally persistent. PBDEs show endocrine disruption, neurotoxicity, and lower birth weight in infants, and their human body burden has become a public health concern. The infants' exposure begins in the prenatal period and continues via breast milk ingestion, although, little is known about the factors that may influence this exposure. In this study, PBDE levels in Brazilian breast milk were assessed in 200 lactating women. The risk assessment of infants' exposure to PBDE was performed through the estimated daily intake (EDI) calculation. The geometric mean (GM) of ∑PBDEs levels was 2.33 (0.14-6.05) ng/g wet weight. At least one PBDE congener was detected in the samples, and the 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) showed a 100% of detection rate (GM of 1.05 ng/g). Location of residence, maternal level education, monthly salary, and race were positively associated with PBDE levels (p < 0.05). The EDI of BDE-47 was higher in Belo Horizonte (8.29 ng/kg/day) than in Viçosa (6.36 ng/kg/day), as well as for the ∑PBDEs (19.77 versus 12.78 ng/kg/day) (p < 0.05). Taking the high detection rate of PBDEs in breast milk and their toxicity, continuous studies on infant exposure, fetal growth, and child neurodevelopment are requested.

A fast-multiclass method for the determination of 21 endocrine disruptors in human urine by using vortex-assisted dispersive liquid-liquid microextraction (VADLLME) and LC-MS/MS.

Simplicity, speed, and reduced cost are essential demands for routine analysis in human biomonitoring studies. Moreover, the availability of higher volumes of human specimens is becoming more restrictive due to ethical controls and to the costs associated with sample transportation and storage. Thus, analytical methods requiring much lower sample volumes associated with simultaneous detection capability (multiclass analysis) are with a very high claim. In this sense, the present approach aimed at the development of a method for preconcentration and simultaneous determination of four classes of endocrine disruptors (seven bisphenols, seven parabens, five benzophenones, and two antimicrobials) in the urine. The approach is based on vortex-assisted dispersive liquid-liquid microextraction (VADLLME) and high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). After optimization of the significant parameters of VADLLME extraction, the proposed procedure showed to be simple, fast, sensitive, requiring only 1.0 mL of urine, 400 µL of organic solvents with a total stirring time of 20 s. Moreover, a variation of inter-day and between-day runs were lower than 10.0% and 11.0%, respectively. Finally, the proposed method was successfully applied to the analysis of 50 urine samples of Brazilian pregnant women to establish reference ranges.

A critical viewpoint on current issues, limitations, and future research needs on micro- and nanoplastic studies: From the detection to the toxicological assessment.

Increasing scientific attention on the presence of micro- and nanoplastics (MNPs) in the environments and their potential toxic effects on humans and the ecosystems is evident. Accordingly, the number of publications on this topic has increased substantially from only 5 in 2010 to more than 850 in 2019. Thus, this critical review aimed at providing state-of-the-art information on the existing methods for characterization and detection of MNPs in various matrices, as well as the reported toxic effects of MNPs in both in vivo and in vitro systems, anticipating challenges and providing future needs to improve the current scientific knowledge. We performed a systematic search of recent literature on available methodologies for the characterization/detection of MNPs in different samples, and the summary of such protocols is provided. Also, the existing procedures for in vitro and in vivo toxicity evaluation of MNPs were critically described. The results of our search revealed that quite a great deal of effort had been made to detect, characterize, and quantify the fate and effect of MNPs. However, we are still far from a complete understanding of behaviors of MNPs in the environments and biological systems. Thus, there is a need to advance the existing protocols to improve data accuracy. Besides, more studies that focus on uptake kinetics, accumulation, and biodistribution of MNPs in biological systems are required.

An overview of the current progress, challenges, and prospects of human biomonitoring and exposome studies.

Human Biomonitoring (HB), the process for determining whether and to what extent chemical substances penetrated our bodies, serves as a useful tool to quantify human exposure to pollutants. In cases of nutrition and physiologic status, HB plays a critical role in the identification of excess or deficiency of essential nutrients. In pollutant HB studies, levels of substances measured in body fluids (blood, urine, and breast milk) or tissues (hair, nails or teeth) aid in the identification of potential health risks or associated adverse effects. However, even as a widespread practice in several countries, most HB studies reflect exposure to a single compound or mixtures which are measured at a single time point in lifecycle. On the other hand, throughout an individual's lifespan, the contact with different physical, chemical, and social stressors occurs at varying intensities, differing times and durations. Further, the interaction between stressors and body receptors leads to dynamic responses of the entire biological system including proteome, metabolome, transcriptome, and adductome. Bearing this in mind, a relatively new vision in exposure science, defined as the exposome, is postulated to expand the traditional practice of measuring a single exposure to one or few chemicals at one-time point to an approach that addresses measures of exposure to multiple stressors throughout the lifespan. With the exposome concept, the science of exposure advances to an Environment-Wide Association Perspective, which might exhibit a stronger relationship with good health or disease conditions for an individual (phenotype). Thus, this critical review focused on the current progress of HB and exposome investigations, anticipating some challenges, strategies, and future needs to be taken into account for designing future surveys.

In vitro enantioselective human liver microsomal metabolism and prediction of in vivo pharmacokinetic parameters of tetrabenazine by DLLME-CE.

A new capillary electrophoresis method for the enantioselective analysis of cis- and trans- dihydrotetrabenazine (diHTBZ) after in vitro metabolism by human liver microsomes (HLMs) was developed. The chiral electrophoretic separations were performed by using tris-phosphate buffer (pH 2.5) containing 1% (w/v) carboxymethyl-ß-CD as background electrolyte with an applied voltage of +15kV and capillary temperature kept at 15°C. Dispersive liquid-liquid microextraction was employed to extract the analytes from HLMs. Dichloromethane was used as extraction solvent (75µL) and acetone as disperser solvent (150µL). The method was validated according to official guidelines and showed to be linear over the concentration range of 0.29-19.57µmolL(-1) (r=0.9955) for each metabolite enantiomer. Within- and between-day precision and accuracy evaluated by relative standard deviation and relative error were lower than 15% for all enantiomers. The stability assay showed that the analytes kept stable under handling, storage and in metabolism conditions. After method validation, an enantioselective in vitro metabolism and in vivo pharmacokinetic prediction was carried out. This study showed a stereoselective metabolism and the observed kinetic profile indicated a substrate inhibition behavior. DiHTBZ enantiomers were catalyzed mainly by CYP2C19 and the predicted clearance suggests that liver metabolism is the main route for TBZ elimination which supports the literature data.

A new enantioselective CE method for determination of oxcarbazepine and licarbazepine after fungal biotransformation.

The present work describes, for the first time, the simultaneous separation of oxcarbazepine (OXC) and its active metabolite 10-hydroxy-10,11-dihydrocarbamazepine (licarbazepine, Lic) by chiral CE. The developed method was employed to monitor the enantioselective biotransformation of OXC into its active metabolite by fungi. The electrophoretic separations were performed using 10 mmol/L of a Tris-phosphate buffer solution (pH 2.5) containing 1% w/v of ß-CD phosphate sodium salt (P-ß-CD) as running electrolyte, -20 kV of applied voltage and a 15°C capillary temperature. The method was linear over the concentration range of 1000-30 000 ng/mL for OXC and 75-900 ng/mL for each Lic enantiomer (r ≥ 0.9952). Within-day precision and accuracy evaluated by RSD and relative errors, respectively, were lower than 15% for all analytes. The validated method was used to evaluate the enantioselective biotransformation of OXC, mediated by fungi, into its active metabolite Lic. This study showed that the fungi Glomerella cingulata (VA1) and Beuveria bassiana were able to enantioselectively metabolize the OXC into Lic after 360 h of incubation. Biotransformation by the fungus Beuveria bassiana showed 79% enantiomeric excess for (S)-(+)-Lic, while VA1 gave an enantiomeric excess of 100% for (S)-(+)-Lic. This study opens a new route to the drug (S)-(+)-licarbazepine.

Solid phase microextraction and LC-MS/MS for the determination of paliperidone after stereoselective fungal biotransformation of risperidone.

The present work describes for the first time the use of SPME coupled to LC-MS/MS employing the polar organic mode in a stereoselective fungal biotransformation study to investigate the fungi ability to biotransform the drug risperidone into its chiral and active metabolite 9-hydroxyrisperidone (9-RispOH). The chromatographic separation was performed on a Chiralcel OJ-H column using methanol:ethanol (50:50, v/v) plus 0.2% triethylamine as the mobile phase at a flow rate of 0.8 mL min(-1). The SPME process was performed using a C18 fiber, 30 min of extraction time and 5 min of desorption time in the mobile phase. The method was completely validated and all parameters were in agreement with the literature recommendations. The Cunninghamella echinulata fungus was able to biotransform risperidone into the active metabolite, (+)-9-RispOH, resulting in 100% of enantiomeric excess. The Cunninghamella elegans fungus was also able to stereoselectively biotransform risperidone into (+)- and (-)-9-RispOH enantiomers at different rates.