Brassica oleracea Var italica by-Products Prevent Lipid Accumulation and Cell Death in a Liver Cell Model of Lipid Toxicity.

Obesity, a rising concern in the Eastern world, encompasses several co-morbidities, namely non-alcoholic fatty liver disease (NAFLD). Potential natural-based interventions to decrease the burden of obesity complications are being investigated. Many of the edible parts of plants are not sold for consumption and end up as massive waste, losing nutritional potential. In fact, a sizeable amount of waste is generated within the different steps of the food supply chain, representing a massive loss of both plant material and natural resources. A good example is Brassica by-products (BBPs). The objective of this work was to investigate the effect of three different extracts from broccoli (Brassica oleracea var italica) by-products in an in vitro model of free fatty acid (FFA)-induced lipotoxicity using human hepatoma HepG2 cells. Broccoli leaf, stalk, and inflorescence extracts induced a dose-dependent decrease in the cell viability of HepG2 cells. However, the maximal non-lethal concentrations of leaves, stalks, and inflorescences (10 µg/mL) did not compromise mitochondrial function or neutral lipid accumulation in HepG2 cells. The extracts significantly decreased FFA-induced lipid accumulation in HepG2 cells either in a co-incubation or pre-incubation strategy. The broccoli extracts' capacity to prevent the FFA-induced decrease in catalase activity in HepG2 may explain the observed effects.

Hydropriming and Nutripriming of Bread Wheat Seeds Improved the Flour's Nutritional Value of the First Unprimed Offspring.

Seed hydropriming or nutripriming has been used for wheat biofortification. Previously, the untreated S1 offspring of bread wheat S0 seeds hydro- and nutriprimed with FeSO4.7H2O and/or ZnSO4.7H2O showed improved yield relative to the offspring of untreated S0 seeds. We hypothesize that such improvement would have its origin in the higher quality of S1 seeds resulting from plants whose seeds were primed. In this work, we characterised biochemically the whole-wheat flour of unprimed S1 offspring whose S0 seeds were hydro- and nutriprimed with Fe and/or Zn and compared it to the offspring of untreated S0 seeds (control). We identified and quantified 16 free amino acids and five soluble sugars per offspring using high-performance liquid chromatography and the Association of Official Analytical Chemists (AOAC) methods. The most abundant amino acids were glutamic acid and glutamine, proline, and glycine, presenting their highest contents in the offspring of seeds nutriprimed with 8 ppm Zn (0.351 mmol∙g-1), 8 ppm Fe + 8 ppm Zn (0.199 mmol∙g-1), and (0.135 mmol∙g-1), respectively. The highest contents of glucose (1.91 mg∙g-1 sample), ash (24.90 g∙kg-1 dry matter, DM), and crude protein (209.70 g∙kg-1 DM) were presented by the offspring resulting from 4 ppm Fe + 4 ppm Zn, 8 ppm Zn, and 8 ppm Fe + 8 ppm Zn, respectively. The highest total starch content (630.10 g∙kg-1 DM) was detected in the offspring of seeds soaked in 8 ppm Fe. The nutritional value of the flour of the S1 offspring resulting from nutripriming was significantly higher than the control. Overall, the novelty of our research is that seed priming can improve the quality of the wheat grain and flour, at least till the first offspring, without the need to repeat the presowing treatment. Beyond the study of subsequent generations, the unravelling of transgenerational mechanisms underlying the biochemical improvement of the offspring is approached.

Nutrients, Antinutrients, Phenolic Composition, and Antioxidant Activity of Common Bean Cultivars and their Potential for Food Applications.

Phaseolus vulgaris L. is the most commonly consumed legume in the world, given its high vegetable protein content, phenolic compounds, and antioxidant properties. It also represents one of the most sustainable, low-carbon and sources of food available at present to man. This study aims to identify the nutrients, antinutrients, phenolic composition, and antioxidant profile of 10 common bean cultivars (Arikara yellow, butter, cranberry, red kidney, navy, pinto, black, brown eyed, pink eyed, and tarrestre) from two harvest years, thereby assessing the potential of each cultivar for specific applications in the food industry. Navy and pink eyed beans showed higher potential for enrichment of foodstuffs and gluten-free products due to their higher protein and amino acid contents. Additionally, red kidney, cranberry and Arikara yellow beans had the highest content of phenolic compounds and antioxidant properties, which can act as functional ingredients in food products, thus bringing health benefits. Our study highlights the potential of using specific bean cultivars in the development of nutrient-enriched food and as functional ingredients in diets designed for disease prevention and treatment.

Comparison of near-infrared (NIR) and mid-infrared (MIR) spectroscopy for the determination of nutritional and antinutritional parameters in common beans.

Common beans (Phaseolus vulgaris L.), represent the most consumed legume worldwide and constitute an important source of protein, being also known to contain antinutritional compounds, which compromise nutrients' bioavailability. However, the standard methodologies to assess these constituents are time-consuming and complex. Therefore, the present study evaluated the suitability of near-infrared (NIR) and mid-infrared (MIR) spectroscopies for the development of simple and reliable methods to assess protein, lipids, tannins and phytic acid contents, besides specific amino acids, in whole bean flours. Partial least squares (PLS) regression was used to develop analytical models, and external validation was performed. NIR displayed better performance for the evaluation of protein, lipids, tannins and phytic acid contents, and MIR, for the assessment of specific amino acids. In both techniques, the use of the 1st derivative was the best data treatment. Overall, both techniques represent reliable methods to evaluate the proximate and antinutritional composition of bean flours.

Determination of opiates in whole blood using microextraction by packed sorbent and gas chromatography-tandem mass spectrometry.

In Portugal, and worldwide, the abuse of psychoactive substances is increasing, with a significant incidence of deaths related to their consumption. Opiates are one of the most prevalent group of substances in that context, and they are responsible for a significant impact of the mentioned harms. Therefore, it becomes necessary to equip labs with faster and effective methods to identify and quantify these substances. This work describes the development and validation of a novel analytical method for the simultaneous determination of morphine, codeine and 6-monoacetylmorphine in blood samples by gas chromatography-tandem mass spectrometry (GC-MS/MS), using microextraction by packed sorbent (MEPS) for sample preparation. Before the MEPS procedure, a precipitation step with acetonitrile was performed. The MEPS parameters were optimized using the fractional factorial planning (2k-1) and surface response methodology. The final optimized conditions were: number of strokes (20), amount of formic acid in the washing solution (3.36%), number of washes of the sorbent (1), amount of ammonium hydroxide in the elution solution (2.36%) and number of elution cycles (11). After the extraction procedure, the analytes were derivatized with MSTFA with 5% TMS. Using a sample volume of 250 µL, the method was validated according to internationally accepted standards. The method proved to be linear in the range of 5-1000 ng/mL with coefficients of determination greater than 0.99 for all analytes. Intra-and inter-day precision and accuracy were in accordance with the above-mentioned criteria, presenting coefficients of variation ≤15% and relative errors within a range of ± 15% of the theoretical concentration. The absolute recoveries ranged from 6 to 23%. The validated method was applied to the analysis of real samples, being an advantageous tool for the detection of those substances in blood. This is the first time that GCMS/MS with MEPS was used for the determination of these compounds in biological fluids.

Opioid Use in Pregnant Women and Neonatal Abstinence Syndrome-A Review of the Literature.

Opiate use during pregnancy has been an increasing problem over the last two decades, making it an important social and health concern. The use of such substances may have serious negative outcomes in the newborn, and clinical and cognitive conditions have been reported, including neonatal abstinence syndrome, developmental problems, and lower cognitive performance. These conditions are common when opiates are used during pregnancy, making the prescription of these kinds of drugs problematic. Moreover, the mother may develop opiate addiction, thus, increasing the likelihood of the infant being born with any of those conditions. This paper reviews the use of opiates during pregnancy and focuses mainly on the neonatal abstinence syndrome. First, the commonly prescribed opiates will be identified, namely those usually involved in cases of addiction and/or neonatal abstinence syndrome. Second, published approaches to deal with those problems will be presented and discussed, including the treatment of both the mother and the infant. Finally, we will outline the treatments that are safest and most efficient, and will define future goals, approaches, and research directions for the scientific community regarding this problem.

Alcohol consumption assessment in a student population through combined hair analysis for ethyl glucuronide and fatty acid ethyl esters.

This study aimed to assess alcohol consumption in a university student population though the combined analysis of the alcohol biomarkers ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) in hair samples. A total of 975 hair samples were analysed for EtG and FAEEs using liquid chromatography coupled to mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS/MS), respectively. The results were analysed using the cut-offs proposed by the Society of Hair Testing and receiver operating characteristic (ROC) curve analysis was performed to verify the adequacy of the proposed values for the study population. Good sensitivity and specificity were obtained for both biomarkers, especially for EtG, and a correlation was found with the self-reported alcohol consumption habit. In 56.3% of the abstinent, 65.8% of the moderate and 80.0% of the excessive drinking cases, self-reported alcohol consumption could be confirmed by combined alcohol biomarker analysis. Combined analysis of EtG and FAEEs in hair samples proved to be a valuable tool for the monitoring of alcohol consumption in a student population. For a feasible result interpretation, it is very important to document the use of hair products, cosmetic treatments and washing frequency, and for these to be considered during interpretation. Overall the participants were aware of their consumption pattern, however for doubtful cases and to account for academic calendars, repeated analysis of samples collected at different time frames would be advisable.

Development and validation of a gas chromatography/tandem mass spectrometry method for simultaneous quantitation of several antipsychotics in human plasma and oral fluid.

RATIONALE: Antipsychotic drugs are prescription medications used to treat psychotic disorders, such as schizophrenia, schizoaffective disorder, or psychotic depression. With several antipsychotic drugs currently available all over the world, this class of drugs has quickly gained importance in both the clinical and forensic context. This work describes the development and validation of a methodology for the determination of seven antipsychotic drugs in plasma and oral fluid samples. METHODS: The antipsychotic drugs (chlorpromazine, clozapine, haloperidol, olanzapine, quetiapine, cyamemazine and, levomepromazine) were isolated from 0.2 mL of oral fluid and 0.5 mL of plasma using solid-phase extraction (SPE) following analysis by gas chromatography/tandem mass spectrometry (GC/MS/MS). The method was validated according to the international guidelines in terms of selectivity, linearity, accuracy, precision and recovery. RESULTS: The procedure was linear within 2-600 ng/mL (plasma) and 2-400 ng/mL (oral fluid), the intervals varying according to the compound; a mean R2 value of 0.99 was obtained and the calibrator's accuracy (mean relative error) was within a ±15 % interval for all concentrations. The limits of detection ranged from 1 to 10 ng/mL. Within- and between-run precision and accuracy were acceptable for all studied compounds. The extraction efficiency of the process ranged from 79% to 95%. The method was applied to authentic specimens. CONCLUSIONS: The described method was proven selective and sensitive for the determination of antipsychotics in low sample volumes using SPE and GC/MS/MS. This method was considered suitable not only for routine analysis of patients undergoing antipsychotic treatment (to evaluate compliance), but also in forensic scenarios where the studied compounds may be involved. To the best of our knowledge, this is the first work that reports the determination of antipsychotic drugs in oral fluid using MS/MS.

New grape stems' isolated phenolic compounds modulate reactive oxygen species, glutathione, and lipid peroxidation in vitro: Combined formulations with vitamins C and E.

The antioxidant potential of grape (Vitis vinífera L.) stems has been reported in the last decade although no identification of the individual compounds responsible for such action has been done. In this work, polyphenolic extract of grape stems was processed resorting to semi-preparative HPLC, allowing to obtain 5 purified polyphenols (caftaric acid, malvidin-3-O-glucoside, quercetin-3-O-glucuronide, mailvidin-3-O-(6-O-caffeoyl)-glucoside, and Σ-viniferin), which were fully characterized by HPLC-PDA-ESI-MSn. Isolated compounds were featured on their radical scavenging capacity (DPPH and ABTS), cell viability, anti-inflammatory activity, and capacity to modulate the level of reactive oxygen species, glutathione, lipid peroxidation, and overall oxidative stress in a biological model (human keratinocytes) in vitro, under basal and oxidative conditions. The results obtained noticed the combinations malvidin-3-O-glucoside+Vitamin E and quercetin-3-O-glucuronide+vitamin C as the most effective, allowing to improve the capacity of complete extracts or individual compounds, and being candidates to be used in the development of new functional products.

Evaluating the freezing impact on the proximate composition of immature cowpea (Vigna unguiculata L.) pods: classical versus spectroscopic approaches.

BACKGROUND: Freezing represents a common conservation practice regarding vegetal foodstuffs. Since compositional features need to be monitored during storage, the development of rapid monitoring tools suitable for assessing nutritional characteristics arises as a pertinent issue. In this study, cowpea (Vigna unguiculata L.) pods, both fresh and after 6 and 9 months of freezing at -18 °C, were evaluated by high-performance liquid chromatography for their content of protein as well as of essential and nonessential amino acids, while their Fourier transform infrared spectra in the mid infrared (MIR) and near infrared (NIR) ranges were concomitantly registered to assess the feasibility of this approach for the traceability of these frozen matrices. RESULTS: For the NIR interval, the application of the 1st derivative to the spectral data retrieved the best results, while for lower concentrations the application of the Savitzky-Golay algorithm was indispensable to achieve quantification models for the amino acids. MIR is also suitable for this purpose, though being unable to quantify amino acids with concentrations below 0.07 mmol g-1 dry weight, irrespective of the data treatment used. CONCLUSIONS: The spectroscopic approach constitutes a methodology suitable for monitoring the impact of freezing on the nutritional properties of cowpea pods, allowing accurate quantification of the protein and amino acid contents, while NIR displayed better performance. © 2017 Society of Chemical Industry.

Determination of ethyl glucuronide and fatty acid ethyl esters in hair samples.

Hair testing for alcohol biomarkers is an important tool for monitoring alcohol consumption. We propose two methods for assessing alcohol exposure through combined analysis of ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) species (ethyl myristate, palmitate, stearate and oleate) in hair (30 mg). EtG was analysed by liquid chromatography-tandem mass spectrometry, while FAEEs were analysed by gas chromatography-tandem mass spectrometry using electron impact ionization. Both methods were validated according to internationally accepted guidelines. Linearity was proven between 3 and 500 pg/mg for EtG and 30-5000 pg/mg for FAEEs, and the limits of quantification were 3 pg/mg for EtG and 30 pg/mg for each of the four FAEEs. Precision and accuracy were considered adequate, processed EtG samples were found to be stable for up to 96 h left in the injector and processed FAEEs samples for up to 24 h. Matrix effects were not significant. Both methods were applied to the analysis of 15 authentic samples, using the cut-off values proposed by the Society of Hair Testing for interpretation. The results agreed well with the self-reported alcohol consumption in most cases, and demonstrated the suitability of the methods to be applied in routine analysis of alcohol biomarkers, allowing monitoring consumption using low sample amounts.

Bioanalytical procedures and developments in the determination of alcohol biomarkers in biological specimens.

Excessive alcohol consumption is a global problem, and consequently its evaluation is of great clinical and forensic interest. Alcohol biomarkers have been the focus of several research works in the past decades, with new compounds being studied in more recent years. The main objective of this review is to discuss topics for an analyst to consider when evaluating alcohol consumption through the analysis of alcohol biomarkers in biological specimens. For this, existing alcohol biomarkers will be reviewed, including carbohydrate-deficient transferrin, 5-hydroxytryptophol, ethanol, hemoglobin-associated acetaldehyde, fatty acid ethyl esters, ethyl glucuronide, ethyl sulfate and phosphatidylethanol. Additionally, their potential will be discussed, as well as analytical considerations, main challenges, limitations, data interpretation and existing methodologies for their determination in biological specimens.

Determination of ethyl glucuronide in hair to assess excessive alcohol consumption in a student population.

Hair analysis for ethyl glucuronide (EtG) was used to evaluate the pattern of alcohol consumption amongst the Portuguese university student population. A total of 975 samples were analysed. For data interpretation, the 2014 guidelines from the Society of Hair Testing (SoHT) for the use of alcohol markers in hair for the assessment of both abstinence and chronic excessive alcohol consumption were considered. EtG concentrations were significantly higher in the male population. The effect of hair products and cosmetics was evaluated by analysis of variance (ANOVA), and significant lower concentrations were obtained when conditioner or hair mask was used or when hair was dyed. Based on the analytical data and information obtained in the questionnaires from the participants, receiver operating characteristic (ROC) curves were constructed in order to determine the ideal cut-offs for our study population. Optimal cut-off values were estimated at 7.3 pg/mg for abstinence or rare occasional drinking control and 29.8 pg/mg for excessive consumption. These values are very close to the values suggested by the SoHT, proving their adequacy to the studied population. Overall, the obtained EtG concentrations demonstrate that participants are usually well aware of their consumption pattern, correlating with the self-reported consumed alcohol quantity, consumption habits and excessive consumption close to the time of hair sampling.

Combinatorial delivery of Crizotinib-Palbociclib-Sildenafil using TPGS-PLA micelles for improved cancer treatment.

The co-delivery of multiple chemotherapeutics by micellar delivery systems is a valuable approach to improve cancer treatment since various disease hallmarks can be targeted simultaneously. However, the delivery of multiple drugs requires a nanocarrier structure that can encapsulate various bioactive molecules. In this study, we evaluate the simultaneous encapsulation of a novel triple drug combination in D-α-tocopheryl polyethylene glycol 1000 succinate-poly(lactic acid) (TPGS-PLA) amphiphilic micelles for cancer therapy. The drug mixture involves two anti-tumoral drugs, Crizotinib and Palbociclib combined with Sildenafil, a compound that is capable of increasing drug accumulation in the intracellular compartment. Such combination aims to achieve an enhanced cytotoxic effect in cancer cells. Our results demonstrated that TPGS-PLA copolymers self-assembled into stable nanosized micelles (158.3nm) capable of co-encapsulating the three drugs with high loading efficiency. Triple drug loaded TPGS-PLA micelles were internalized in A549 non-small lung cancer cells and exhibited an improved cytotoxic effect in comparison with single (Crizotinib) or dual (Crizotinib-Palbociclib) drug loaded micelles, indicating the therapeutic potential of the triple co-delivery strategy. These findings demonstrate that TPGS-PLA micelles are suitable carriers for multiple drug delivery and also that this particular drug combination may have potential to improve cancer treatment.

Analysis of Salvinorin A in urine using microextraction in packed syringe and GC-MS/MS.

BACKGROUND: The aim of this work was to develop and validate a method for the determination of Salvinorin A in human urine using microextraction by packed sorbent (MEPS) and GC-MS/MS. RESULTS: The technique uses a sample volume as low as 0.2 ml, and the analyte was extracted using a C18 sorbent. The method showed to be linear between 20 and 1000 ng/ml and presented a LOD of 5 ng/ml. Intra- and inter-day precision and accuracy were acceptable. Absolute recoveries ranged from 71 to 80%. CONCLUSION: GC-MS/MS with MEPS demonstrated to be a fast and simple procedure for the quantification of Salvinorin A in urine. This is the first time that GC-MS/MS with MEPS was used for the determination of this compound in biological fluids. Furthermore, the device could be reused for up to 80 extractions, which accounted for a lower cost of analysis.

Analytical approach to determine biogenic amines in urine using microextraction in packed syringe and liquid chromatography coupled to electrochemical detection.

The goal of this work was to develop and validate an analytical method for the detection and quantification of the biogenic amines serotonin (5-HT), dopamine (DA) and norepinephrine (NE), using microextraction in packed syringe (MEPS) and liquid chromatography coupled to electrochemical detection (HPLC-ED) in urine. The method was validated according to internationally accepted guidelines from the Food and Drug Administration. Linearity was established between 50 and 1000 ng/mL for 5-HT and between 5 and 1000 ng/mL for DA and NE, with determination coefficients (R(2)) >0.99 for all compounds. The limits of quantification and detection were respectively 50 and 20 ng/mL for 5-HT, and 5 and 2 ng/mL for DA and NE. Within- and between-run precision ranged from 0.84 to 9.41%, while accuracy ranged from 0.79 to 12.76% for all compounds. The intermediate precision and accuracy were 1.50-8.36 and 0.54-13.51%, respectively. The method was found suitable for clinical routine analysis of the studied compounds, using a sample volume of 0.5 mL. This is the first study employing a commercially available MEPS column for the simultaneous detection and quantification of 5-HT, DA and NE in urine by coulometric detection.