Exploring tea and herbal infusions consumption patterns and behaviours: The case of Portuguese consumers.

Consumption of tea and herbal infusions (THIs) have a long history in traditional medicine and cultural practices. The health-promoting benefits attributed to THIs are considered influential factors in consumer choices. However, there is limited data on consumer choices and attitudes that might interfere with the positive effects associated with THIs consumption. The aim of this study was to investigate the consumption pattern and behavior of THIs consumers in Portugal, assessing the influence of socio-demographic factors on the selection of THIs products and consumer practices related to these beverages. An online survey was conducted, and from the collected data, 720 responses met the aim of the study and were further analyzed. Most of the respondents were female, 74.4%, belonging to the 40-60 age group (40.6%) and were medium consumers of THIs (47.2%). Green tea was the most consumed type among participants, and its consumption was associated not only with age but also with the pattern of THIs consumption. Despite that, participants preferred herbal infusions, with citronella, chamomile, and lemon verbena being the most consumed types. For certain types of herbal infusions, consumption was associated with age, while other types were preferred by moderate or heavy consumers. Most participants purchased THIs in supermarkets, registered trademark and brand stores, in the form of THIs bag. Light consumers use only bag, while medium/heavy consumers indicated the use of other forms. Almost half of the respondents admitted to not reading the information on product labels before consumption and using THIs after the expiry date, while only one-third of them declared paying attention to the label instructions. This study revealed the impact of socio-demographic factors as age on the consumption patterns and preferences of THIs of consumers. Of concern is the neglect of label usage among Portuguese consumers. This emphasizes the urgency of implementing interventions to guide proper label use and promote good consumption practices to ensure the quality of THIs products.

Toxicity of butylone and its enantiomers to Daphnia magna and its degradation/toxicity potential using advanced oxidation technologies.

Butylone (BTL) is a chiral synthetic cathinone available as a racemate and reported as contaminant in wastewater effluents. However, there are no studies on its impact on ecosystems and possible enantioselectivity in ecotoxicity. This work aimed to evaluate: (i) the possible ecotoxicity of BTL as racemate or its isolated (R)- and (S)- enantiomers using Daphnia magna; and (ii) the efficiency of advanced oxidation technologies (AOTs) in the removal of BTL and reduction of toxic effects caused by wastewaters. Enantiomers of BTL were obtained by liquid chromatography (LC) using a chiral semi-preparative column. Enantiomeric purity of each enantiomer was > 97 %. For toxicity assessment, a 9-day sub-chronic assay was performed with the racemate (at 0.10, 1.0 or 10 µg L-1) or each enantiomer (at 0.10 or 1.0 µg L-1). Changes in morphophysiological, behavioural, biochemical and reproductive endpoints were observed, which were dependent on the form of the substance and life stage of the organism (juvenile or adult). Removal rates of BTL in spiked wastewater (10 µg L-1) treated with different AOTs (ultraviolet, UV; ozonation, O3; and UV/O3) were similar and lower than 29 %. The 48 h D. magna acute toxicity assays demonstrated a reduction in the toxicity of the treated spiked effluents, but no differences were found amongst AOTs treatments. These results warn for the contamination and negative impact of BTL on ecosystems and highlight the need for efficient removal processes.

Evaluation of Antitumor Activity of Xanthones Conjugated with Amino Acids.

Cancer is a complex disease characterized by several alterations, which confer, to the cells, the capacity to proliferate uncontrollably and to resist cellular death. Multiresistance to conventional chemotherapy drugs is often the cause of treatment failure; thus, the search for natural products or their derivatives with therapeutic action is essential. Chiral derivatives of xanthones (CDXs) have shown potential inhibitory activity against the growth of some human tumor cell lines. This work reports the screening of a library of CDXs, through viability assays, in different cancer cell lines: A375-C5, MCF-7, NCI-H460, and HCT-15. CDXs' effect was analyzed based on several parameters of cancer cells, and it was also verified if these compounds were substrates of glycoprotein-P (Pgp), one of the main mechanisms of resistance in cancer therapy. Pgp expression was evaluated in all cell lines, but no expression was observed, except for HCT-15. Also, when a humanized yeast expressing the human gene MDR1 was used, no conclusions could be drawn about CDXs as Pgp substrates. The selected CDXs did not induce significant differences in the metabolic parameters analyzed. These results show that some CDXs present promising antitumor activity, but other mechanisms should be triggered by these compounds.

Chromatographic Methods for Detection and Quantification of Pyrrolizidine Alkaloids in Flora, Herbal Medicines, and Food: An Overview.

Pyrrolizidine alkaloids (PAs) are natural toxins produced by some plants that gained special interest due to their potential hazardous effects in humans and animals. These substances have been found in wild flora, herbal medicines and food products raising health concerns. Recently, maximum concentration levels of PAs were established for some food products; however, maximum daily intake frequently surpasses the upper limit set by the competent authorities posing a health risk. Given the scarcity or absence of occurrence data on PAs in many products, there is an urgent need to measure their levels and establish safety intake levels. Analytical methods have been reported to detect and quantify PAs in different matrices. The commonly used chromatographic methodologies provides accurate and reliable results. Analytical methods include diverse steps as extraction and sample preparation procedures that are critical for sensitivity and selectivity of the analytical method. Great efforts have been directed toward optimization of extraction procedures, clean up and chromatographic conditions to improve recovery, reduce matrix effects, and achieve low limits of detection and quantification. Therefore, this paper aims to give a general overview about the occurrence of PAs in flora, herbal medicines, and foodstuff; and discuss the different chromatographic methodologies used for PAs analysis, namely extraction and sample preparation procedures and chromatographic conditions.

Synthesis and Anti-Inflammatory Evaluation of a Library of Chiral Derivatives of Xanthones Conjugated with Proteinogenic Amino Acids.

In recent decades, the relationship between drug chirality and biological activity has been assuming enormous importance in medicinal chemistry. Particularly, chiral derivatives of xanthones (CDXs) have interesting biological activities, including enantioselective anti-inflammatory activity. Herein, the synthesis of a library of CDXs is described, by coupling a carboxyxanthone (1) with both enantiomers of proteinogenic amino esters as chiral building blocks (2-31), following the chiral pool strategy. The coupling reactions were performed at room temperature with good yields (from 44 to 99.9%) and very high enantiomeric purity, with most of them presenting an enantiomeric ratio close to 100%. To afford the respective amino acid derivatives (32-61), the ester group of the CDXs was hydrolyzed in mild alkaline conditions. Consequently, in this work, sixty new derivatives of CDXs were synthetized. The cytocompatibility and anti-inflammatory activity in the presence of M1 macrophages were studied for forty-four of the new synthesized CDXs. A significant decrease in the levels of a proinflammatory cytokine targeted in the treatment of several inflammatory diseases, namely interleukin 6 (IL-6), was achieved in the presence of many CDXs. The amino ester of L-tyrosine (X1AELT) was the most effective in reducing IL-6 production (52.2 ± 13.2%) by LPS-stimulated macrophages. Moreover, it was ≈1.2 times better than the D-enantiomer. Indeed, enantioselectivity was observed for the majority of the tested compounds. Thus, their evaluation as promising anti-inflammatory drugs should be considered.

Toxicity of the 3,4-Methylenedioxymethamphetamine and Its Enantiomers to Daphnia magna after Isolation by Semipreparative Chromatography.

MDMA (3,4-methylenedioxymethamphetamine) is a chiral psychoactive recreational drug sold in illicit markets as racemate. Studies on the impact of MDMA on aquatic organisms are scarce. While enantioselectivity in toxicity in animals and humans has been reported, none is reported on aquatic organisms. This study aimed to investigate the ecotoxicological effects of MDMA and its enantiomers in Daphnia magna. For that, enantiomers (enantiomeric purity > 97%) were separated by liquid chromatography using a homemade semipreparative chiral column. Daphnids were exposed to three concentrations of (R,S)-MDMA (0.1, 1.0 and 10.0 µg L-1) and two concentrations of (R)- and (S)-enantiomers (0.1 and 1.0 µg L-1) over the course of 8 days. Morphophysiological responses were dependent on the substance form and daphnia development stage, and they were overall not affected by the (R)-enantiomer. Changes in swimming behaviour were observed for both the racemate and its enantiomers, but enantioselective effects were not observed. Reproductive or biochemical changes were not observed for enantiomers whereas a significant decrease in acetylcholinesterase and catalase activity was noted at the highest concentration of (R,S)-MDMA (10 µg L-1). Overall, this study showed that sub-chronic exposure to MDMA racemate and its enantiomers can interfere with morphophysiological and swimming behaviour of D. magna. In general, the (R)-enantiomer demonstrated less toxicity than the (S)-enantiomer.

Echinacea purpurea Fractions Represent Promising Plant-Based Anti-Inflammatory Formulations.

Echinacea purpurea is traditionally used in the treatment of inflammatory diseases. Therefore, we investigated the anti-inflammatory capacity of E. purpurea dichloromethanolic (DE) and ethanolic extracts obtained from flowers and roots (R). To identify the class of compounds responsible for the strongest bioactivity, the extracts were fractionated into phenol/carboxylic acid (F1) and alkylamide fraction (F2). The chemical fingerprint of bioactive compounds in the fractions was evaluated by LC-HRMS. E. purpurea extracts and fractions significantly reduced pro-inflammatory cytokines (interleukin 6 and/or tumor necrosis factor) and reactive oxygen and nitrogen species (ROS/RNS) production by lipopolysaccharide-stimulated primary human monocyte-derived macrophages. Dichloromethanolic extract obtained from roots (DE-R) demonstrated the strongest anti-inflammatory activity. Moreover, fractions exhibited greater anti-inflammatory activity than whole extract. Indeed, alkylamides must be the main compounds responsible for the anti-inflammatory activity of extracts; thus, the fractions presenting high content of these compounds presented greater bioactivity. It was demonstrated that alkylamides exert their anti-inflammatory activity through the downregulation of the phosphorylation of p38, ERK 1/2, STAT 3, and/or NF-κB signaling pathways, and/or downregulation of cyclooxygenase 2 expression. E. purpurea extracts and fractions, mainly DE-R-F2, are promising and powerful plant-based anti-inflammatory formulations that can be further used as a basis for the treatment of inflammatory diseases.

On the Bioactivity of Echinacea purpurea Extracts to Modulate the Production of Inflammatory Mediators.

Inflammatory diseases are the focus of several clinical studies, due to limitations and serious side effects of available therapies. Plant-based drugs (e.g., salicylic acid, morphine) have become landmarks in the pharmaceutical field. Therefore, we investigated the immunomodulatory effects of flowers, leaves, and roots from Echinacea purpurea. Ethanolic (EE) and dichloromethanolic extracts (DE) were obtained using the Accelerated Solvent Extractor and aqueous extracts (AE) were prepared under stirring. Their chemical fingerprint was evaluated by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The pro- and anti-inflammatory effects, as well as the reduction in intracellular reactive oxygen and nitrogen species (ROS/RNS), of the different extracts were evaluated using non-stimulated and lipopolysaccharide-stimulated macrophages. Interestingly, AE were able to stimulate macrophages to produce pro-inflammatory cytokines (tumor necrosis factor -TNF-α, interleukin -IL-1ß, and IL-6), and to generate ROS/RNS. Conversely, under an inflammatory scenario, all extracts reduced the amount of pro-inflammatory mediators. DE, alkylamides-enriched extracts, showed the strongest anti-inflammatory activity. Moreover, E. purpurea extracts demonstrated generally a more robust anti-inflammatory activity than clinically used anti-inflammatory drugs (dexamethasone, diclofenac, salicylic acid, and celecoxib). Therefore, E. purpurea extracts may be used to develop new effective therapeutic formulations for disorders in which the immune system is either overactive or impaired.

Quantification of a Sulfated Marine-Inspired Antifouling Compound in Several Aqueous Matrices: Biodegradation Studies and Leaching Assays from Polydimethylsiloxane Coatings.

The development of marine-inspired compounds as non-toxic antifouling (AF) agents has been pursued in the last years. Sulfur is the third most common element in seawater. Sulfur is present in oxygenated seawater as sulfate anion (SO42-), which is the most stable combination of sulfur in seawater, and several promising AF secondary metabolites with sulfate groups have been described. However, sulfated compounds proved to be an analytical challenge to quantify by HPLC. Taking these facts into consideration, this work presents the development and validation of a method for the quantification of gallic acid persulfate (GAP) in seawater and ultrapure water matrix, based on hydrophilic interaction liquid chromatography (HILIC). This method was used to evaluate GAP stability following several abiotic and biotic degradation assays, and to quantify its release in seawater from room-temperature-vulcanizing polydimethylsiloxane commercial coating. GAP was very stable in several water matrices, even at different pH values and in the presence/absence of marine microorganisms and presented a leaching value lower than 0.5%. This work discloses HILIC as an analytical method to overcome the difficulties in quantifying sulfated compounds in water matrices and highlights the potential of GAP as a promising long-lasting coating.

Analytical Methods for Determination of BPA Released from Dental Resin Composites and Related Materials: A Systematic Review.

Knowing the impacts of bisphenol A (BPA) on human health, this systematic review aimed to gather the analytical methods for the quantification of BPA release of BPA in dental materials in in vitro and in vivo (biological fluids) studies. A brief critical discussion of the impacts of BPA on human health and the possible association with BPA in dental materials was also presented. The research was carried out by three independent researchers, (according to PRISMA guidelines) in PUBMED and SCOPUS databases, by searching for specific keywords and articles published between January 2011 and February 2022. Seventeen articles met the eligibility criteria and were included in this systematic review: 10 in vitro and 7 in vivo. In in vitro studies, the highest amounts of BPA released were from flowable to conventional resins, followed by resin-modified glass ionomer. In contrast, the smallest amount was released from "BPA-free" composites and CAD-CAM blocks. Regarding in vivo studies, a higher concentration of BPA were found in saliva than urine or blood. The best analytical method for trace quantifying BPA is LC-MS/MS (Liquid Chromatography with Tandem Mass Spectrometry) due to its selectivity, low quantification limits, and the unequivocal identification. However, further studies are required to develop faster and more sensitive methods, in order to obtain more reliable results.

Microfluidic mixing system for precise PLGA-PEG nanoparticles size control.

In this study, a microfluidic device was employed to produce polymeric nanoparticles (NPs) with well-controlled sizes. The influence of several parameters in the synthesis process, namely, polymer concentration, flow rate and flow rate ratio between the aqueous and organic solutions was investigated. To evaluate the NPs size effect, three diameters were selected (30, 50 and 70 nm). Their cytocompatibility was demonstrated on endothelial cells and macrophages. Additionally, their efficacy to act as drug carriers was assessed in an in vitro inflammatory scenario. NPs loaded and released diclofenac (DCF) in a size-dependent profile (smaller sizes presented lower DCF content and higher release rate). Moreover, 30 nm NPs were the most effective in reducing prostaglandin E2 concentration. Therefore, this study demonstrates that microfluidics can generate stable NPs with controlled sizes, high monodispersity and enhanced batch-to-batch reproducibility. Indeed, NPs size is a crucial parameter for drug encapsulation, release and overall biological efficacy.

Ketamine and Norketamine: Enantioresolution and Enantioselective Aquatic Ecotoxicity Studies.

Ketamine is a chiral drug used for various clinical purposes but often misused. It is metabolized to norketamine, an active chiral metabolite. Both substances have been detected in environmental matrices, but studies about their enantioselective toxic effects are scarce. In the present study, the enantiomers of ketamine and norketamine were separated by a semipreparative enantioselective liquid chromatography method, and their toxicity was investigated in different aquatic organisms. The enantioseparation was achieved using a homemade semipreparative chiral column. Optimized conditions allowed the recovery of compounds with enantiomeric purity higher than 99%, except for (R)-ketamine (97%). The absolute configuration of the enantiomers was achieved by experimental electronic circular dichroism (ECD). The ecotoxicity assays were performed with the microcrustacean Daphnia magna and the protozoan Tetrahymena thermophila using Toxkit MicroBioTests. Different concentrations were tested (0.1-10 000 µg/L) to include environmental levels (~0.5-~100 µg/L), for racemates (R,S) and the isolated enantiomers (R or S) of ketamine and norketamine. No toxicity was observed in either organism at environmental levels. However, at greater concentrations, (R,S)-ketamine presented higher mortality for D. magna compared with its metabolite (R,S)-norketamine (85 and 20%, respectively), and the (S)-ketamine enantiomer showed higher toxicity than the (R)-ketamine enantiomer. In addition, (S)-ketamine also presented higher growth inhibition than (R)-ketamine for T. thermophila at the highest concentrations (5000 and 10 000 µg/L). Contrary to D. magna, growth inhibition was observed for both enantiomers of norketamine and in the same magnitude order of the (S)-ketamine enantiomer. The results showed that the 2 organisms had different susceptibilities to norketamine and that the toxicity of ketamine at high concentrations is enantioselective for both organisms. Environ Toxicol Chem 2022;41:569-579. © 2020 SETAC.

Development and validation of a liquid chromatography method using UV/fluorescence detection for the quantitative determination of metabolites of the kynurenine pathway in human urine: Application to patients with heart failure.

Recent evidence indicates the relevant role of the tryptophan (TRP) metabolites in the pathophysiology of cardiovascular diseases via inflammatory and oxidative stress mechanisms. Therefore, quantification of TRP and its metabolites in biological samples can be a powerful tool to elucidate the disease mechanisms. The aim of this work was to develop and validate a liquid chromatography with ultraviolet (UV) and fluorescence detection (FD) (LCUV/FD) method for the quantification of TRP and its metabolites (L-kynurenine (KYN) and kynurenic acid (KA)) in urine samples from heart failure (HF) patients. Biochemical parameters and inflammatory markers were quantified, and data correlated with urinary concentrations of TRP and its metabolites. Optimized chromatographic conditions were achieved using a Luna® 3 µm PFP(2) analytical column, a mobile phase of 20 mM of ammonium formate in ultra-pure water (with 0.01 % of formic acid), acetonitrile and ethanol (95/2/3, v/v/v), a flow rate of 0.7 mL/min and a column oven temperature set at 25 °C. The method was validated according to the European Medicines Agency (EMA) guidelines and showed to be linear (r2 >0.99), accurate (82-116%) and precise (%RSD below 15 %). The limits of quantification varied between 50 and 125 ng/mL. The method was applied to the quantification of TRP, KYN and KA in healthy volunteers and male HF patients. The results obtained through this pilot study (small group of patients) showed a relationship between biochemical parameters, inflammatory markers and changes in the concentration of TRP, KYN and KA. The KYN/TRP and KA/KYN ratios were calculated. Results support the hypothesis that KYN/TRP ratio is related with enzymatic activity and that KA/KYN ratio can be a good neuroprotection indicator. The potential of the LCUV/FD method for the monitoring of the selected compounds in cardiac patients was also demonstrated.

Sardine Roe as a Source of Lipids To Produce Liposomes.

Sea-derived materials have promising applications in the medical, pharmaceutical, and biotechnological fields. Fish roe, for example, is a highly nutritional product, presenting diverse beneficial effects on human health. Therefore, this work explored extracts of sardine (Sardina pilchardus) roe, due to the well-known health benefits of this fish, to produce novel and promising delivery systems. After morphological, histological, and histochemical characterizations of sardine roe, their lipids were extracted using two different approaches, namely, Bligh and Dyer (BD) and methyl-tert-butyl ether (MTBE) methods. Gas chromatography/mass spectrometry analyses demonstrated that lipid extracts contain several fatty acids, such as ω3 polyunsaturated fatty acids. The lipids, especially phospholipids, were used to produce multilamellar liposomes (MLVs). These delivery systems presented size heterogeneity, a negative surface charge, and the ability to control the release of the encapsulated anti-inflammatory drug, namely, celecoxib. Biological assays indicated that MLVs produced with MTBE lipidic extracts presented a better cytocompatibility than those obtained by the BD method. This can be further improved if the lipid extracts are processed by chemical extraction. Therefore, sardine roe-derived lipids can produce drug-delivery systems with the potential to be applied in the biomedical field.

Influence of PDLA nanoparticles size on drug release and interaction with cells.

Polymeric nanoparticles (NPs) are strong candidates for the development of systemic and targeted drug delivery applications. Their size is a determinant property since it defines the NP-cell interactions, drug loading capacity, and release kinetics. Herein, poly(d,l-lactic acid) (PDLA) NPs were produced by the nanoprecipitation method, in which the influence of type and concentration of surfactant as well as PDLA concentration were assessed. The adjustment of these parameters allowed the successful production of NPs with defined medium sizes, ranging from 80 to 460 nm. The surface charge of the different NPs populations was consistently negative. Prednisolone was effectively entrapped and released from NPs with statistically different medium sizes (i.e., 80 or 120 nm). Release profiles indicate that these systems were able to deliver appropriate amounts of drug with potential applicability in the treatment of inflammatory conditions. Both NPs populations were cytocompatible with human endothelial and fibroblastic cells, in the range of concentrations tested (0.187-0.784 mg/mL). However, confocal microscopy revealed that within the range of sizes tested in our experiments, NPs presenting a medium size of 120 nm were able to be internalized in endothelial cells. In summary, this study demonstrates the optimization of the processing conditions to obtain PDLA NPs with narrow size ranges, and with promising performance for the treatment of inflammatory diseases. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 482-493, 2019.

Synthesis and characterization of chitosan-grafted-polycaprolactone micelles for modulate intestinal paclitaxel delivery.

In this work, self-assembled amphiphilic micelles based on chitosan (CS) and polycaprolactone (PCL) were produced and used as carriers of paclitaxel (PTX) to improve its intestinal pharmacokinetic profile. Chitosan-grafted-polycaprolactone (CS-g-PCL) was synthesized through a carbodiimide reaction by amidation and confirmed by Fourier transform infrared spectroscopy (FTIR), hydrogen nuclear magnetic resonance analysis (1H NMR), and contact angle evaluation. Micelles were produced by solvent evaporation method, and the critical micelle concentration was investigated by conductimetry. The obtained micelles were of 408-nm mean particle size, narrow size distribution (polydispersity index of 0.335) and presented positive surface charge around 30 mV. The morphology of micelles assessed by transmission electron microscopy (TEM) revealed round and smooth surface, in agreement with dynamic light scattering measurements. The association efficiency determined by high-performance liquid chromatography (HPLC) was as high as 82%. The in vitro cytotoxicity of the unloaded and PTX-loaded micelles was tested against Caco-2 and HT29-MTX intestinal epithelial cells, resulting in the absence of cell toxicity for all formulations. Moreover, the permeability of PTX-loaded micelles in Caco-2 monolayer and Caco-2/HT29-MTX co-culture model was determined. Results showed that the permeability of PTX was higher in Caco-2/HT29-MTX co-culture model compared with Caco-2 monolayer due to the mucoadhesive character of micelles, acting as a platform to deliver PTX at the sites of absorption. Therefore, it can be concluded that the PTX-loaded CS-g-PCL micelles, employed for the first time as PTX carriers, may be a potential drug carrier for the intestinal delivery of hydrophobic drugs, particularly anticancer agents.

Liquid chromatographic methods for the therapeutic drug monitoring of methotrexate as clinical decision support for personalized medicine: A brief review.

Methotrexate (MTX) is an antifolate drug used for several diseases. Depending on the disease, MTX can be administered at low dose (LDMTX) in some autoimmune diseases, like rheumatoid arthritis, or at high dose (HDMTX) in some cancers, such as acute lymphoblastic leukemia. After absorption, MTX is metabolized in the liver to 7-hydroxymethotrexate and in the intestine to 2,4-diamino-N10-methylpteroic acid (DAMPA). Moreover, inside red blood cells, MTX is converted to active metabolites, MTX polyglutamates (MTXPGs), contributing to its pharmacodynamics. Owing to its narrow therapeutic range, and inter- and intra-patient variability, either noneffectiveness and/or toxicity may occur. Because of the existence of a relationship between drug therapeutic outcome and its systemic concentration, therapeutic drug monitoring (TDM) may ensure the effectiveness and safety of MTX use. In order to monitor the optimization of patient clinical response profile, several analytical methods have been described for TDM in biological samples. These include liquid chromatography (LC) coupled with ultraviolet detection, fluorescence detection or mass spectrometry, each one presenting advantages and drawbacks. This paper reviews the most commonly used techniques for sample preparation and critically discusses the current LC methods applied for the TDM of MTX in biological samples, at LDMTX and HDMTX.

Development and validation of a rapid reversed-phase HPLC method for the quantification of monoclonal antibody bevacizumab from polyester-based nanoparticles.

Bevacizumab is a powerful human monoclonal antibody approved worldwide for treatment of several types of cancer and ocular diseases due to its potential as antiangiogenic drug. Nowadays, in order to improve the monoclonal antibody-based therapy, attempts have been focused in the formulation of these biomacromolecules into nanoparticles. Thus, the aim of this work was to develop and validate a reversed-phase high-performance liquid chromatography with fluorescence detection method for the determination of bevacizumab from nanoparticulate systems, according to the International Conference on Harmonization guidelines. Chromatographic analysis were performed on a RP-C8 column with a mobile phase composed by water-0.1% (v/v) TFA and acetonitrile-0.1% (v/v) TFA in gradient mode at a flow rate of 1mLmin-1. Results showed that the proposed method is specific, linear in the range of 10-100µgmL-1 (r2=0.9997), accurate (recovery rate 100.50±0.85%), precise at the intraday and inter-day (relative standard deviation less than 1.79%) and robust. The detection and quantification limits were calculated by specific linear calibration curve with less concentrated standard (range of 1-20µgmL-1). The LOD was 2.16µgmL-1 and LOQ was 6.55µgmL-1. This method was also successfully used, for the first time, to quantify and compare the content of bevacizumab encapsulated into poly(lactic-co-glycolic acid)-based nanoparticles before and after lyophilization.

Quantification of alprenolol and propranolol in human plasma using a two-dimensional liquid chromatography (2D-LC).

A new two-dimensional liquid chromatography (2D-LC) method using a column switching valve, with a restricted-access media (RAM) column in the first dimension was developed and validated for the quantification of two ß-blockers in human plasma. Several parameters, such as sample collection, mobile phase composition and flow rate for sample cleanup, transference and analytical separation of the ß-blockers were investigated and optimized. The developed method allowed for the simultaneous pre-treatment and quantification of alprenolol (ALP) and propranolol (PRO) in human plasma in less than 25min. The method consisted in the injection of 100µL of plasma samples on the RAM alkyl-diol-silica column (Lichrospher® RP-18 ADS, 25µm) with water/acetonitrile (98:2, v/v; at a flow rate of 2.0mL/min) and then transferred (via a six-port valve) to the analytical column (Luna PFP (2), 150×4.6mm ID, 100Å, 3µm) with 0.1% (v/v) triethylamine in water acidified with trifluoroacetic acid (pH=3)/acetonitrile (74:26, v/v) at a flow rate of 0.6mL/min in a back-flush mode. The column oven temperature was optimized to 42°C and the fluorescence detector set at 280nm and 310nm (excitation and emission, respectively). The method was validated according to the European Medicines Agency's guidelines and was linear (r2>0.999) over a dynamic range of 5.0 - 200ng/mL. Recoveries ranged from 90.2 and 107% and the lower limit of quantification was 5.0ng/mL for both compounds. Precision was expressed as a percentage of relative standard deviation and did not exceed 11%. Finally, the method was successfully applied to determine the plasma concentration of PRO in four healthy volunteers.

New trends in sample preparation techniques for environmental analysis.

Environmental samples include a wide variety of complex matrices, with low concentrations of analytes and presence of several interferences. Sample preparation is a critical step and the main source of uncertainties in the analysis of environmental samples, and it is usually laborious, high cost, time consuming, and polluting. In this context, there is increasing interest in developing faster, cost-effective, and environmentally friendly sample preparation techniques. Recently, new methods have been developed and optimized in order to miniaturize extraction steps, to reduce solvent consumption or become solventless, and to automate systems. This review attempts to present an overview of the fundamentals, procedure, and application of the most recently developed sample preparation techniques for the extraction, cleanup, and concentration of organic pollutants from environmental samples. These techniques include: solid phase microextraction, on-line solid phase extraction, microextraction by packed sorbent, dispersive liquid-liquid microextraction, and QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe).