Biocompatible metal-organic frameworks as promising platforms to eradicate HIV reservoirs ex vivo in people living with HIV.

The HIV attacks the immune system provoking an infection that is considered a global health challenge. Despite antiretroviral treatments being effective in reducing the plasma viral load in the blood to undetectable levels in people living with HIV (PLWH), the disease is not cured and has become chronic. This happens because of the existence of anatomical and cellular viral reservoirs, mainly located in the lymph nodes and gastrointestinal tract, which are composed of infected CD4+ T cells with a resting memory phenotype and inaccessible to antiretroviral therapy. Herein, a new therapeutic strategy based on nanotechnology is presented. Different combinations of antiretroviral drugs (bictegravir/tenofovir/emtricitabine and nevirapine/tenofovir/emtricitabine) and toll-like receptor agonists were encapsulated into metal-organic frameworks (MOFs) PCN-224 and ZIF-8. The encapsulation efficiencies of all the drugs, as well as their release rate from the carriers, were measured. In vitro studies about the cell viability, the hemocompatibility, and the platelet aggregation of the MOFs were carried out. Epifluorescence microscopy assays confirmed the ability of ZIF-8 to target a carboxyfluorescein probe inside HeLa cell lines and PBMCs. These results pave the way for the use of these structures to eliminate latent HIV reservoirs from anatomical compartments through the activation of innate immune cells, and a higher efficacy of the triplet combinations of antiretroviral drugs.

Bioavailability of flumequine and diclofenac in mice exposed to a metal-drug chemical cocktail. Evaluation of the protective role of selenium.

BACKGROUND AND PURPOSE: Organisms, including humans, are subjected to the simultaneous action of a wide variety of pollutants, the effects of which should not be considered in isolation, as many synergies and antagonisms have been found between many of them. Therefore, this work proposes an in vivo study to evaluate the effect of certain metal contaminants on the bioavailability and metabolism of pharmacologically active compounds. Because the most frequent entry vector is through ingestion, the influence of the gut microbiota and the possible protective effects of selenium has been additionally evaluated. EXPERIMENTAL APPROACH: A controlled exposure experiment in mammals (Mus musculus) to a "chemical cocktail" consisting of metals and pharmaceuticals (diclofenac and flumequine). The presence of selenium has also been evaluated as an antagonist. Mouse plasma samples were measured by UPLC-QTOF. A targeted search of 48 metabolites was also performed. KEY RESULTS: Metals significantly affected the FMQ plasma levels when the gut microbiota was depleted. Hydroxy FMQ decreased if metals were present. Selenium minimized this decrease. The 3-hydroxy DCF metabolite was not found in any case. Changes in some metabolic pathways are discussed. CONCLUSIONS AND IMPLICATIONS: The presence of metals in the mouse diet as well as the prior treatment of mice with an antibiotic mixture (Abxs), which deplete the gut microbiota, has a decisive effect on the bioavailability and metabolism of the tested pharmaceuticals and dietary selenium minimize some of their effects.

An improved microfluidic device to enhance the enrichment factors in liquid phase microextraction: application to the simultaneous extraction of polar and non-polar acids in biological samples.

A new microfluidic device to enhance the enrichment factor in miniaturized systems is proposed. The microfluidic system was design for liquid phase microextractions, and it was applied to the simultaneous extraction of acidic compounds of a wide range of polarity (0.5 < log P < 3). The device operated under stagnant acceptor phase conditions and all the operational parameters involved were optimized. Tributyl phosphate was found to be a new highly efficient supported liquid membrane to simultaneously extract analytes of very different polarities. The optimal donor and acceptor phase were pH 2 and pH 13, respectively. The donor flow rate and the extraction time were investigated simultaneously, offering great versatility with high enrichment factors (EFs). Limits of quantitation were within 0.02 and 0.09 µg mL-1 for all compounds at 10 µL min-1 as donor flow rate and 20-min extractions, offering EFs between 11 and 18 with only 200-µL sample volume consumption. The method was successfully applied to human urine samples, observing recoveries between 47 and 90% for all compounds. This new proposed microfluidic system increases the wide range of applications, especially when the analytes are present in lower concentrations in the sample.

Bioaccumulation and biochemical responses in the peppery furrow shell Scrobicularia plana exposed to a pharmaceutical cocktail at sub-lethal concentrations.

Pharmaceutical drugs in the aquatic medium may pose significant risk to non-target organisms. In this study, the potential toxicity of a mixture of three compounds commonly detected in marine waters (ibuprofen, ciprofloxacin and flumequine) was assessed, by studying bioaccumulation, oxidative stress and neurotoxicity parameters (catalase CAT, superoxide dismutase SOD, glutathione reductase GR, glutathione S-transferase GST, lipid peroxidation LPO, glutathione peroxidase GPX, metallothionein MT and acetylcholinesterase AChE) in the clam Scrobicularia plana. Temporal evolution of selected endpoints was evaluated throughout an exposure period (1, 7 and 21 days) followed by a depuration phase. The accumulation of all drugs was fast, however clams showed the ability to control the internal content of drugs, keeping their concentration constant throughout the exposure and reducing their content after 7 days of depuration. The induction of biochemical alterations (SOD, CAT, LPO, MT, AChE) was observed in gills and digestive gland probably related to an imbalance in the redox state of clams as a consequence of the exposure to the drug mixture. These alterations were also maintained at the end of the depuration week when the high levels of SOD, CAT, GST and LPO indicated the persistence of oxidative stress and damage to lipids despite the fact that clams were no longer exposed to the mixture.

Monitoring of pharmaceuticals in aquatic biota (Procambarus clarkii) of the Doñana National Park (Spain).

In this work the presence of different pharmaceuticals at Doñana National Park (Spain) and their main entry sources (input source or entry points) have been stated over the 2011-2016 years period. Twenty-three selected pharmaceuticals (corresponding to eight therapeutic families) were evaluated in crayfish and water samples from Doñana National Park (Spain) (six sampling points selected in order to cover different possible pollution sources into and surrounding the Park). The multiresidue determination was carried out using enzymatic-microwave assisted extraction prior to high performance liquid chromatography mass spectrometry detection. Sulphonamides (sulfadiazine, sulfamerazine, sulfamethazine, and sulfamethoxazole); trimethoprim, an antibiotic that is frequently co-administered with sulfamethoxazole; amphenicols (chloramphenicol, florfenicol and thiamphenicol); fluoroquinolones (ciprofloxacin, enrofloxacin, flumequine, danofloxacin, gatifloxacin, norfloxacin, marbofloxacin and grepafloxacin); penicillins (amoxicillin); tetracyclines (chlortetracycline and oxytetracycline); non-steroidal anti-inflammatory drugs (salicylic acid and ibuprofen); beta-blocker drugs (atenolol); and antiepileptics (carbamazepine) were analysed. Ciprofloxacin, ibuprofen, salicylic acid, flumequine, and carbamazepine were detected and/or quantified at some of the selected sampling points. A clear ecotoxicological risk to the ecosystem was demonstrated from the occurrence of ciprofloxacin in samples obtained after the punctual and massive presence of people inside the Park. Furthermore, flumequine and carbamazepine have been detected in Procambarus clarkii specimens in concentrations around 30 ng g-1 and 14 ng g-1, respectively, and their occurrence in the specimens could indicate the persistence of the discharge sources. The main source of pharmaceuticals into the Park might be the livestock farming activities, and the influence of urban wastewaters from surrounding villages does not seem to be very important.

Assessment of pharmaceutical mixture (ibuprofen, ciprofloxacin and flumequine) effects to the crayfish Procambarus clarkii: A multilevel analysis (biochemical, transcriptional and proteomic approaches).

The knowledge about the effects of pharmaceuticals on aquatic organisms has been increasing in the last decade. However, due to the variety of compounds presents in the aquatic medium, exposure scenarios and exposed organisms, there are still many gaps in the knowledge on how mixtures of such bioactive compounds affect exposed non target organisms. The crayfish Procambarus clarkii was used to analyze the toxicity effects of mixtures of ciprofloxacin, flumequine and ibuprofen at low and high concentrations (10 and 100 µg/L) over 21 days of exposure and to assess the recovery capacity of the organism after a depuration phase following exposure during additional 7 days in clean water. The crayfish accumulated the three compounds throughout the entire exposure in the hepatopancreas. The exposure to the mixture altered the abundance of proteins associated with different cells functions such as biotransformation and detoxification processes (i.e. catalase and glutathione transferase), carbohydrate metabolism and immune responses. Additionally changes in expression of genes encoding antioxidant enzymes and in activity of the corresponding enzymes (i.e. superoxide dismutase, glutathione peroxidase and glutathione transferase) were reported. Alterations at different levels of biological organization did not run in parallel under all circumstances and can be related to changes in the redox status of the target tissue. No differences were observed between control and exposed organisms for most of selected endpoints after a week of depuration, indicating that exposure to the drug mixture did not produce permanent damage in the hepatopancreas of P. clarkii.

Uptake study in Juncus sp. and Salicornia europaea of six pharmaceuticals by liquid chromatography quadrupole time-of-flight mass spectrometry.

In this work, eight plants of Juncus sp. and ten of Salicornia europaea were used for an uptake assay of pharmaceuticals (flumequine, cirpofloxacin, enrofloxacin, carbamazepine, diclofenac and ibuprofen) by irrigation at three concentration levels: 10 ng mL-1 (low level); 700 ng mL-1 (medium level) and 10 µg mL-1 (high level). Two plants irrigated with pharmaceutical-free water were set up as controls. For each level, two plants were watered every day with 50 mL (Juncus sp.) and every two days with 20 mL (Salicornia europaea) of aqueous solutions containing all the analytes at the described concentrations. Plants irrigated at 10 µg mL-1 were significantly the most affected, whereas the rest of the plants remained, in general, largely displayed no apparent physiological effects throughout the 30 days (Juncus sp.) and 21 days (Salicornia europaea) assays. Leaves and stems were cut every seven days and roots were collected at the end of the assay. The samples were lyophilized, submitted to a microwave assisted extraction using 5 mL of acetonitrile:water mixture (1:1, v/v) and they were analyzed (in triplicate) in a liquid chromatography-quadrupole time of flight mass spectrometry instrument. Most of the analytes were quantified in many of the samples corresponding to the three exposure levels with the highest concentrations obtained at high exposure levels. Ibuprofen was not detected in any sample and enrofloxacin, ciprofloxacin and diclofenac were not detected in the samples from Salicornia europaea.

Comparison of three electromembrane-based extraction systems for NSAIDs analysis in human urine samples.

A comparative study on the extraction efficiency of five non-steroidal anti-inflammatories was carried out using three different electromembrane extraction (EME) devices with different geometries. The employed setups were (a) a hollow fiber configuration (HF-EME), (b) a microfluidic device that allows working in semi-dynamic mode (µF-EME), and (c) a static miniaturized flat membrane device (FM-EME). Each system was applied to the extraction of salicylic acid (SAC), ketoprofen (KTP), naproxen (NAX), diclofenac (DIC), and ibuprofen (IBU) and subsequent determination by high-performance liquid chromatography with UV and fluorescence detection (HPLC/UV-DAD-FLD). Voltage, pH composition, and extraction time were optimized for all devices. Additionally, volume ratio was investigated for HF-EME and FM-EME and flow rate for the microfluidic device. HF-EME provides the best result in terms of sensitivity with a limit of detection (LOD) between 0.1 and 1.5 ng mL-1 for SAC and KTP, respectively, while LODs for µF-EME were between 100 ng mL-1 and 400 ng mL-1 for SAC and DIC, respectively; however, a lower amount of sample was required. Finally, the obtained results, in terms of enrichment factors and extraction recoveries, were discussed to establish the advantages and disadvantages of each device. The proposed EME methods were successfully applied to the determination of the target analytes in fortified human urine samples. Graphical abstract.

Lacosamide intake during pregnancy increases the incidence of foetal malformations and symptoms associated with schizophrenia in the offspring of mice.

The use of first and second generation antiepileptic drugs during pregnancy doubles the risk of major congenital malformations and other teratogenic defects. Lacosamide (LCM) is a third-generation antiepileptic drug that interacts with collapsing response mediator protein 2, a protein that has been associated with neurodevelopmental diseases like schizophrenia. The aim of this study was to test the potential teratogenic effects of LCM on developing embryos and its effects on behavioural/histological alterations in adult mice. We administered LCM to pregnant mice, assessing its presence, and that of related compounds, in the mothers' serum and in embryonic tissues using liquid chromatography coupled to quadrupole/time of flight mass spectrometry detection. Embryo morphology was evaluated, and immunohistochemistry was performed on adult offspring. Behavioural studies were carried out during the first two postnatal weeks and on adult mice. We found a high incidence of embryonic lethality and malformations in mice exposed to LCM during embryonic development. Neonatal mice born to dams treated with LCM during gestation displayed clear psychomotor delay and behavioural and morphological alterations in the prefrontal cortex, hippocampus and amygdala that were associated with behaviours associated with schizophrenia spectrum disorders in adulthood. We conclude that LCM and its metabolites may have teratogenic effects on the developing embryos, reflected in embryonic lethality and malformations, as well as behavioural and histological alterations in adult mice that resemble those presented by patients with schizophrenia.

A Method for the Determination of Veterinary Drugs from Different Therapeutic Classes in Animal Urine.

A rapid, precise and robust HPLC separation procedure has been developed and optimized for the determination of a series of drugs of different therapeutic classes: chlortetracycline, oxitetracycline, cefoperazone, diclofenac, tiamphenicol, marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin and flumequine. The chromatographic method used a monolithic C18 column and both diode array and fluorescence detection. This procedure was validated for the analysis of drugs in cow urine, using a simple and fast procedure with methanol/acetonitrile, allowing the simultaneous and efficient extraction of most of the studied drugs. The proposed method was successfully applied to the determination of enrofloxacin in cow urine, collected after the administration of this antibiotic.

Simultaneous determination of metformin and glimepiride in human serum by ultra high performance liquid chromatography quadrupole time of flight mass spectrometry detection.

This work proposes a simple method for the simultaneous determination of two antidiabetic compounds, metformin and glimepiride, by ultra-high performance liquid chromatography using quadrupole time of flight mass spectrometry detection with electrospray ionization. The method was validated and shown to be linear, selective, accurate and precise. The chromatographic separation was performed using a BEH C18 column (50 mm x 2.1 mm, 1.7 µm particle size). The mobile phase was composed by 0.05% (v/v) aqueous formic acid solution and acetonitrile using a gradient elution program, at a flow rate of 0.3 mL/min. Linearity range for metformin was 1.7-100 µg·L-1, using propranolol as internal standard and 3.3-100 µg·L-1 for glimepiride using glibenclamide as internal standard. Limits of detection and lower limits of quantitation were 0.4 µg·L-1 and 1.7 µg·L-1 for metformin and 0.7 and 3.3 µg·L-1 for glimepiride, respectively. The method was used for the simultaneous determination of these compounds in human serum samples with lower limits of detection and quantitation than serum levels reported in previous works that allows its applicability in therapy drug monitoring.

A New Microchip Design. A Versatile Combination of Electromembrane Extraction and Liquid-Phase Microextraction in a Single Chip Device.

For the first time, a novel and versatile microfluidic device was developed to achieve the possibility of combining different extraction principles using a miniaturized approach for the extraction of different classes of analytes. This novel microchip is composed of a sandwich of three poly(methyl methacrylate) (PMMA) layers. Four channels allowed the combination of electromembrane extraction (EME) and liquid-phase microextraction (LPME) in three different ways: (I) EME and LPME, (II) EME and EME, or (III) LPME and LPME. The microchip can be used either (a) using a common acceptor phase (for both extractions) for the simultaneous extraction of drugs from different nature in a single step, or (b) a common sample solution (for both extractions) and two acceptor solutions for simultaneous drug separation. In this work, the performance of this novel microchip was demonstrated by simultaneous integration of EME and LPME using a common acceptor phase for both extractions. This configuration reduces the time of analysis allowing direct analysis in a single chip. The microchip was tested for extracting two different classes of analytes: five fluoroquinolones and four parabens as model analytes. All effective variables were optimized for EME and LPME. Under the optimized conditions, the reusable microchip enables simultaneous µ-EME/LPME with extraction efficiencies over 77% in only 8 min extraction and sample volume consumption lower than 40 µL. The optimized procedure was successfully applied to urine samples obtaining recoveries over 90% for all analytes.

Multiresidue determination of 21 pharmaceuticals in crayfish (Procambarus clarkii) using enzymatic microwave-assisted liquid extraction and ultrahigh-performance liquid chromatography-triple quadrupole mass spectrometry analysis.

In this paper, a multiresidue enzymatic-microwave assisted extraction prior to ultrahigh performance liquid chromatography and triple quadrupole mass spectrometry analysis has been developed for the determination of 21 pharmaceuticals in crayfish (Procambarus Clarkii) samples. The analysed compounds corresponding to 6 therapeutic families were: fluoroquinolones (ciprofloxacin, danofloxacin, enrofloxacin, flumequine, gatifloxacin, grepafloxacin, marbofloxacin and norfloxacin); tetracyclines (chlortetracycline and oxytetracycline); sulphonamides (sulfamethoxazole, sulfadiazine, sulfamethazine, sulfamerazine); penicillins (amoxicillin); anfenicols (chloramphenicol, thiamphenicol and florfenicol); non-steroidal anti-inflammatory drugs (ibuprofen and salicylic acid) and trimethoprim an antibiotic that is frequently co-administered with sulfamethoxazole. The main factors affecting the extraction efficiency were optimized for 0.5 g of lyophilized tissue. The enzymatic microwave extraction was carried out using an extraction time of 5 min with 5 mL of an acetonitrile: water (1:1, v/v) mixture, 50 µL of Proteinase-K solution and 5 µL of formic acid at 50 W. After centrifugation, the liquid extract was evaporated and the residue was reconstituted with 1 mL of 0.1% (v/v) formic acid. Chromatographic and MS parameters, in both positive and negative ionization modes, were also optimized. The mobile phase used consisted on a mixture of 0.1% (v/v) formic acid aqueous solution and acetonitrile in gradient elution mode at a 0.4 mL min-1 flow rate. The proposed method was validated and recoveries over 70% were obtained for all the analytes with detection limits in the 0.6-12 ng g-1 range. The proposed method was successfully applied to crayfish specimens from Doñana National Park, Spain.

Recent trends in capillary electrophoresis for complex samples analysis: A review.

CE has been a continuously evolving analytical methodology since its first introduction in the 1980s of the last century. The development of new CE separation procedures, the coupling of these systems to more sensitive and versatile detection systems, and the advances in miniaturization technology have allowed the application of CE to the resolution of new and complex analytical problems, overcoming the traditional disadvantages associated with this method. In the present work, different recent trends in CE and their application to the determination of high complexity samples (as biological fluids, individual cells, etc.) will be reviewed: capillary modification by different types of coatings, microfluidic CE, and online microextraction CE. The main advantages and disadvantages of the different proposed approaches will be discussed with examples of most recent applications.

Liquid chromatography quadrupole time-of-flight mass spectrometry determination of six pharmaceuticals in vegetal biota. Uptake study in Lavandula dentata.

A procedure based on microwave assisted extraction for the determination of 6 pharmaceuticals in samples of Lavandula dentata, Salicornia ramosissima and Juncus sp. by liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOF/MS) was optimized and validated. Best results were obtained using microwave assisted extraction of 1.0g of homogeneous lyophilized samples and 5mL of a mixture ACN:H2O (1:1 v/v) as extracting solvent. Analytical recoveries ranged from 60 to 107% with relative standard deviation (RSD) lower than 15%. Limits of quantitation (LOQ) for the 6 pharmaceuticals flumequine (FLM), carbamazepine (CBZ), ciprofloxacin (CPR), enrofloxacin (ENR), diclofenac (DCL), and ibuprofen (IBU) were in the range 20.8-125ngg-1. The method was satisfactory applied for an uptake study in Lavandula dentata samples finding quantifying concentrations of FLM and CBZ in roots, leaf and stem.

Recent advances in sample pre-treatment for emerging methods in proteomic analysis.

The objective of proteomics is the study of proteins (including their structure elucidation, the characterization of their role in biochemical process, the detection and identification of anomalous modifications in their structures and behavior, etc.) from both a qualitative and quantitative point of view. These studies have to face several difficulties as (a) the intrinsic complexity of protein molecules, (b) the low concentrations of the studied proteins, (c) the complexity of the biological samples (which could lead to interferences from the components of these matrixes), etc. Thus, the sample preparation procedure has a critical importance in order to obtain good separations and sensitivity in the results. In this paper, recent sample treatment methodologies for proteomic studies are reviewed and discussed. These methods include recent innovations in nanoparticle enrichment pre-treatment (for both the selective pre-concentration of the studied protein and the microwave-assisted digestion of the sample), treatments for tissue imaging (based on surface analysis of the studied biological tissues) and protein microarrays analysis (which allows the simultaneous determination of a high number of different proteins or associated species on a glass slide support).

Easy, fast and environmental friendly method for the simultaneous extraction of the 16 EPA PAHs using magnetic molecular imprinted polymers (mag-MIPs).

An easy and environmental friendly method, based on the use of magnetic molecular imprinted polymers (mag-MIPs) is proposed for the simultaneous extraction of the 16 U.S. EPA polycyclic aromatic hydrocarbons (PAHs) priority pollutants. The mag-MIPs based extraction protocol is simple, more sensitive and low organic solvent consuming compared to official methods and also adequate for those PAHs more retained in the particulate matter. The new proposed extraction method followed by HPLC determination has been validated and applied to different types of water samples: tap water, river water, lake water and mineral water.

New nanostructured support for carrier-mediated electromembrane extraction of high polar compounds.

A new support has been proposed to be used for carrier-mediated electromembrane extraction purposes. The new support (Tiss®-OH) is a 100µm thickness sheet nanofiber membrane manufactured by electrospinning and composed by acrylic nanofibers. It has been used in an electromembrane extraction (EME) combined with a HPLC procedure using diode array detection. The proposed method has been used for the extraction of four high polarity acidic compounds: nicotinic acid, amoxicillin, hippuric acid and salicylic acid. Analytes were extracted from an aqueous sample solution (pH 4) (donor phase) using a Tiss®-OH sheet that supports a 5% (w/v) Aliquat®336 in 1-octanol liquid membrane. Aqueous solution (pH 6) was used as acceptor phase. The electrical field was generated from a d.c. electrical current of 100V through two spiral shaped platinum wires placed into donor and acceptor phases. Analytes were extracted in 10min with recoveries in the 60-85% range. The proposed EME procedure has been successfully applied to the determination of the target analytes in human urine samples.

Electromembrane extraction for the determination of parabens in water samples.

To our knowledge, for the first time an electromembrane extraction combined with a high-performance liquid chromatography procedure using diode-array detection has been developed for the determination of five of the most widely used parabens: ethyl 4-hydroxybenzoate, propyl 4-hydroxybenzoate, butyl 4-hydroxybenzoate, isobutyl 4-hydroxybenzoate, and benzyl 4-hydroxybenzoate. Parabens were extracted from pH 4 aqueous sample solutions with use of an Accurel® S6/2 polypropylene hollow fiber that supports a liquid membrane of 1-octanol to a pH 12 aqueous acceptor solution placed inside the lumen of the hollow fiber. An electric current of 30 V was applied over the supported liquid membrane by means of platinum wires placed in the donor and acceptor phases. Parabens were extracted in 40 min with enrichment factors in the 30-49 range. The procedure has detection limits between 0.98 and 1.43 µg L(-1). The method was applied to the determination of parabens in surface environmental waters with excellent results.

New developments in microextraction techniques in bioanalysis. A review.

In recent years, the interest in new extraction methods with lower sample volume requirements, simpler equipment and handling, and lower reagent consumption, has led to the development of a series of microextraction methods based on extraction phases in the microliter order. Nowadays, many references can be found for several of these methods, which imply a wide range of applications referred to both the analyte and the sample nature. In this paper, recent developments in both well-established microextraction techniques (solid phase microextraction, hollow-fiber liquid phase microextraction, dispersive liquid-liquid microextraction, etc.) and recently appeared microextraction procedures (nanoextraction systems, microchip devices, etc.) for the clinical analysis of biological samples will be reviewed and discussed.