Design and validation of recombinant protein standards for quantitative Western blot analysis of cannabinoid CB1 receptor density in cell membranes: an alternative to radioligand binding methods.

BACKGROUND: Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen. RESULTS: Here we generated highly soluble and stable recombinant protein constructs GST-CB1414-472 and GST-CB1414-442 containing much of the human CB1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443-473 of the mouse CB1 receptor that corresponds to residues 442-472 in the human homolog. Estimated values of CB1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay. CONCLUSIONS: Collectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints.

Solid-phase synthesis of imprinted nanoparticles as artificial antibodies against the C-terminus of the cannabinoid CB1 receptor: exploring a viable alternative for bioanalysis.

The production of artificial anti-CB1 antibodies in nanoparticle format is described using the solid-phase imprinting approach. Instead of whole protein imprinting, a linear C-terminus sequence of the receptor comprising 15 amino acids (458-KVTMSVSTDTSAEAL-472) has been used as template, in accordance with the epitope imprinting approach. This sequence is located intracellularly, and it is involved in coupling to Gi/o proteins, being responsible for CB1 receptor desensitisation and internalisation. Developed molecularly imprinted materials were found to be in the nanometre scale, with a particle size of 126.4 ± 10.5 nm at pH 3 (25 ºC) and spherical shape. It was also observed that the size was sensible to temperature changes being reduced to 106.3 ± 15.2 nm at 35 °C. Lower critical solution temperature of this polymer was found to be ≈ 33.4 °C. The affinity and selectivity of the artificial antibody were assessed through dot blot and Western blot experiments. For the latter, recombinant fusion proteins GST-CB1414-472 and GST-CB1414-442 were produced to work respectively as target and negative control proteins. The control protein did not carry the target epitope for being devoid of last 30 amino acids at the C-terminus. The results demonstrated that the anti-CB1 material recognised selectively the target protein, thanks to the presence of the 15-amino acid sequence selected as epitope, which revealed that binding occurred at the C-terminus of the receptor itself. The methodology presented may pave the way for the development of novel imprinted nanomaterials for other proteins included in the superfamily of the G-protein-coupled receptors (GPCR).

Determination of mercury(ii) in water at sub-nanomolar levels by laser ablation-ICPMS analysis of screen printed electrodes used as a portable voltammetric preconcentration system.

Environmental pollution by mercury in ambient water samples is a recognized problem worldwide. Sample preservation and transport to the laboratory lead to uncertain analytical results. This study outlines the development of a procedure for on-site electrodeposition of mercury from water samples on a screen-printed gold electrode (SPGE) using portable voltammetric techniques. Once in the laboratory, Hg is analyzed by laser ablation inductively coupled plasma-mass spectrometry (LA-ICPMS) in order to ensure that the required sensitivity and precision levels for environmental sample analysis are reached. A new ablation chamber was intentionally designed for the analysis of SPGE's gold electrode. This cell has a small internal volume of 15 cm3 and the SPGE device perfectly fits inside. This design assures signal stability, avoids elemental fractionation and reduces wash-out time to a few seconds, reducing the analysis time considerably. The proposed method is capable of measuring dissolved mercury at the ng L-1 level (quantification limit 200 ng L-1) with good precision (RSD < 7.6%). The proposed method was tested with the NCS ZC 76303 (NIM-GBW08603) Mercury in water Certified Reference Material.

Iniferter-mediated grafting of molecularly imprinted polymers on porous silica beads for the enantiomeric resolution of drugs.

A surface-imprinted chiral stationary phase for the enantiomeric resolution of the antidepressant drug, citalopram, is presented in this work. N, N'-diethylaminodithiocarbamoylpropyl(trimethoxy)silane has been used as silane iniferter for the surface functionalization of the solid silica support. A molecularly imprinted polymer thin film, in the nm scale, was then grafted on the silanized silica using itaconic acid as the functional monomer and ethylene glycol dimethacrylate as the cross-linker in the presence of the template S-citalopram. The total monomer amount was calculated to obtain the desired thickness. Non-imprinted stationary phases were prepared similarly in the absence of S-citalopram. Characterization of the materials was carried out by scanning electron microscopy, thermogravimetric analysis, elemental analysis and Fourier transform infrared spectroscopy. Stationary phases have been applied to the chromatographic separation of the target. Conditions for best chromatographic resolution of the enantiomers were optimized, and it was found that a mobile phase consisting of a mixture of formate buffer (40 mM, pH 3) and acetonitrile (30:70 v/v) at 40 °C provided best results. Binding behaviour of the developed material was finally assessed by batch rebinding experiments. The obtained binding isotherm was fitted to different binding models being the Freundlich-Langmuir model, the one that best fitted the experimental data. The developed material has shown high selectivity for the target enantiomer, and the stationary phase could be undoubtedly exploited for chiral separation of the drug.

Revealing novel biomarkers for diagnosing chronic kidney disease in pediatric patients.

Pediatric chronic kidney disease (CKD) is a clinical condition characterized by progressive renal function deterioration. CKD diagnosis is based on glomerular filtration rate, but its reliability is limited, especially at the early stages. New potential biomarkers (citrulline (CIT), symmetric dimethylarginine (SDMA), S-adenosylmethionine (SAM), n-butyrylcarnitine (nC4), cis-4-decenoylcarnitine, sphingosine-1-phosphate and bilirubin) in addition to creatinine (CNN) have been proposed for early diagnosis. To verify the clinical value of these biomarkers we performed a comprehensive targeted metabolomics study on a representative cohort of CKD and healthy pediatric patients. Sixty-seven children with CKD and forty-five healthy children have been enrolled in the study. Targeted metabolomics based on liquid chromatography-triple quadrupole mass spectrometry has been used for serum and plasma samples analysis. Univariate data analysis showed statistically significant differences (p < 0.05) in the concentration of CNN, CIT, SDMA, and nC4 among healthy and CKD pediatric patients. The predictive ability of the proposed biomarkers was also confirmed through specificity and sensitivity expressed in Receiver Operating Characteristic curves (AUC = 0.909). In the group of early CKD pediatric patients, AUC of 0.831 was obtained, improving the diagnostic reliability of CNN alone. Moreover, the models built on combined CIT, nC4, SDMA, and CNN allowed to distinguish CKD patients from healthy control regardless of blood matrix type (serum or plasma). Our data demonstrate potential biomarkers in the diagnosis of early CKD stages.

SPARC-mediated long-term retention of nab-paclitaxel in pediatric sarcomas.

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein overexpressed by several cancers. Because SPARC shows high binding affinity to albumin, we reasoned that pediatric sarcoma xenografts expressing SPARC would show enhanced uptake and accumulation of nanoparticle albumin-bound (nab)-paclitaxel, a potent anticancer drug formulation. We first evaluated the expression of SPARC in patient-derived xenografts (PDXs) of Ewing sarcoma, rhabdomyosarcoma and osteosarcoma, finding variable SPARC gene expression that correlated well with SPARC protein measured by immunoblotting. We revealed that the activity of the fusion gene chimera EWSR1-FLI1, the genetic driver of Ewing sarcoma, leads to lower expression of the gene SPARC in these tumors, likely due to enriched acetylation marks of the histone H3 lysine 27 at regions including the SPARC promoter and potential enhancers. Then, we used SPARC-edited Ewing sarcoma cells (A673 line) to demonstrate that SPARC knocked down (KD) cells accumulated significantly less amount of nab-paclitaxel in vitro than SPARC wild type (WT) cells. In vivo, SPARC KD and SPARC WT subcutaneous xenografts in mice achieved similar maximum intratumoral concentrations of nab-paclitaxel, though drug clearance from SPARC WT tumors was significantly slower. We confirmed such SPARC-mediated long-term intratumoral accumulation of nab-paclitaxel in Ewing sarcoma PDX with high expression of SPARC, which accumulated significantly more nab-paclitaxel than SPARC-low PDX. SPARC-high PDX responded better to nab-paclitaxel than SPARC-low tumors, although these results should be taken cautiously, given that the PDXs were established from different patients that could have specific determinants predisposing response to paclitaxel. In addition, SPARC KD Ewing sarcoma xenografts responded better to soluble docetaxel and paclitaxel than to nab-paclitaxel, while SPARC WT ones showed similar response to soluble and albumin-carried drugs. Overall, our results show that pediatric sarcomas expressing SPARC accumulate nab-paclitaxel for longer periods of time, which could have clinical implications for chemotherapy efficacy.

Particle Analysis for the Detection of Gunshot Residue (GSR) in Nasal Samples Using Scanning Laser Ablation and Inductively Coupled Plasma-Mass Spectrometry (SLA-ICPMS).

Currently, aluminum stub with carbon adhesive devices are used to collect inorganic gunshot residues (GSR) from the hands of a shooter. In an ideal shooting case, the gunshot particles do not persist for more than 2 h in the hands of the shooter, provided that the hands have not been washed. However, for forensic analysis and inference, the extended persistence of GSR would be desirable. This study investigates a novel GSR sampling and detection protocol. Sampling was performed in the nostrils using swab devices impregnated in ethylenediaminetetraacetic acid (EDTA). The GSRs persisted for longer periods in nasal mucus than on the hands, and particles were detected 6 h after shooting occurred. The analytical determination was conducted by scanning laser ablation-inductively coupled plasma-mass spectrometry (SLA-ICPMS) which enable the identification of the number of particles and their elemental composition. Seventeen isotope signals corresponding to 13 C, 205 Tl and 15 analytes that are usually associated with the composition of GSR residues were monitored: 27 Al, 29 Si, 31 P, 33 S, 35 Cl, 39 K, 44 Ca, 57 Fe, 60 Ni, 63 Cu, 66 Zn, 118 Sn, 121 Sb, 137 Ba, and 208 Pb. The SLA technique enabled the reduction of the swab analysis time to 40 min. The effectiveness of this methodology was evaluated with two types of firearms: a pistol and a shotgun. The results indicated that the methodology proposed for the analysis of the nasal GSR was effective and that it can improve or complement the forensic analyses and inferences presented in a court.

Fungicide distribution in vitiviniculture ecosystems according to different application strategies to reduce environmental impact.

Systematic fungicides treatments in vine-growing European ecosystems have been conducted for decades. The goal of this study was to determine the mobility and persistence of 20 fungicides used in two viticultural zones in Atlantic and Mediterranean climates, from the moment of their application until their distribution throughout different compartments of the ecosystem: soil, water, grapes, musts and wines. This study also sought to obtain valuable information to reduce the usage of these products without affecting the health of the vines. For this purpose, different phytosanitary treatments were applied, using dosing criteria based on data provided by meteorological stations, degree-day accumulation, phenological state, and growers' criteria. The observed differences between studied geographical areas were not significant with regard to chemical accumulation in the soil and water; however, they were significantly different regarding to grapes, musts, and wines.

Simultaneous determination of citalopram, fluoxetine and their main metabolites in human urine samples by solid-phase microextraction coupled with high-performance liquid chromatography.

A liquid chromatography method was developed for the determination of some frequently prescribed selective serotonin re-uptake inhibitors (SSRI) - citalopram and fluoxetine - and its main metabolites - demethylcitalopram, didemethylcitalopram and norfluoxetine - in human urine samples, using a previous stage of solid-phase microextraction. All the extraction parameters influencing adsorption (extraction time, temperature, pH, ion strength and organic modifier addition) and desorption (desorption time and desorption solvent mixture composition) of the analytes on the fiber have been studied. A satisfactory reproducibility for extraction from urine samples (R.S.D.<10%) was obtained. The linearity for urine ranged from 0.05 to 2 mg l(-1) with limits of detection close to 0.01 mg l(-1), which cover the typical urinary concentrations obtained for citalopram, fluoxetine and their metabolites.

Determination of fluoxetine, norfluoxetine and their enantiomers in rat plasma and brain samples by liquid chromatography with fluorescence detection.

Fluoxetine (FLX) and norfluoxetine (NFLX) racemic mixtures were determined by reversed-phase liquid chromatography with fluorescence detection (lambda(exc)=227 nm, lambda(em)=305 nm). The calibration curves prepared from drug-free plasma and brain were linear in the range of 5-1000 ng ml(-1) and 100-40,000 ng g(-1) for doped samples, with detection limits of 3.2 and 2.1 ng ml(-1) in plasma and 31.5 and 26.1 ng g(-1) in brain tissue for FLX and NFLX, respectively. Enantiomer determination was carried out through normal phase HPLC-FD (lambda(exc)=224 nm, lambda(em)=336 nm) after precolumn chiral derivatization with R-1-(1-naphthyl)ethyl isocyanate. Standard curves also prepared in a drug-free matrix were linear for each enantiomer over the range of 2-1000 ng ml(-1) and 20-7000 ng g(-1) with detection limits for the four compounds ranging between 0.2 and 0.5 ng ml(-1) in plasma and between 3.0 and 8.2 ng g(-1) in brain tissue. In both methods the analytes were isolated from the biological matrix by a new solid-phase extraction procedure with recovery in plasma and brain over 90 and 87%, respectively. The repeatability of this extraction procedure was satisfactory within-day and between-day with CV<9.1%. This study also offered the opportunity to obtain an assessment of the potential relationships between the concentration of individual enantiomers of FLX and NFLX in plasma and brain tissue after chronic treatment with racemic FLX at a dose intended to mimic the human plasma concentration of FLX in standard clinical conditions, and therefore should make for more reliable extrapolation of neurochemical findings in other species.

Molecularly imprinted nanoparticles grafted to porous silica as chiral selectors in liquid chromatography.

The work presented here explores the grafting of molecularly imprinted nanoparticles (MIN) on silica beads for the development of new chiral stationary phases (CSP). Both solid-phase imprinting and precipitation polymerisation were tested for MIN synthesis though the latter approach was the only one that provided efficient chiral selectors. MIN particles were prepared by iniferter polymerisation initiated by UV radiation, using itaconic acid as functional monomer and ethylene dimethacrylate as cross-linker. This resulted in particles with an average size of 249.0±4.0nm which were covalently immobilised onto chromatographic silica beads. The resultant CSP based on the composite silica beads-MIN was capable of resolving the racemate of the antidepressant drug citalopram and also separating its major metabolites by liquid chromatography, with better efficiency and peak symmetry than other MIP based CSP. The methodology presented here allowed for the quantification of the pharmacologically active enantiomer (+)-(S)-citalopram (SCIT) and its main metabolites (+)-(S)-desmethylcitalopram (SDCIT) and (+)-(S)-didesmethylcitalopram (SDDCIT) in urine, registering mean recoveries that ranged from 91.5 to 103.7% with RSD values that were below 10% in all tested concentration levels (0.1, 0.75 and 5µgmL-1), which confirmed method suitability for the intended application.

A novel strategy for Cr(III) and Cr(VI) analysis in dietary supplements by speciated isotope dilution mass spectrometry.

In recent years, Cr speciation in dietary supplements has become decisive in the evaluation of their health risks. Despite being an beneficial micronutrient, Cr(III) can be toxic at living organisms at high concentrations, while Cr(VI) is known to be highly toxic and carcinogenic. The main objective of this work was to optimize an analytical methodology for the extraction and accurate quantification of Cr(III) and Cr(VI) in dietary supplements. The extraction of Cr species was carried out with 50mM EDTA solution on a hotplate under optimized conditions. Special attention was paid to bidirectional species transformations. No noticeable oxidation of Cr(III) into Cr(VI) was observed and the reduction to Cr(III) only occurred at very high Cr(VI) concentrations. Cr(III) as Cr(EDTA)(-) complex was chromatographically separated from Cr(VI), retained as CrO4(2-), on an anion exchange column coupled to inductively coupled plasma mass spectrometry (LC-ICP-MS). The limit of quantification (0.08µgg(-1)) was below the limit established for Cr enriched yeasts by the European Union. Eleven dietary supplements were analyzed and Cr(III) and Cr(VI) quantification was carried out by external calibration monitoring (52)Cr isotope and by speciated isotope dilution mass spectrometry (SIDMS) adding (50)Cr(III) and (53)Cr(VI) spikes. Total Cr was also quantified by ICP-MS and mass balance between total Cr and the sum of Cr(III) and Cr(VI) was achieved. In eight of the eleven tested supplements Cr(III) calculated amounts were higher than those indicated by the manufacturer, but only one of them exceeded the 250µgday(-1) recommended by World Health Organization (WHO). In contrast, it is worth noting that Cr(VI) amounts beyond the recommendations of the European Union for Cr enriched yeasts were found in five supplements. These results revealed that more accurate and rigorous quality assurance protocols should be applied to the testing of the final products, including the analysis of both Cr(III) and Cr(VI).

Water compatible stir-bar devices imprinted with underivatised glyphosate for selective sample clean-up.

This paper reports the development of stir bars with a new MIP based coating, for the selective sorptive extraction of the herbicide glyphosate (GLYP). Molecular imprinting of the polymer has directly been carried out employing underivatised GLYP as the template molecule. Due to the poor solubility of the target compound in organic solvents, the MIP methodology has been optimised for rebinding in aqueous media, being the synthesis and the rebinding steps carried out in water:methanol mixtures and pure aqueous media. The coating has been developed by radical polymerisation initiated by UV energy, using N-allylthiourea and 2-dimethyl aminoethyl methacrylate as functional monomers and ethylene glycol dimethacrylate as the cross-linker. Mechanical stability of the coating has been improved using 1,3-divinyltetramethyldisiloxane in the polymerisation mixture. Under the optimised conditions, the MIP has demonstrated excellent selectivity for the target compound in the presence of structural analogues, including its major metabolites. The applicability of the proposed method to real matrices has also been assessed using river water and soil samples. Registered mean recoveries ranged from 90.6 to 97.3% and RSD values were below 5% in all cases, what confirmed the suitability of the described methodology for the selective extraction and quantification of GLYP.

Molecularly imprinted polymers as a tool for the study of the 4-ethylphenol metabolic pathway in red wines.

A molecularly imprinted polymer (MIP) based methodology is described here for the determination of compounds that belong to the 4-ethylphenol (4EP) metabolic pathway in red wines. To this end, two MIP materials have been developed: a 4EP MIP as a class-selective material to extract phenols that belong to the 4EP metabolic pathway and a coumaric acid (CA) imprinted polymer as a MIP-based stationary phase capable of selectively separating these phenols on HPLC analysis, obtaining clean chromatograms. 4-vinyl pyridine and ethylene glycol dimethacrylate were respectively used as functional monomer and cross-linker for both MIPs. Once polymer compositions were optimised, the 4EP MIP was packed into SPE cartridges for wine sample clean-up and CA MIP was packed into HPLC columns to chromatographically separate the compounds present in the eluates obtained after SPE extraction. The accuracy of the proposed method was tested spiking wine samples with known concentrations of target compounds and subsequently, analytes were quantified by the standard addition method. Registered mean recoveries ranged from 95.2 to 109.2% and RSD values were below 10% in most cases. The described methodology was found to be suitable for the selective extraction and quantification of the compounds that belong to the 4EP metabolic pathway in red wines with minimal matrix effects and could be undoubtedly exploited to monitor 4EP and its precursors in wines.

Direct potentiometric quantification of histamine using solid-phase imprinted nanoparticles as recognition elements.

A new potentiometric sensor based on molecularly imprinted nanoparticles produced via the solid-phase imprinting method was developed. For histamine quantification, the nanoparticles were incorporated within a membrane, which was then used to fabricate an ion-selective electrode. The use of nanoparticles with high affinity and specificity allowed for label-free detection/quantification of histamine in real samples with short response times. The sensor could selectively quantify histamine in presence of other biogenic amines in real wine and fish matrices. The limit of detection achieved was 1.12×10(-6)molL(-1), with a linear range between 10(-6) and 10(-2)molL(-1) and a response time below 20s, making the sensor as developed a promising tool for direct quantification of histamine in the food industry.

Conductance based sensing and analysis of soluble phosphates in wastewater.

The current standard method used for measuring soluble phosphate in environmental water samples is based on a colourimetric approach, developed in the early 1960s. In order to provide an alternative, label free sensing solution, a molecularly imprinted polymer (MIP) was designed to function as a phosphate receptor. A combination of functional monomer (N-allylthiourea), cross-linker and monomer/template ratios were optimised in order to maximise the binding capacity for phosphate. When produced in membrane format, the MIP's ability to produce a reversible change in conductance in the presence of phosphate was explored for fabrication of a sensor which was able to selectively detect the presence of phosphate compared to sulphate, nitrate and chloride. In wastewater samples the sensor had a limit of detection of 0.16 mg P/l, and a linear range between 0.66 and 8 mg P/l. This is below the minimum monitoring level (1 mg P/l) as required by current legislation for wastewater discharges, making the sensor as developed promising for direct quantification of phosphate in environmental monitoring applications.

Characterisation of the flavour profile from Graciano Vitis vinifera wine variety by a novel dual stir bar sorptive extraction methodology coupled to thermal desorption and gas chromatography-mass spectrometry.

The aim of this work was to develop a new analytical technique for the study of the organoleptic compounds (flavour profile) of the Graciano Vitis vinifera wine variety. The cv. Graciano is a singular variety of red grapes with its origins in La Rioja and Navarra (northern Spain). This variety transfers an intense red colour, aroma and high acidity to musts and provides greater longevity and, consequently, a better capacity for ageing wine. A new dual-stir bar sorptive extraction approach coupled with thermal desorption (TD) and GC-MS has been used to extract the volatile and semivolatile compounds. In this extraction step, the optimal values for the experimental variables were obtained through the Response Surface Methodology (RSM). Full scan chromatogram data were evaluated with two deconvolution software tools, and the results were compared. The volatile and semivolatile components were identified with an MS match ≥80%. As a result, the flavour metabolome of the Graciano Vitis vinifera wine variety was obtained, and 205 metabolites were identified using different databases. These metabolites were grouped into esters, acids, alcohols, nitrogen compounds, furans, lactones, ketones, aldehydes, phenols, terpenes, norisoprenoids, sulphur compounds, acetals and pyrans. The majority of the metabolites observed had already been reported in the literature; however, this work also identified new, previously unreported metabolites in red wines, which may be characteristic of the Graciano variety.

Enantioselective extraction of (+)-(S)-citalopram and its main metabolites using a tailor-made stir bar chiral imprinted polymer for their LC-ESI-MS/MS quantitation in urine samples.

This paper reports the application of a chiral imprinted polymer (CIP)-coated stir bar for the selective extraction of (+)-(S)-citalopram (SCIT) and its main metabolites, (+)-(S)-desmethylcitalopram (SDCIT) and (+)-(S)-didesmethylcitalopram (SDDCIT), from urine samples. The developed device has been demonstrated to be capable of selectively extracting the three target analytes from urine samples without saturating the imprinted sites. A CIP-coated stir bar sorptive extraction procedure (CIP-SBSE) is proposed for the isolation of SCIT, SDCIT and SDDCIT followed by their subsequent analysis using liquid chromatography ion trap mass spectrometry (LC-ITMS). Deuterated SCIT-d6 was used as an internal standard. The method was validated using a standard procedure, which revealed that a quantification of 5 ng mL(-1) was obtained in urine samples and that the accuracy and precision were within the established values while no matrix effect was observed.

Rational design and chromatographic evaluation of histamine imprinted polymers optimised for solid-phase extraction of wine samples.

This article reports on the computational design, development and application of a molecularly imprinted polymer (MIP) with specific affinity towards histamine. Computational modelling was used to screen a monomer library in order to select the monomers able to form the strongest complex with the target analyte. These were subsequently used for MIP synthesis by radical polymerisation initiated by UV. MIPs were then evaluated by liquid chromatography and solid phase extraction (SPE) and best MIP behaviour was observed when itaconic acid was used as functional monomer. Finally, after optimisation of the polymer composition, MIPs were used as adsorbents for SPE and clean-up of histamine in wine samples. The proposed histamine extraction method with the MIP-SPE cartridge was found to be reproducible (<5% RSD) and accurate (93-99% recovery) and provided clear wine extracts. The described methodology is simple and fast and is suitable for the selective histamine extraction and its subsequent quantification by HPLC-DAD from complex matrices such as wine samples.

Screening and quantification of antipsychotic drugs in human brain tissue by liquid chromatography-tandem mass spectrometry: application to postmortem diagnostics of forensic interest.

A quantitative LC-MS/MS method has been developed for the simultaneous determination of 17 antipsychotic drugs in human postmortem brain tissue. Sample preparation was performed using Hybrid Solid Phase Extraction-Precipitation technology for the removal of endogenous protein and phospholipid interferences. The chromatographic separation was performed for 16 min on a C8 column, which used a gradient elution of formate ammonium and acetonitrile, and a flow rate gradient. Triple quadrupole mass spectrometry was employed to generate tandem mass spectrometric (MS/MS) data of the target analytes to select the ion m/z signals. Quantitation of the analytes was performed by operating in the dynamic multiple reaction monitoring (dMRM) mode using an electrospray ionization interface. Calibration curves prepared in the spiked brain tissue were linear in the range 20-8000 ng/g (r(2)>0.993) for all drugs (except olanzapine). Within- and between-day coefficients of variation were lower than 25% for all drugs at the LOQ. The LOQ in the matrix ranged between 2 ng/g and 80 ng/g. The method was successfully applied to the unequivocal identification and accurate quantification of antipsychotic drugs in human postmortem brain tissues: therefore, this method can be used in forensic investigations.