Markov state modelling reveals heterogeneous drug-inhibition mechanism of Calmodulin.

Calmodulin (CaM) is a calcium sensor which binds and regulates a wide range of target-proteins. This implicitly enables the concentration of calcium to influence many downstream physiological responses, including muscle contraction, learning and depression. The antipsychotic drug trifluoperazine (TFP) is a known CaM inhibitor. By binding to various sites, TFP prevents CaM from associating to target-proteins. However, the molecular and state-dependent mechanisms behind CaM inhibition by drugs such as TFP are largely unknown. Here, we build a Markov state model (MSM) from adaptively sampled molecular dynamics simulations and reveal the structural and dynamical features behind the inhibitory mechanism of TFP-binding to the C-terminal domain of CaM. We specifically identify three major TFP binding-modes from the MSM macrostates, and distinguish their effect on CaM conformation by using a systematic analysis protocol based on biophysical descriptors and tools from machine learning. The results show that depending on the binding orientation, TFP effectively stabilizes features of the calcium-unbound CaM, either affecting the CaM hydrophobic binding pocket, the calcium binding sites or the secondary structure content in the bound domain. The conclusions drawn from this work may in the future serve to formulate a complete model of pharmacological modulation of CaM, which furthers our understanding of how these drugs affect signaling pathways as well as associated diseases.

Highly conserved protein Rv1211 in Mycobacterium tuberculosis is a natively unfolded protein that binds to a calmodulin antagonist, trifluoperazine.

Rv1211 is a conserved hypothetical protein in Mycobacterium tuberculosis and is required for the growth and pathogenesis of the bacteria. The protein has been suggested as a calmodulin-like calcium-binding protein with an EF-hand motif and as a target of trifluoperazine, a calmodulin antagonist in eukaryotes that inhibits mycobacterial growth. Here, we expressed the recombinant protein of Rv1211 and performed structural and biochemical studies of Rv1211 and its interaction with Ca2+ or trifluoperazine. Surprisingly, Rv1211 exhibited an elution property typical of a natively unfolded protein. Subsequent circular dichroism experiments with temperature elevation and trifluoroethanol treatment showed that Rv1211 has unfolded structure. Additional NMR experiment confirmed the unfolded state of the protein and further showed that it does not bind to Ca2+. Still, Rv1211 did bind to trifluoperazine, as evidenced by the two-dimensional NMR spectra of 15N-labeled Rv1211. However, there were no peak shifts upon binding, showing that Rv1211 retained its unfolded state even after the trifluoperazine binding. The residues involved in the binding were clustered in the C-terminal region, as identified by the sequence assignment. Isothermal titration calorimetry showed that the Kd of trifluoperazine-Rv1211 binding is 41 µM and that the stoichiometry is 1 : 2 (Rv1211: trifluoperazine). Our results argue against the suggestion of Rv1211 as a Ca2+-binding calmodulin-like protein, and show that Rv1211 is a natively unfolded protein that binds to trifluoperazine. In addition, our results suggest the evidence of the "Fuzziness" in the Rv1211-trifluoperazine interaction that differs from the conventional binding-induced folding of natively unfolded proteins.

Design of Inhibitors of the Intrinsically Disordered Protein NUPR1: Balance between Drug Affinity and Target Function.

Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their physiological ubiquity and their prevalence in various diseases, including cancer. NUPR1 is an IDP that localizes throughout the whole cell, and is involved in the development and progression of several tumors. We have previously repurposed trifluoperazine (TFP) as a drug targeting NUPR1 and, by using a ligand-based approach, designed the drug ZZW-115 starting from the TFP scaffold. Such derivative compound hinders the development of pancreatic ductal adenocarcinoma (PDAC) in mice, by hampering nuclear translocation of NUPR1. Aiming to further improve the activity of ZZW-115, here we have used an indirect drug design approach to modify its chemical features, by changing the substituent attached to the piperazine ring. As a result, we have synthesized a series of compounds based on the same chemical scaffold. Isothermal titration calorimetry (ITC) showed that, with the exception of the compound preserving the same chemical moiety at the end of the alkyl chain as ZZW-115, an increase of the length by a single methylene group (i.e., ethyl to propyl) significantly decreased the affinity towards NUPR1 measured in vitro, whereas maintaining the same length of the alkyl chain and adding heterocycles favored the binding affinity. However, small improvements of the compound affinity towards NUPR1, as measured by ITC, did not result in a corresponding improvement in their inhibitory properties and in cellulo functions, as proved by measuring three different biological effects: hindrance of the nuclear translocation of the protein, sensitization of cells against DNA damage mediated by NUPR1, and prevention of cancer cell growth. Our findings suggest that a delicate compromise between favoring ligand affinity and controlling protein function may be required to successfully design drugs against NUPR1, and likely other IDPs.

Identifying FDA-approved drugs with multimodal properties against COVID-19 using a data-driven approach and a lung organoid model of SARS-CoV-2 entry.

BACKGROUND: Vaccination programs have been launched worldwide to halt the spread of COVID-19. However, the identification of existing, safe compounds with combined treatment and prophylactic properties would be beneficial to individuals who are waiting to be vaccinated, particularly in less economically developed countries, where vaccine availability may be initially limited. METHODS: We used a data-driven approach, combining results from the screening of a large transcriptomic database (L1000) and molecular docking analyses, with in vitro tests using a lung organoid model of SARS-CoV-2 entry, to identify drugs with putative multimodal properties against COVID-19. RESULTS: Out of thousands of FDA-approved drugs considered, we observed that atorvastatin was the most promising candidate, as its effects negatively correlated with the transcriptional changes associated with infection. Atorvastatin was further predicted to bind to SARS-CoV-2's main protease and RNA-dependent RNA polymerase, and was shown to inhibit viral entry in our lung organoid model. CONCLUSIONS: Small clinical studies reported that general statin use, and specifically, atorvastatin use, are associated with protective effects against COVID-19. Our study corroborrates these findings and supports the investigation of atorvastatin in larger clinical studies. Ultimately, our framework demonstrates one promising way to fast-track the identification of compounds for COVID-19, which could similarly be applied when tackling future pandemics.

Characterizing the interactions of the antipsychotic drug trifluoperazine with bovine serum albumin: Probing the drug-protein and drug-drug interactions using multi-spectroscopic approaches.

Trifluoperazine is a potent antipsychotic drug used in the treatment of neurological disorders. The usage of trifluoperazine is often found to be associated with more adverse side effects as compared to other low-potency antipsychotic agents. Plasma proteins play an inevitable role in determining the pharmacokinetic properties of a drug. Hence, this study was conducted with an aim to characterize the interactions of trifluoperazine with bovine serum albumin and determine the influence of other small molecules on its interaction with serum albumin. Trifluoperazine bound to BSA at two independent sites with Kd values of 9.5 and 172.6 µM. Förster resonance energy transfer and computational docking analysis revealed that both the binding sites of trifluoperazine were located closer to TRP 213 in subdomain IIA of BSA. Evaluation of trifluoperazine-BSA interactions at three different temperatures indicated that there was a stable complex formation between the two molecules at the ground state and that the static quenching mechanism was predominant behind these interactions. Binding studies in the presence of pharmaceutically relevant drugs indicated that warfarin, paracetamol, and caffeine negatively influenced the binding of trifluoperazine on BSA. Lastly, Fourier transformed infrared spectroscopy and circular dichroism spectroscopy indicated that the binding of trifluoperazine induced a conformational change by reducing the α-helical content of BSA. The study implicates that the small molecules which prefer binding to the Sudlow site I of BSA might compete with trifluoperazine for its binding site thereby increasing the concentration of free trifluoperazine in the plasma which could lead to adverse side effects in patients.

Ligand-based design identifies a potent NUPR1 inhibitor exerting anticancer activity via necroptosis.

Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their prevalence in various diseases including cancer. Drug development targeting IDPs is challenging because they have dynamical structure features and conventional drug design is not applicable. NUPR1 is an IDP playing an important role in pancreatic cancer. We previously reported that Trifluoperazine (TFP), an antipsychotic agent, was capable of binding to NUPR1 and inhibiting tumors growth. Unfortunately, TFP showed strong central nervous system side-effects. In this work, we undertook a multidisciplinary approach to optimize TFP, based on the synergy of computer modeling, chemical synthesis, and a variety of biophysical, biochemical and biological evaluations. A family of TFP-derived compounds was produced and the most active one, named ZZW-115, showed a dose-dependent tumor regression with no neurological effects and induced cell death mainly by necroptosis. This study opens a new perspective for drug development against IDPs, demonstrating the possibility of successful ligand-based drug design for such challenging targets.

Photochemical and Pharmacokinetic Characterization of Orally Administered Chemicals to Evaluate Phototoxic Risk.

This study aimed to verify the applicability of a proposed photosafety screening system based on a reactive oxygen species (ROS) assay and a cassette-dosing pharmacokinetic (PK) study to chemicals with wide structural diversity. The orally taken chemicals, erythromycin, gatifloxacin, 8-methoxypsoralen (MOP), pirfenidone (PFD), trifluoperazine (TFP), and voriconazole (VRZ), were selected as test compounds. The ROS assay was conducted to evaluate their photoreactivity, and all test compounds excluding erythromycin generated significant ROS under simulated sunlight exposure. According to the ROS data, TFP had potent photoreactivity, and the photoreactivity of 4 other compounds was judged to be moderate. Regarding the oral cassette-dosing PK test in rats, the skin deposition of MOP, PFD, and VRZ was relatively high, and gatifloxacin and TFP exhibited moderate skin deposition properties. Based on the ROS and PK data of test compounds, PFD and TFP were judged to be potent phototoxic compounds, and MOP and VRZ were deduced to have phototoxic risk. The predicted phototoxic risk of test compounds by proposed screening was mostly in agreement with observed in vivo phototoxicity in the rat skin. The proposed screening system could provide reliable photosafety information on orally administered compounds with wide structural diversity.

Role of Overexpressed Transcription Factor FOXO1 in Fatal Cardiovascular Septal Defects in Patau Syndrome: Molecular and Therapeutic Strategies.

Patau Syndrome (PS), characterized as a lethal disease, allows less than 15% survival over the first year of life. Most deaths owe to brain and heart disorders, more so due to septal defects because of altered gene regulations. We ascertained the cytogenetic basis of PS first, followed by molecular analysis and docking studies. Thirty-seven PS cases were referred from the Department of Pediatrics, King Abdulaziz University Hospital to the Center of Excellence in Genomic Medicine Research, Jeddah during 2008 to 2018. Cytogenetic analyses were performed by standard G-band method and trisomy13 were found in all the PS cases. Studies have suggested that genes of chromosome 13 and other chromosomes are associated with PS. We, therefore, did molecular pathway analysis, gene interaction, and ontology studies to identify their associations. Genomic analysis revealed important chr13 genes such as FOXO1, Col4A1, HMGBB1, FLT1, EFNB2, EDNRB, GAS6, TNFSF1, STARD13, TRPC4, TUBA3C, and TUBA3D, and their regulatory partners on other chromosomes associated with cardiovascular disorders, atrial and ventricular septal defects. There is strong indication of involving FOXO1 (Forkhead Box O1) gene-a strong transcription factor present on chr13, interacting with many septal defects link genes. The study was extended using molecular docking to find a potential drug lead for overexpressed FOXO1 inhibition. The phenothiazine and trifluoperazine showed efficiency to inhibit overexpressed FOXO1 protein, and could be potential drugs for PS/trisomy13 after validation.

Synergistic Enhancement of Enzyme Performance and Resilience via Orthogonal Peptide-Protein Chemistry Enabled Multilayer Construction.

Protein immobilization is critical to utilize their unique functions in diverse applications. Herein, we report that orthogonal peptide-protein chemistry enabled multilayer construction can facilitate the incorporation of various folded structural domains, including calmodulin in different states, affibody, and dihydrofolate reductase (DHFR). An extended conformation is found to be the most advantageous for steady film growth. The resulting protein thin films exhibit sensitive and selective responsive behaviors to biosignals, such as Ca2+, trifluoperazine, and nicotinamide adenine dinucleotide phosphate (NADPH), and fully maintain the catalytic activity of DHFR. The approach is applicable to different substrates such as hydrophobic gold and hydrophilic silica microparticles. The DHFR enzyme can be immobilized onto silica microparticles with tunable amounts. The multilayer setup exhibits a synergistic enhancement of DHFR activity with increasing numbers of bilayers and also makes the embedded DHFR more resilient to lyophilization. Therefore, this is a convenient and versatile method for protein immobilization with potential benefits of synergistic enhancement in enzyme performance and resilience.

Inhibitor and peptide binding to calmodulin characterized by high pressure Fourier transform infrared spectroscopy and Förster resonance energy transfer.

We compare the binding of an inhibitor with that of a natural peptide to Ca2+ saturated calmodulin (holo-CaM). As inhibitor we have chosen trifluoperazine (TFP) that is inducing a huge conformational change of holo-CaM from the open dumbbell-shaped to the closed globular conformation upon binding. On the other hand, melittin is used as model peptide, which is a well-known natural binding partner of holo-CaM. The experiments are carried out as a function of pressure to reveal the contribution of volume or packing effects to the stability of the calmodulin-ligand complexes. From high-pressure Fourier transform infrared (FTIR) spectroscopy, we find that the holo-CaM/TFP complex has a much higher pressure stability than the holo-CaM/melittin complex. Although the analysis of the secondary structure of holo-CaM (without and with ligand) indicates no major changes up to several kbar, pressure-induced exposure of α-helices to water is most pronounced for holo-CaM without ligand, followed by holo-CaM/melittin and then holo-CaM/TFP. Moreover, structural pressure resistance of the holo-CaM/TFP complex in comparison with the holo-CaM/melittin complex is also clearly visible by higher Ca2+ affinity. Förster resonance energy transfer (FRET) from the Tyr residues of holo-CaM to the Trp residue of melittin even suggests some partial dissociation of the complex under pressure which points to void volumes at the protein-ligand interface and to electrostatic binding. Thus, all results of this study show that the inhibitor TFP binds to holo-CaM with higher packing density than the peptide melittin enabling a favorable volume contribution to the inhibitor efficiency.

Reprint of: A chemical screen identifies trifluoperazine as an inhibitor of glioblastoma growth.

Glioblastoma (GBM) is regarded as the most common malignant brain tumor but treatment options are limited. Thus, there is an unmet clinical need for compounds and corresponding targets that could inhibit GBM growth. We screened a library of 80 dopaminergic ligands with the aim of identifying compounds capable of inhibiting GBM cell line proliferation and survival. Out of 45 active compounds, 8 were further validated. We found that the dopamine receptor D2 antagonist trifluoperazine 2HCl inhibits growth and proliferation of GBM cells in a dose dependent manner. Trifluoperazine's inhibition of GBM cells is cell line dependent and correlates with variations in dopamine receptor expression profile. We conclude that components of the dopamine receptor signaling pathways are potential targets for pharmacological interventions of GBM growth.

A high pressure study of calmodulin-ligand interactions using small-angle X-ray and elastic incoherent neutron scattering.

Calmodulin (CaM) is a Ca2+ sensor and mediates Ca2+ signaling through binding of numerous target ligands. The binding of ligands by Ca2+-saturated CaM (holo-CaM) is governed by attractive hydrophobic and electrostatic interactions that are weakened under high pressure in aqueous solutions. Moreover, the potential formation of void volumes upon ligand binding creates a further source of pressure sensitivity. Hence, high pressure is a suitable thermodynamic variable to probe protein-ligand interactions. In this study, we compare the binding of two different ligands to holo-CaM as a function of pressure by using X-ray and neutron scattering techniques. The two ligands are the farnesylated hypervariable region (HVR) of the K-Ras4B protein, which is a natural binding partner of holo-CaM, and the antagonist trifluoperazine (TFP), which is known to inhibit holo-CaM activity. From small-angle X-ray scattering experiments performed up to 3000 bar, we observe a pressure-induced partial unfolding of the free holo-CaM in the absence of ligands, where the two lobes of the dumbbell-shaped protein are slightly swelled. In contrast, upon binding TFP, holo-CaM forms a closed globular conformation, which is pressure stable at least up to 3000 bar. The HVR of K-Ras4B shows a different binding behavior, and the data suggest the dissociation of the holo-CaM/HVR complex under high pressure, probably due to a less dense protein contact of the HVR as compared to TFP. The elastic incoherent neutron scattering experiments corroborate these findings. Below 2000 bar, pressure induces enhanced atomic fluctuations in both holo-CaM/ligand complexes, but those of the holo-CaM/HVR complex seem to be larger. Thus, the inhibition of holo-CaM by TFP is supported by a low-volume ligand binding, albeit this is not associated with a rigidification of the complex structure on the sub-ns Å-scale.

Interaction of Antipsychotic Drugs with Sucrase, Kinetics and Structural Study.

BACKGROUND: In patients with the Congenital Sucrase-Isomaltase Deficiency (CSID), who lack intestinal sucrase-isomaltase enzyme, a suspension of yeast sucrase is applied as a drug to compensate the enzyme deficiency. While antipsychotic drugs are used for the treatment of schizophrenia, administering multiple drugs at the same time may counteract each other. METHODS: In this study, the interaction between trifluoperazine and haloperidol as antipsychotic drugs on oral drug yeast sucrase was investigated. In this regard, the kinetic parameters of enzyme were determined in the presence or absence of the drugs. The kinetic parameters of the drugs such as Ki and IC50 were also calculated. Lineweaver - Burk plot was used to reveal the type of inhibition. RESULTS: The results showed that both drugs could reduce sucrase activity and decrease the Vmax of the enzyme by non-competitive inhibition. The IC50 and Ki values of the drugs were determined to be 0.7 and 0.068 mM and 0.45 and 0.063 mM for haloperidol and trifluoperazine, respectively. The results suggested that trifluoperazine binds to the enzyme with higher affinity than haloperidol. Fluorescence measurement was used for conformational investigations of the drugs and sucrase interaction. It was shown that the drugs bind to free enzyme and enzyme-substrate complex which are accompanied with hyperchromicity. This suggests that tryptophan residues of the enzyme transferred to hydrophobic medium after binding of the drugs to the enzyme. CONCLUSION: The finding of this research revealed that both trifluoperazine and haloperidol could inhibit sucrase in non-competitive manner. The kinetic parameters and conformational changes due to binding of trifluoperazine to the enzyme were different from that of haloperidol.

Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma.

Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the 'fuzzy' interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs.

Trifluoperazine blocks the human cardiac sodium channel, Nav1.5, independent of calmodulin.

Trifluoperazine is a phenothiazine derivative which is mainly used in the management of schizophrenia and also acts as a calmodulin inhibitor. We used the whole-cell patch-clamp technique to study the effects of trifluoperazine on human Nav1.5 (hNav1.5) currents expressed in HEK293 cells. The 50% inhibitory concentration of trifluoperazine was 15.5 ± 0.3 µM and the Hill coefficient was 2.7 ± 0.1. The effects of trifluoperazine on hNav1.5 were completely and repeatedly reversible after washout. Trifluoperazine caused depolarizing shifts in the activation and hyperpolarizing shifts in the steady-state inactivation of hNav1.5. Trifluoperazine also showed strong use-dependent inhibition of hNav1.5. The blockade of hNav1.5 currents by trifluoperazine was not affected by the whole cell dialysis of the calmodulin inhibitory peptide. Our results indicated that trifluoperazine blocks hNav1.5 current in concentration-, state- and use-dependent manners rather than via calmodulin inhibition.

Ganoderic acid B's influence towards the therapeutic window of trifluoperazine (TFP).

BACKGROUND: Ganoderic acid B is an important bioactive ingredient isolated from Ganoderma lucidum, and exhibits various pharmacological activities. AIMS: To investigate the influence of Ganoderic acid B towards the therapeutic window of trifluoperazine (TFP). METHODS: In vitro human liver microsomes (HLMs) incubation system was used to determine the inhibition of Ganoderic acid B towards the glucuronidation of trifluoperazine (TFP). RESULTS: Ganoderic acid B exerted concentration-dependent inhibition towards the glucuronidation of TFP. Furthermore, Dixon plot was used to determine the inhibition type. The intersection point was located in the second quadrant in Dixon plot, indicating the competitive inhibition of Ganoderic acid B towards TFP glucuronidation. Through fitting the data using competitive nonlinear fitting equation, the inhibition kinetic parameter was calculated to be 56.7 uM. CONCLUSION: All this data indicated the potential influence of Ganoderic acid B-containing herbs towards therapeutic window of TFP. Given that the glucuronidation reaction of TFP is the probe reaction of UGT1A4, the data obtained from the present study also indicated the potential influence of Ganoderic acid-containing herbs towards the therapeutic window of drugs mainly undergoing UGT1A4-mediated metabolism.

Opposing orientations of the anti-psychotic drug trifluoperazine selected by alternate conformations of M144 in calmodulin.

The anti-psychotic drug trifluoperazine (TFP) is an antagonist observed to bind to calcium-saturated calmodulin ((Ca(2+) )4 -CaM) at ratios of 1:1 (1CTR), 2:1 (1A29), and 4:1 (1LIN). Each structure contains one TFP bound in the hydrophobic cleft of the C-domain of CaM. However, the orientation of the trifluoromethyl (CF3 ) moiety differs among them: it is buried in the C-domain cleft of 1A29 and 1LIN, but protrudes from 1CTR. We report a 2.0 Å resolution crystallographic structure (4RJD) of TFP bound to the (Ca(2+) )-saturated C-domain of CaM (CaMC ). The asymmetric unit contains two molecules of (Ca(2+) )2 -CaMC . Chain backbones were nearly identical, but the orientation of TFP in the cleft of Chain A matched 1A29/1LIN, while TFP bound to Chain B matched 1CTR. This was accommodated by a flip of the M144 sidechain and small changes in sidechains of M109 and M145. Docking simulations suggested that the rotamer conformation of M144 determined the orientation of TFP within the cleft of (Ca(2+) )2 -CaMC . Chains A and B show that the open cleft of (Ca(2+) )2 -CaMC is promiscuous in accepting TFP in reversed directions under the same crystallization conditions. Observing multiple orientations of an antagonist bound to a single protein highlights the challenge of designing highly specific pharmaceuticals, and may have importance for QSAR of other CF3 -containing drugs such as fluoxetine (anti-depressant) or efavirenz (reverse transcriptase inhibitor). This study emphasizes that a single structure of a complex represents an energetically accessible state, but does not necessarily show the full range of energetically equivalent states.

Molecular structure and vibrational analysis of Trifluoperazine by FT-IR, FT-Raman and UV-Vis spectroscopies combined with DFT calculations.

The complete vibrational assignment and analysis of the fundamental vibrational modes of Trifluoperazine (TFZ) was carried out using the experimental FT-IR, FT-Raman and UV-Vis data and quantum chemical studies. The observed vibrational data were compared with the wavenumbers derived theoretically for the optimized geometry of the compound from the DFT-B3LYP gradient calculations employing 6-31G (d,p) basis set. Thermodynamic properties like entropy, heat capacity and enthalpy have been calculated for the molecule. The HOMO-LUMO energy gap has been calculated. The intramolecular contacts have been interpreted using natural bond orbital (NBO) and natural localized molecular orbital (NLMO) analysis. Important non-linear properties such as first hyperpolarizability of TFZ have been computed using B3LYP quantum chemical calculation.

Molecularly imprinted solid-phase extraction for selective trace analysis of trifluoperazine.

In this study, a novel sample clean-up technique based on the molecularly imprinted solid-phase extraction procedure is described for the determination of trifluoperazine (TFP) in biological fluids. The water-compatible molecularly imprinted polymers (MIPs) were prepared by using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and TFP as the template molecule. The novel imprinted polymer was used as a solid-phase extraction sorbent for the extraction of TFP from human serum and urine samples. Various parameters affecting the extraction efficiency of the polymer were evaluated. The selectivity of MIPs was evaluated by checking several substances with molecular structures similar to the template. The limits of detection and quantification for TFP in urine samples were 0.06 and 0.2 µg/L, respectively. These limits for TFP in serum samples were 0.15 and 0.4 µg/L, respectively. The recovery values for serum and urine samples were higher than 92 and 93%, respectively.

Room temperature phosphorimetric determination of bromate in flour based on energy transfer.

Determination of bromate ions in contaminated flour samples by using a room temperature phosphorescence (RTP) optosensor is described. The optosensor is based on the non-radiative energy transfer from α-bromonaphthalene (a phosphorescent molecule insensitive to the presence of the analyte) acting as donor, to an energy acceptor bromate-sensitive molecule (trifluoperazine hydrochloride). The RTP emission of the selected donor greatly overlaps with the absorption spectrum of the acceptor, resulting in a decrease of the measured signal as the concentration of bromate ions increases. A simple and general procedure is proposed to carry out the incorporation of both the donor and acceptor molecules in an appropriate solid material (sensing phase) through the co-immobilization of the species in a sol-gel inorganic matrix. The optimum amounts of the sol-gel precursors, including silica precursors, type of catalysis, and concentrations of donor and acceptor molecules, have been evaluated in order to obtain the best analytical features of the proposed optosensor for bromate determination. The highly stable developed sensing phase shows a selective and reversible response towards bromate even in presence of dissolved oxygen (a well-known quencher of the RTP). The calibration graphs were linear up to 200 mg L(-1), with a detection limit for bromate dissolved in aqueous medium of 0.2 mg L(-1). Sample throughput of the proposed optosensor was about 18 measurements h(-1). Application of the developed sensing phase was successfully proved for the detection of bromate ions in commercial flours, obtaining good recoveries.