A Small Footprint and Robust Interface for Solid Phase Microextraction and Mass Spectrometry Based on Vibrating Sharp-Edge Spray Ionization.

Combining solid phase microextraction (SPME) and mass spectrometry (MS) analysis has become increasingly important to many bioanalytical, environmental, and forensic applications due to its simplicity, rapid analysis, and capability of reducing matrix effects for complex samples. To further promote the adoption of SPME-MS based analysis and expand its application scope calls for efficient and convenient interfaces that couple the SPME sample handling with the efficient analyte ionization for MS. Here, we report a novel interface that integrates both the desorption and the ionization steps in one device based on the capillary vibrating sharp-edge spray ionization (cVSSI) method. We demonstrated that the cVSSI is capable of nebulizing liquid samples in a pulled-tip glass capillary with a battery powered function generator. The cVSSI device allows the insertion of a SPME probe into the spray capillary for desorption and then direct nebulization of the desorption solvent in situ. With the integrated interface, we have demonstrated rapid MS analysis of drug compounds from serum samples. Quantitative determination of various drug compounds including metoprolol, pindolol, acebutolol, oxprenolol, capecitabine, and irinotecan was achieved with good linearity (R2 = 0.97-0.99) and limit of detection ranging from 0.25 to 0.59 ng/mL without using a high voltage source. Only 3.5 µL of desorption solvent and 3 min desorption time were needed for the present method. Overall, we demonstrated a portable SPME-MS interface featuring high sensitivity, short analysis time, small footprint, and low cost, which makes it an attractive method for many applications requiring sample cleanup including drug compound monitoring, environmental sample analysis, and forensic sample analysis.

Kinetics, mechanism and toxicity of intermediates of solar light induced photocatalytic degradation of pindolol: Experimental and computational modeling approach.

In this work, we have investigated the stability of pindolol (PIN), a non-selective ß1-blocker detected in the river and wastewater of hospitals, in water solution under solar irradiation. Further, detailed insights into the stability of PIN were obtained by the density functional theory (DFT) calculations and molecular dynamics simulations. The kinetics of PIN photocatalytic degradation and mineralization has been studied using four commercial photocatalysts ZnO and TiO2 (P25, Hombikat, and Wackherr). It was found that the major role in degradation of PIN play the reactive hydroxyl radicals. The structures of degradation intermediates were suggested by LC-ESI-MS/MS and DFT calculations. Also, DFT calculations were used to refine molecular structures of intermediates and obtain their geometries. Toxicity of PIN and its mixtures formed during photocatalytic degradation were investigated using mammalian cell lines (H-4-II-E, HT-29, and MRC-5). The H-4-II-E cell line was the most sensitive to PIN and its photodegradation mixtures. The computational results were combined with the experimental data on the amounts of degradation intermediates for determination of the intermediates that were principally responsible for the toxicity. Intermediate with two hydroxyl groups, positioned on indole ring in meta and para positions, was proposed as the one with the highest contribution to toxicity.

Pharmacological Analysis and Structure Determination of 7-Methylcyanopindolol-Bound ß1-Adrenergic Receptor.

Comparisons between structures of the ß1-adrenergic receptor (AR) bound to either agonists, partial agonists, or weak partial agonists led to the proposal that rotamer changes of Ser(5.46), coupled to a contraction of the binding pocket, are sufficient to increase the probability of receptor activation. (RS)-4-[3-(tert-butylamino)-2-hydroxypropoxy]-1H-indole-2-carbonitrile (cyanopindolol) is a weak partial agonist of ß1AR and, based on the hypothesis above, we predicted that the addition of a methyl group to form 4-[(2S)-3-(tert-butylamino)-2-hydroxypropoxy]-7-methyl-1H-indole-2-carbonitrile (7-methylcyanopindolol) would dramatically reduce its efficacy. An eight-step synthesis of 7-methylcyanopindolol was developed and its pharmacology was analyzed. 7-Methylcyanopindolol bound with similar affinity to cyanopindolol to both ß1AR and ß2AR. As predicted, the efficacy of 7-methylcyanopindolol was reduced significantly compared with cyanopindolol, acting as a very weak partial agonist of turkey ß1AR and an inverse agonist of human ß2AR. The structure of 7-methylcyanopindolol-bound ß1AR was determined to 2.4-Å resolution and found to be virtually identical to the structure of cyanopindolol-bound ß1AR. The major differences in the orthosteric binding pocket are that it has expanded by 0.3 Å in 7-methylcyanopindolol-bound ß1AR and the hydroxyl group of Ser(5.46) is positioned 0.8 Å further from the ligand, with respect to the position of the Ser(5.46) side chain in cyanopindolol-bound ß1AR. Thus, the molecular basis for the reduction in efficacy of 7-methylcyanopindolol compared with cyanopindolol may be regarded as the opposite of the mechanism proposed for the increase in efficacy of agonists compared with antagonists.

Sustained and controlled release of lipophilic drugs from a self-assembling amphiphilic peptide hydrogel.

Materials which undergo self-assembly to form supramolecular structures can provide alternative strategies to drug loading problems in controlled release application. RADA 16 is a simple and versatile self-assembling peptide with a designed structure formed of two distinct surfaces, one hydrophilic and one hydrophobic that are positioned in such a well-ordered fashion allowing precise assembly into a predetermined organization. A "smart" architecture in nanostructures can represent a good opportunity to use RADA16 as a carrier system for hydrophobic drugs solving problems of drugs delivery. In this work, we have investigated the diffusion properties of Pindolol, Quinine and Timolol maleate from RADA16 in PBS and in BSS-PLUS at 37°C. A sustained, controlled, reproducible and efficient drug release has been detected for all the systems, which allows to understand the dependence of release kinetics on the physicochemical characteristics of RADA16 structural and chemical properties of the selected drugs and the nature of solvents used. For the analysis various physicochemical characterization techniques were used in order to investigate the state of the peptide before and after the drugs were added. Not only does RADA16 optimise drug performance, but it can also provide a solution for drug delivery issues associated with lipophilic drugs.

Intermolecular complexation of low-molecular-weight succinoglycans directs solubility enhancement of pindolol.

The low-molecular-weight succinoglycans isolated from Sinorhizobium meliloti are repeating octasaccharide units consisting of monomers, dimers, and trimers. Pindolol is a beta-blocker used to treat cardiovascular disorders. We investigated the formation of complexes between pindolol and low-molecular-weight succinoglycan monomers (SGs). Even though SGs have a linear structure, the solubility of pindolol in the presence of SGs was increased up to 7-fold compared with methyl-ß-cyclodextrin reported as the best solubilizer of pindolol. Complexation of SGs with pindolol was confirmed by nuclear magnetic resonance, Fourier-transform infrared spectroscopy, differential scanning calorimetry, and scanning electron microscopy. Formation constants of complexes were determined from phase solubility diagrams. Conformation of complex was suggested based on a molecular docking study. The present study indicated that formation of pindolol/SGs complexes not only resulted in increased pindolol solubility but also could be useful for improving its clinical application as it did not affect cell viability.

Chiral separations by non-aqueous capillary electrophoresis in DMSO-based background electrolytes.

Capillary electrophoresis (CE) is a powerful technique for enantioseparations due to its high separation efficiency, high versatility, speed of analysis and low consumption of samples and reagents. Non-aqueous capillary electrophoresis (NACE) appears as a promising technique to perform enantioseparations when the drugs, chiral selectors or samples are non-water soluble. Chiral separations have been performed by NACE mainly using alcoholic solvents as BGEs, with problems of current breakdowns and changes in the BGE composition, due to their high volatility. In this work, the suitability of DMSO as BGE in NACE has been evaluated. Different experimental variables affecting the enantioresolution of three drugs have been evaluated, finally achieving complete enantioresolution of two drugs (verapamil, Rs=1.5 and pindolol, Rs=2.0) and partial resolution of the third one (fenfluramine, Rs=1.2). DMSO has been demonstrated to be a good alternative to methanolic BGEs in NACE.

Rational synthesis of pindolol imprinted polymer by non-covalent protocol based on computational approach.

Pindolol (PDL) is a potent and specific adrenoreceptor blocking agent. It is widely used in the treatment of hypertension, cardiac arrhythmia and angina pectoris. Molecularly imprinted polymers (MIPs) are synthetic receptors having potential applications in drug delivery systems and devices such as diagnostic sensors. In the present work, ab initio quantum mechanical simulations and computational screening were used to identify functional monomer having best interactions with PDL. A virtual library of 16 functional monomers was built and the possible minimum energy conformation of the monomers and PDL were calculated using Hartree-Fock (HF) method for the synthesis of PDL imprinted polymer. The interaction energy between functional monomer and the template were corrected by means of basis set superposition error (BSSE) in all pre-polymerization complexes. The hydrogen bonding between PDL and functional monomer was evaluated by changes in bond lengths before and after complex formation. The virtual template-monomer complex with highest interaction energy is more stable during the polymerization and leads to high selectivity and specificity toward the template. The interaction energy of PDL was found to be the highest with itaconic acid followed by 4-vinyl pyridine and least with acrylonitrile. Taking a spectroscopic viewpoint, results obtained from analysis of the harmonic infrared spectrum were examined. Red and blue shifts related to the stretching frequencies of either donors or acceptors of protons were identified and compared experimentally. Stoichiometric mole ratio of template to functional monomer was optimized and confirmed by UV visible spectra titrations. The theoretical results were correlated by evaluation of binding parameters of MIPs. The experimental binding results were in good agreement with theoretical computations.

Structural basis for molecular recognition at serotonin receptors.

Serotonin or 5-hydroxytryptamine (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. We report the crystal structures of the human 5-HT1B G protein-coupled receptor bound to the agonist antimigraine medications ergotamine and dihydroergotamine. The structures reveal similar binding modes for these ligands, which occupy the orthosteric pocket and an extended binding pocket close to the extracellular loops. The orthosteric pocket is formed by residues conserved in the 5-HT receptor family, clarifying the family-wide agonist activity of 5-HT. Compared with the structure of the 5-HT2B receptor, the 5-HT1B receptor displays a 3 angstrom outward shift at the extracellular end of helix V, resulting in a more open extended pocket that explains subtype selectivity. Together with docking and mutagenesis studies, these structures provide a comprehensive structural basis for understanding receptor-ligand interactions and designing subtype-selective serotonergic drugs.

Kinetic capillary electrophoresis with mass-spectrometry detection (KCE-MS) facilitates label-free solution-based kinetic analysis of protein-small molecule binding.

Tandem tracker: Here we introduce a method for studying the kinetics of protein-small-molecule interactions based on kinetic capillary electrophoresis (KCE) separation and MS detection. Due to the variety of KCE methods and MS modes available, the KCE-MS tandem is a highly versatile platform for label-free, solution-based kinetic studies of affinity interactions.

Synthesis and characterization of high-affinity 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-labeled fluorescent ligands for human ß-adrenoceptors.

The growing practice of exploiting noninvasive fluorescence-based techniques to study G protein-coupled receptor pharmacology at the single cell and single molecule level demands the availability of high-quality fluorescent ligands. To this end, this study evaluated a new series of red-emitting ligands for the human ß-adrenoceptor family. Upon the basis of the orthosteric ligands propranolol, alprenolol, and pindolol, the synthesized linker-modified congeners were coupled to the commercially available fluorophore BODIPY 630/650-X. This yielded high-affinity ß-adrenoceptor fluorescent ligands for both the propranolol and alprenolol derivatives; however, the pindolol-based products displayed lower affinity. A fluorescent diethylene glycol linked propranolol derivative (18a) had the highest affinity (log K(D) of -9.53 and -8.46 as an antagonist of functional ß2- and ß1-mediated responses, respectively). Imaging studies with this compound further confirmed that it can be employed to selectively label the human ß2-adrenoceptor in single living cells, with receptor-associated binding prevented by preincubation with the nonfluorescent ß2-selective antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118551) ( J. Cardiovasc. Pharmacol.1983, 5, 430-437. ).

1H and 13C NMR characteristics of ß-blockers.

The ß-blockers are important drugs and decades of clinical experience proved their high medical status. However, to the best of our knowledge, there is no complete assignment of (1)H and (13)C NMR resonances of popular representatives: acebutolol, alpenolol, pindolol, timolol and propranolol and the published NMR data on carvedilol and atenolol are incorrect. Therefore, (1)H and (13)C NMR spectroscopy was applied for the characterization of a series of ß-adrenolytics: carvedilol (1), pindolol (2), alprenolol (3), acebutolol (4), atenolol (5), propranolol (6) and timolol (7). Two-dimensional NMR experiments (COSY, HMQC, HMBC, NOESY) allowed the unequivocal assignment of (1)H and (13)C spectra for solution (DMSO-d(6) ). Salts and bases can be easily distinguished based on (13)C chemical shifts which are within 65.0-65.5 ppm (OC2) and 46.9-47.0 (NC3) for hydrochlorides and larger, ca. 68.4 ppm (OC2) and 50.3-52.6 (NC3) for bases. NMR data of 1-7 should be included in pharmacopoeias.

Electrically assisted capillary liquid chromatography using a silica monolithic column.

A silica monolithic capillary column was linked to an open capillary of the same internal diameter via a Teflon sleeve to form a duplex column to investigate the combination of chromatography and electrophoresis in the mode of electrically assisted capillary liquid chromatography (eCLC). Using a commercial CE instrument with an 8.5 cm long, 100 microm i.d. reversed phase silica monolithic section and a window 1.5 cm beyond the end of this in a 21.5 cm open section, a minimum plate height of 9 microm was obtained in capillary liquid chromatography (CLC) mode at a low driving pressure of 50 psi. In eCLC mode, high speed and high resolution separations of acidic and basic compounds were achieved with selectivity tuning based on the flexible combination of pressure (0-100 psi) and voltage. Taking advantage of the excellent permeability of silica monolithic columns, use of a step flow gradient enabled elution of compounds with different charge state.

X-ray structure breakthroughs in the GPCR transmembrane region.

G-protein-coupled receptor (GPCR) proteins [Lundstrom KH, Chiu ML, editors. G protein-coupled receptors in drug discovery. CRC Press; 2006] are the single largest drug target, representing 25-50% of marketed drugs [Overington JP, Al-Lazikani B, Hopkins AL. How many drug targets are there? Nat Rev Drug Discov 2006;5(12):993-6; Parrill AL. Crystal structures of a second G protein-coupled receptor: triumphs and implications. ChemMedChem 2008;3:1021-3]. While there are six subclasses of GPCR proteins, the hallmark of all GPCR proteins is the transmembrane-spanning region. The general architecture of this transmembrane (TM) region has been known for some time to contain seven alpha-helices. From a drug discovery and design perspective, structural information of the GPCRs has been sought as a tool for structure-based drug design. The advances in the past decade of technologies for structure-based design have proven to be useful in a number of areas. Invoking these approaches for GPCR targets has remained challenging. Until recently, the most closely related structures available for GPCR modeling have been those of bovine rhodopsin. While a representative of class A GPCRs, bovine rhodopsin is not a ligand-activated GPCR and is fairly distant in sequence homology to other class A GPCRs. Thus, there is a variable degree of uncertainty in the use of the rhodopsin X-ray structure as a template for homology modeling of other GPCR targets. Recent publications of X-ray structures of class A GPCRs now offer the opportunity to better understand the molecular mechanism of action at the atomic level, to deploy X-ray structures directly for their use in structure-based design, and to provide more promising templates for many other ligand-mediated GPCRs. We summarize herein some of the recent findings in this area and provide an initial perspective of the emerging opportunities, possible limitations, and remaining questions. Other aspects of the recent X-ray structures are described by Weis and Kobilka [Weis WI, Kobilka BK. Structural insights into G-protein-coupled receptor activation. Curr Opin Struct Biol 2008;18:734-40] and Mustafi and Palczewski [Mustafi D, Palczewski K. Topology of class A G protein-coupled receptors: insights gained from crystal structures of rhodopsins, adrenergic and adenosine receptors. Mol Pharmacol 2009;75:1-12].

A study of the structure of the pindolol based on infrared spectroscopy and natural bond orbital theory.

Beta-adrenoceptor-blocking agents (beta-blockers) are on the list of the top selling drugs. Pindolol is a representative of this type of compound, either from the structural point of view, or as reference for comparison of the pharmacokinetic properties of the beta-blockers. A study of the pindolol structure based on infrared spectroscopy and natural bond orbital (NBO) theory is the main aim of the present research. FTIR spectra of the solid pindolol were recorded from 4000 to 400cm(-1), at temperatures between 25 and -170 degrees C. For spectral interpretation, the theoretical vibrational spectra of the conformer present in the solid was obtained at the B3LYP/6-31G* level of theory. NBO analysis of the reference conformer, before and after optimization, was carried out at the same level of theory referred above. Characteristic absorption vibrational bands of the spectra of solid pindolol and of the isolated conformer were identified. Intra- and intermolecular interactions in pindolol were confirmed by the frequency shift of the vibrational modes and by the NBO theory. A detailed molecular picture of pindolol and of its intermolecular interactions was obtained from spectroscopy and NBO theory. The combination of both methods gives a deeper insight into the structure.

Preparation and evaluation of propranolol molecularly imprinted solid-phase microextraction fiber for trace analysis of beta-blockers in urine and plasma samples.

An improved multiple co-polymerization technique was developed to prepare a novel molecularly imprinted polymer (MIP)-coated solid-phase microextraction (SPME) fiber with propranolol as template. Investigation was performed for the characteristics and application of the fibers. The MIP coating was highly crosslinked and porous with the average thickness of only 25.0 microm. Consequently, the adsorption and desorption of beta-blockers within the MIP coating could be achieved quickly. The specific selectivity was discovered with the MIP-coated fibers to propranolol and its structural analogues such as atenolol, pindolol, and alprenolol. In contrast, only non-specific adsorption could be shown with the non-imprinted polymer (NIP)-coated fibers, and the extraction efficiencies of propranolol and pindolol with the MIP-coated fibers were higher markedly than that with the commercial SPME fibers. A MIP-coated SPME coupled with high-performance liquid chromatography (HPLC) method for propranolol and pindolol determination was developed under the optimized extraction conditions. Linear ranges for propranolol and pindolol were 20-1000 microg L(-1) and detection limits were 3.8 and 6.9 microg L(-1), respectively. Propranolol and pindolol in the spiked human urine and plasma samples, extracted with organic solvent firstly, could be simultaneous monitored with satisfactory recoveries through this method.

Structure of a beta1-adrenergic G-protein-coupled receptor.

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 A resolution crystal structure of a beta(1)-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane alpha-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the beta(1)-adrenergic receptor and binding of carazolol to the beta(2)-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the beta(2)-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.

Effects of alcohols on CE enantioseparation of basic drugs with native gamma-CD as chiral selector.

The effects of alcohol on the CE enantioseparation of selected basic drugs with gamma-CD as the chiral selector was investigated. The enantioseparation behavior of the analytes with gamma-CD in the absence and presence of different alcohols specifically methanol, ethanol, 2-propanol (IPA), and 2-methyl-2-propanol (TBA), the relationship of enantiomeric resolution (R(s)) values with either hydrophobicity or bulkiness of the alcohols, as well as the effect of these alcohols on interaction of the analytes with gamma-CD were studied. Results showed that hydrophobicity and/or bulkiness of alcohols have an influence on the enantioresolution of most of the analytes based on the relatively high correlation coefficients (R) obtained between R(s) versus log P and between R(s) versus ovality (i.e., parameter to indicate bulkiness of a molecule). Comparison of the values of the average binding constants obtained for each enantiomeric pair in the presence and absence of 5% IPA showed that alcohols can increase, decrease, or give a minimal effect on the analyte-gamma-CD interaction depending on the analyte. Furthermore, the significant enhancement in the enantioresolution of both propranolol and pindolol in the presence of either IPA or TBA led to the baseline enantioresolution of both drugs using 35 mM gamma-CD.

Study on chiral resolution of three beta-blockers by affinity electrokinetic chromatography.

The chiral resolution of three beta-blockers including propranolol, pindolol and oxprenolol, was studied by affinity electrokinetic chromatography. The effect of various chiral selectors and some key parameters including buffer pH, buffer concentration, capillary temperature and applied voltage were carefully studied, respectively. At optimum condition, based on the signal-to-noise ratio of 3, the detection limits for the simple resolution and chiral resolution were found to be 1.0x10(-5) and 4.0 x 10(-5)M, respectively. In addition, the interactions of these beta-blockers with bovine serum albumin (BSA) were studied and the binding constant (K(a)) between BSA and each of beta-blockers were calculated. Based on linear correlation coefficient, it can be concluded that the binding ratio of pindolol (oxprenolol) combining with BSA is 1:1, and that the binding number of propranolol interacting with BSA deviates one.

Thermodynamic study of sublimation, solubility, solvation, and distribution processes of atenolol and pindolol.

Temperature dependency of saturated vapor pressure, thermochemical, and thermodynamic characteristics of sublimation, as well as fusion and vaporization processes, for atenolol and pindolol was studied. Specific and nonspecific energetic terms in the crystal lattices were estimated. Temperature dependencies of solubility in buffer (pH 7.4), in n-octanol, and in n-hexane were measured. Thermodynamic functions of solubility, solvation, and transfer processes were deduced. It was found that transfer processes for the drugs moving from water into n-octanol are determined mainly by the entropy term. Distribution coefficients of the compounds in the water/n-octanol system were derived and compared with analogous coefficients calculated from saturation solubility data in the individual solvents. The impact of mutual dissolution of water and n-octanol molecules in the water/n-octanol system on distribution coefficients is discussed.

Application of liquid chromatography-tandem mass spectrometry method for the analysis of new nonselective beta-adrenergic blocker 1-(1-H-indol-4-yloxy)-3-{[2-(2-methoxy phenoxy)ethylo]amino}propan-2-ol (2F109) in rat plasma.

A sensitive and specific liquid chromatography electrospray ionization-tandem mass spectrometry method for the enantioselective determination of the novel beta-adrenolytic compound, 1-(1-H-indol-4-yloxy)-3-{[2-(2-methoxyphenoxy)ethylo]amino} propan-2-ol, in rat plasma has been developed and validated. Chromatography was performed on a reversed-phase Chiralcel OD-RH analytical column (150x4.6 mm, 5 microm, Daicel Chemical Industries, Tokyo, Japan) with isocratic elution using a mobile phase containing acetonitrile and water with 0.01% formic acid. Detection was achieved by an Applied Biosystems MDS Sciex (Concord, Ontario, Canada) API 2000 triple quadrupole mass spectrometer. Electrospray ionization (ESI) was used for ion production. The limit of detection in the MRM mode was found to be 1.25 ng/ml. The limit of quantification of both enantiomers was 2.5 ng/ml. The precision and accuracy for both intra- and inter-day determination of 2F109 enantiomers ranged from 2.6 to 12% and from 89.1 to 107.1%. This analytical method allowed us to carry out pharmacokinetic studies in rats. Our findings demonstrate that 2F109 shows stereoselective disposition in rat plasma after i.v. administration. The terminal half-lives of (+)-(R)-2F109 and (-)-(S)-2F109 were 33.5 and 42.6 min, respectively. The AUC0-inf of (+)-(R)-2F109 exceeded that of (-)-(S)-2F109.