Rapid and sensitive determination of acetylsalicylic acid and salicylic acid in plasma using liquid chromatography–tandem mass spectrometry: application to pharmacokinetic study

A simple and sensitive analytical method using liquid chromatography–tandem mass spectrometry (LC/MS/MS) for determination of acetylsalicylic acid (aspirin, ASA) and its major metabolite, salicylic acid (SA), in animal plasma has been developed and validated. Both ASA and SA in plasma samples containing potassium fluoride were extracted using acetonitrile (protein precipitation) with 0.1% formic acid in it. 6‐Methoxysalicylic acid was used as the internal standard (IS). The compounds were separated on a reversed‐phase column. The multiple reaction monitoring mode was used with ion transitions of m/z 178.9 → 136.8, 137.0 → 93.0 and 167.0 → 123.0 for ASA, SA and IS, respectively. The lower limits of quantification for ASA and SA were 3 and 30 ng/mL, respectively. The developed method was successfully applied for the evaluation of pharmacokinetics of ASA and SA after p.o. and i.v. administration of 1 mg/kg to rats. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis,self‐assembly and adsorption of PEO–PLA block copolymers onto colloidal polystyrene

A series of amphiphilic block copolymers composed of poly(ethylene oxide) and poly(lactide) were synthesized and their solution properties studied using static and dynamic light scattering. These materials self‐assemble in aqueous media with the hydrodynamic radius increasing with increasing hydrophobic fraction in the copolymer. To ascertain the potential for use of these materials as degradable coatings in delivery applications, block copolymers of varying compositions were adsorbed onto a series of colloidal polystyrene particles with varying radii, and the thickness of the adsorbed layer was determined from changes in the hydrodynamic size. The adlayer thicknesses ranged from 3 to 14 nm with varying block copolymer compositions, and colloid radii. The trends fit well with theoretical models for adlayer thickness, with the exception of the smallest colloids. In these systems, we propose that the colloids may become encapsulated into the block copolymer assembly. © 2007 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 244–252, 2008     

A General Approach to Aza‐Heterocycles by Means of Domino Sequences Driven by Hydroformylation

The development of hydroformylative domino reactions of easily accessible vinyl acetamides is described. Extremely regioselective hydroformylation of terminal double bounds provides a transient N‐acyliminium that can be trapped by various nucleophiles to give several aza‐heterocylic scaffolds in a diastereoselective manner.     

Water cavitation of hydrogels

Using the cavitation rheology (CR) technique developed in our labs, we show that fluids with negligible interfacial tensions with a surrounding material can be used to induce an elastic, cavitation instability in that material. We do this by changing the cavitation media from air, which was demonstrated to induce cavitation at the tip of a syringe needle in previous studies, to water, which has a negligible surface tension with the surrounding poly(vinyl alcohol) hydrogel material. In this case, the critical pressure in which this instability occurs can be directly related to the elastic modulus of the surrounding network and is shown to be nearly independent of length scale. This independence of size scale has important implications in the use of CR for the characterization of mechanical properties from molecular to macroscopic length scales. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 1423–1427, 2010     

Watching solvent friction impede ultrafast barrier crossings: a direct test of Kramers theory

A systematic investigation of the solvent's dynamic influence on activated barrier crossings on an electronic ground state is performed using ultrafast two-dimensional infrared chemical exchange spectroscopy. These measurements facilitate a direct comparison with the widely adopted Kramers theory of condensed phase reaction kinetics, and for the first time avoid the significant complication of electronic excitation to probe directly in the time domain a ground electronic state reaction with a well-defined transition state. The picosecond timescale interconversion between two stable isomers of the metal carbonyl complex Co(2)(CO)(8) in a series of linear alkane solvents shows negligible energetic variation with solvent carbon chain length, providing an exclusive probe of the effects of solvent friction. Relative to the linear alkane series, cyclohexane does alter the potential energy surface by preferentially stabilizing one of the isomers. Despite this pronounced modification of the reaction barrier energetics, combination of experiment and computation enables the removal of the nondynamical barrier contribution to the rate constant, isolating the dynamical influence of solvent friction. The experimental data, supported with quantum and classical computations, show agreement with a simple Markovian Kramers theory for the isomerization rate constant's dependence on solvent viscosity.     

Immobilization of biotinylated biomolecules onto electropolymerized poly(pyrrole-nitrilotriacetic acid)–Cu2+ film

A novel and simple immobilization strategy for biotinylated biological macromolecules onto electropolymerized poly(pyrrole-nitrilotriacetic acid)(NTA)–Cu²⁺ films without avidin as connecting bridge is reported. After complexation of Cu²⁺ by the polymerized NTA chelator, biotinylated biomolecules were immobilized by coordination of the biotin groups on the NTA–Cu²⁺ complex. The anchoring of biotinylated glucose oxidase was demonstrated by fluorescent characterization via FITC-labeled avidin and amperometric measurement of glucose. The resulting calibration curve led to a sensitivity and maximum current density values of 0.6 mA mol^?1 L cm^? 2 and 13.2 μA cm^? 2, respectively. Thus, biotinylated polyphenol oxidase was fixed leading to a catechol sensor with a sensitivity of 656 mA mol^?1 L cm^? 2 and maximum current density of 25.4 μA cm^? 2. This system was also applied to the efficient immobilization of biotinylated DNA, illustrated by impedimetric detection of the formation of the DNA duplex.     

Ozone-induced dissociation on a modified tandem linear ion-trap: Observations of different reactivity for isomeric lipids

Ozone-induced dissociation (OzID) exploits the gas-phase reaction between mass-selected lipid ions and ozone vapor to determine the position(s) of unsaturation. In this contribution, we describe the modification of a tandem linear ion-trap mass spectrometer specifically for OzID analyses wherein ozone vapor is supplied to the collision cell. This instrumental configuration provides spatial separation between mass-selection, the ozonolysis reaction, and mass-analysis steps in the OzID process and thus delivers significant enhancements in speed and sensitivity (ca. 30-fold). These improvements allow spectra revealing the double-bond position(s) within unsaturated lipids to be acquired within 1 s: significantly enhancing the utility of OzID in high-throughput lipidomic protocols. The stable ozone concentration afforded by this modified instrument also allows direct comparison of relative reactivity of isomeric lipids and reveals reactivity trends related to (1) double-bond position, (2) substitution position on the glycerol backbone, and (3) stereochemistry. For cis- and trans-isomers, differences were also observed in the branching ratio of product ions arising from the gas-phase ozonolysis reaction, suggesting that relative ion abundances could be exploited as markers for double-bond geometry. Additional activation energy applied to mass-selected lipid ions during injection into the collision cell (with ozone present) was found to yield spectra containing both OzID and classical-CID fragment ions. This combination CID-OzID acquisition on an ostensibly simple monounsaturated phosphatidylcholine within a cow brain lipid extract provided evidence for up to four structurally distinct phospholipids differing in both double-bond position and sn-substitution.     

Gas-phase acidities of cysteine-polyglycine peptides: The effect of the cysteine position

The sequence and conformational effects on the gas-phase acidities of peptides have been studied by using two pairs of isomeric cysteine-polyglycine peptides, CysGly_3,4NH₂ and Gly_3,4CysNH₂. The extended Cooks kinetic method was employed to determine the gas-phase acidities using a triple quadrupole mass spectrometer with an electrospray ionization source. The ion activation was achieved via collision-induced dissociation experiments. The deprotonation enthalpies (Δ_acid H) were determined to be 323.9 ± 2.5 kcal/mol (CysGly₃NH₂), 319.2 ± 2.3 kcal/mol (CysGly₄NH₂), 333.8 ± 2.1 kcal/mol (Gly₃CysNH₂), and 321.9 ± 2.8 kcal/mol (Gly₄CysNH₂), respectively. The corresponding deprotonation entropies (Δ_acid S) of the peptides were estimated. The gas-phase acidities (Δ_acid G) were derived to be 318.4 ± 2.5 kcal/mol (CysGly₃NH₂), 314.9 ± 2.3 kcal/mol (CysGly₄NH₂), 327.5 ± 2.1 kcal/mol (Gly₃CysNH₂), and 317.4 ± 2.8 kcal/mol (Gly₄CysNH₂), respectively. Conformations and energetic information of the neutral and anionic peptides were calculated through simulated annealing (Tripos), geometry optimization (AM1), and single point energy calculations (B3LYP/6-31+G(d)), respectively. Both neutral and deprotonated peptides adopt many possible conformations of similar energies. All neutral peptides are mainly random coils. The two C-cysteine anionic peptides, Gly_3,4(Cys-H)⁻NH₂, are also random coils. The two N-cysteine anionic peptides, (Cys-H)⁻Gly_3,4NH₂, may exist in both random coils and stretched helices. The two N-cysteine peptides, CysGly₃NH₂ and CysGly₄NH₂, are significantly more acidic than the corresponding C-terminal cysteine ones, Gly₃CysNH₂ and Gly₄CysNH₂. The stronger acidities of the former may come from the greater stability of the thiolate anion resulting from the interaction with the helix-macrodipole, in addition to the hydrogen bonding interactions.     

Analytical and preparative separation of PEGylated lysozyme for the characterization of chromatography media

The effect of PEGylation on cation exchange chromatography was studied with poly(ethylene glycol) of different chain lengths (5 kDa, 10 kDa and 30 kDa) using lysozyme as a model system. A stable binding via reduction of a Schiff base was formed during random PEGylation on lysine residues with methoxy-PEG-aldehyde. A purification method for PEGylated proteins using cation exchange chromatography was developed, and different isoforms of mono-PEGylated lysozyme were isolated. TSKgel SP-5PW and Toyopearl GigaCap S-650M showed the best performance of all tested cation exchange resins, and the separation of PEGylated lysozyme could be also scaled up to semi-preparative level. Size-exclusion chromatography, SDS-PAGE and MALDI-TOF mass spectrometry were used for analysis. Separated mono-PEGylated lysozyme of different sizes was used to determine dynamic binding capacities (DBC) and selectivity of cation exchange chromatography resins. An optimization of binding conditions resulted in a more than 20-fold increase of DBC for Toyopearl GigaCap S-650M with 30 kDa mono-PEGylated lysozyme.     

Structural study of acetogenins by tandem mass spectrometry under high and low collision energy

Collision‐induced dissociation experiments of seven annonaceous acetogenins were carried out under high and low collision energy conditions. Each compound was studied as protonated or deprotonated and lithium‐ or sodium‐ cationized molecules, using ElectroSpray Ionisation (ESI) with a hybrid linear trap/orbitrap mass spectrometer (LTQ‐Orbitrap®). The same ion species were studied with a Matrix‐Assisted Laser Desorption Ionisation (MALDI) tandem mass spectrometer in a high collision energy regime (1 or 2 keV). Although each of the techniques showed some limitations in the detection of functional groups, unambiguous structural identification of the acetogenins was obtained. MALDI ToF‐ToF has the advantage over ESI‐based methods to provide mass spectra rich in informative fragments which allows the confirmation of some functional groups position. By contrast, ESI‐LTQ‐Orbitrap® analysis has the advantage over MALDI that the mass spectra are relatively simple with only fragments close to the functional groups. However, this technique needs to carry out experiments both in negative and positive ionization modes. Copyright © 2010 John Wiley & Sons, Ltd.