Investigation of EDTA anticoagulant in plasma to improve the throughput of liquid chromatography/tandem mass spectrometric assays

In this study, EDTA and heparin are compared as anticoagulants with respect to their efficiency in preventing clot formation in plasma samples that were subsequently analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). A pilot in vivo pharmacokinetic study for the drug chlorpheniramine was conducted in which both EDTA and heparin plasma samples were collected simultaneously. All conditions except the anticoagulant were held constant during the pharmacokinetic study. Bioanalytical results were compared from samples transferred by manual pipette and by an automated liquid handler workstation. The concentration of chlorpheniramine in samples was determined by LC/MS/MS. Results from the analysis of variances (ANOVA) of log-transformed plasma chlorpheniramine concentrations were used to calculate 90% confidence intervals for the ratio least-squares mean values for anticoagulants and for transfer methods. Analytical concentrations of the drug chlorpheniramine were equivalent in heparin- and EDTA-containing plasma. Results suggest that the failure rate for transfer of EDTA plasma (50 micro L by automated workstation or manually) is less than that for heparinized plasma. As a consequence of these results, the vast majority of plasma samples in our laboratories are now collected in EDTA, which allows for use of automated sample transfer resulting in a three-fold timesaving over manual transfer using a single-channel pipette. The ability to use automation has resulted in improved efficiency and cost savings.     

The mass spectra of some selenoureas and selenothiocarbamic esters

The mass spectra of five selenoureas and two selenothiocarbamic esters are reported and discussed. Fragmentation modes are compared with those reported for the ureas, thioureas, carbamates and thiocarbamates.     

Inclusion of quaternary ammonium compounds by calixarenes

In weakly polar solvents, strong association occurs between calixarene anions and tetraalkylammonium cations, with the magnitude of the observed equilibrium constants depending upon the charge on the anion, the solvent, the ring size of the calixarene and the nature of the alkyl group of the cation. Large upfield shifts of the methyl resonances of the [(CH₃)₄N]⁺ cation in solutions of [(CH₃)₄N]₂[p-t-butylcalix[6]arene-2H] indicate cation inclusion in a structure which is possibly identical with that found for the solid salt by X-ray crystallography. This shows one of the cations to be included within a partial cone structure of a hinged 3-up, 3-down conformation of the calixarene. The functionalised tetramethylammonium ions, choline and acetyl choline, are also strongly included by various calixarene anions but attempts to detect significant modification of the reactivity of acetyl choline resulting from inclusion have not been successful.This paper is dedicated to the commemorative issue on the 50th anniversary of calixarenes.     

Bis-phosphites and bis-phosphinites based on distally-functionalised calix[4]arenes: coordination chemistry and use in rhodium-catalysed, low-pressure olefin hydroformylation

Six calix[4]arenes each bearing two non-cyclic PR2 units attached at distal phenolic oxygen atoms, p-Bu t-calix[4]arene-25,27-(OPR2)2-26,28-(OR')2(R = OPh; R'= Prn, L1; R = OPh; R'= CH2CO2Et, L2; R= OPh; R'= CO2 cholesteryl, L3; R = Ph; R'= Prn, 4; R = Ph; R'= CH2CO2Et, L5; R = Ph; R'= CO2cholesteryl, L6) have been synthesized and their coordinative properties investigated. The diphosphites L1-L3, where the P centres are separated by 12 bonds, readily form chelate complexes provided the complexation reaction is achieved either by using a starting complex that possesses good leaving groups or by operating under high dilution in order to avoid oligomer formation. Thus, the cationic complexes [Rh(COD)L1]BF4 and [Rh(COD)L3]BF4 were both formed in high yield by reacting the appropriate diphosphite with either [Rh(COD)(THF)2]BF4 or [Rh(COD)2]BF4. At high dilution, reaction of L3 with the neutral complex [PdCl2(COD)] afforded the chelate complex [PdCl2L3] in 90% yield. The reaction of one equiv. of L1 with [Rh(acac)(CO)2] resulted in the formation of [Rh(acac)L1] without requiring high dilution conditions. When the latter reaction was carried out with 0.5 equiv. of L1, the bimetallic complex [{Rh(acac)(CO)}2(eta]1-P,eta1-P'-L1)] was formed instead. Reaction at high dilution of with the cyclometallated complex [Pd(o-C6H4CH2NMe2)(THF)2]BF4 gave the expected chelate complex [Pd(o-C6H4CH2NMe2)]BF4. The latter slowly converts in solution to an oligomer in which the ligand behaves as a (eta1-P,eta1-P') bridging ligand, thus leading to a less strained structure. All six ligands, when mixed with [Rh(acac)CO2], effectively catalyse the hydroformylation of octene and styrene. In the hydroformylation of octene, the linear aldehyde selectivities observed with L2 and L3 are significantly higher (linear : branched =ca. 10) than those obtained with the other 4 ligands of this study and also with respect to PPh3. Molecular modelling shows that the lower rim substituents of and form tighter pockets about the metal centre than do the other ligands and so sterically favour the formation of Rh(n-alkyl) intermediates over that of Rh(i-alkyl) ones. In styrene hydroformylation, all ligands result in the formation of unusually high amounts of the linear aldehyde, the b : l ratios being all close to 65 : 35. The highest activities were found when using an L/Rh ratio of 1/1.     

Multiple sample amplification and genotyping integrated on a single electronic microarray

We report a novel method that allows simultaneous in situ amplification and then genotyping of single nucleotide polymorphism (SNP) for multiple samples on a single electronic microarray. The locus coding for one of the common inherited thrombosis risk factors, Factor V Leiden (FVL), was chosen as a model system for SNP analysis. This method combines strand displacement amplification (SDA) with electrophoretic movement and concentration of DNA on electronic microarrays to provide a single platform for DNA amplification and analysis. The method includes: electronic anchoring of allele-specific SDA amplifiable primers (APs) and a nonamplifiable primer (NAP) to different electrodes, electronic hybridization of genomic DNA from different samples to those primers, in situ amplification of target DNA, and genotyping of FVL. Compared to previous anchored SDA methods, the addition of a NAP improves detection signals by at least 20-fold. The sensitivity of this method is dependent on the amplification time. Using this method, nine different genomic DNA samples with known FVL genotypes were amplified and correctly genotyped on a single electronic microarray without any contamination between samples. The present method could streamline development of nucleic acid-based assays in applications of molecular diagnostic, point-of-care testing, and forensic detection, which often require the capability to analyze multiple samples efficiently.     

Two novel syntheses of the histamine H3 antagonist thioperamide

The previously described route for the synthesis of the histamine H₃ antagonist thioperamide 3 has been improved considerably. Furthermore, two straightforward novel synthetic routes towards 3 are described herein. The last synthetic route (Scheme 3) is preferable as it is very suitable for the production of multigram quantities of thioperamide 3.     

Identification of oxidation products of solanesol produced during air sampling for tobacco smoke by electrospray mass spectrometry and HPLC

Solanesol, a 45-carbon, trisesquiterpenoid alcohol found in tobacco leaves and tobacco smoke, has been used as a quantitative marker for tobacco smoke for years. However, solanesol appears to be unreliable as a quantitative marker for tobacco smoke during environmental air sampling because it can be degraded substantially when present as a component of tobacco smoke and by as much as 100% when present as pure solanesol on fortified filters during air sampling. Since there is strong evidence that ozone is the agent responsible for the degradation, solanesol appears to be unreliable as a quantitative marker during indoor air sampling when indoor levels of ozone are greater than about 15 ppb. The degree of loss of pure solanesol is directly proportional to the concentration of ozone and the length of the sampling period and depends on the type of 37 mm membrane filter used for air sampling (PTFE or quartz fiber). While the degree of loss of solanesol is inversely proportional to the relative humidity of the air at a sampling rate of 1.7 L min(-1), the degree of loss is virtually independent of relative humidity at a lower sampling rate; i.e., 0.25 L min(-1). A curve of loss of solanesol on a filter versus concentration of ozone from an ozone generator is virtually identical to a curve segment based on atmospheric ozone under the same conditions of air sampling. Oxidation of solanesol by ozone to approximately 25 to 60% completion produces at least three series of products for a total of at least 26 compounds: (1) isoprenoid acetones, (2)omega-hydroxyisoprenoid acetaldehydes, and (3) isoprenoid oxoaldehydes. All products in each series were tentatively identified as their derivatives with 2-(p-aminophenyl)ethanol (APE) by electrospray mass spectrometry (ES-MS). Ten ozonation products were detected as their 2,4-dinitrophenylhydrazine derivatives by HPLC at 360 nm: 4-oxopentanal and nine isoprenoid acetones (acetone, 6-methyl-5-hepten-2-one, geranylacetone, farnesylacetone, tetraprenylacetone, geranylfarnesylacetone, farnesylfarnesylacetone, farnesylgeranylgeranylacetone and bombiprenone.     

Conformational control of oxidation sites, spin states and orbital occupancy in nickel porphyrins

Ni(II) porphyrin π cation radicals are known to undergo an internal electronic isomerization to L₂Ni(III) cations upon complexation with ligands (L). Additional examples of the Ni(II) to Ni(III) conversion are presented for flexible, 'planar' NiOEP (2,3,7,8,12,13,17,18-octaethylporphyrin) and NiT(Pr)P (5,10,15,20-tetra-n-propylporphyrin) in which the Ni(III) orbital occupancy, d _z2 or d _x2-y2, is determined by the ligand field strength of the axial ligands (pyridine, imidazole, or cyanide). In contrast to these results, the nonplanar NiOETPP (2,3,7,8,12,13,17,18-octaethyl-5,10,15,20-tetraphenylporphyrin), which is easily oxidized because of its saddle-shape, yields a complex postulated to be a high spin Ni(II) π cation radical, based on crystallographic and optical data for (imidazole)2NiOETPP⁺ClO^4-, in which the electron of high spin Ni(II) in the d _x2-y2 orbital is antiferromagnetically coupled to the unpaired electron of the porphyrin radical leaving one electron in the Ni(II) d _z2 orbital, i.e. a pseudo Ni(III). The sterically encumbered, nonplanar NiT(t-Bu)P (5,10,15,20-tetra-tertiary-butylporphyrin) yields Ni(III) complexes when ligated by pyridine, imidazole or cyanide, but in all cases only the Ni(III) d _z2 orbital is occupied as evidenced by EPR spectroscopy. This anomalous chemistry is attributed to the fact that the macrocycle of NiT(t-Bu)P is so sterically constrained that it cannot readily expand to accommodate the longer equatorial Ni—N distances required by population of the d _x2-y2 orbital in Ni(III) or high spin Ni(II). Further support for this postulate derives from NiD(t-Bu)P (5,10-di-tertiary-butylporphyrin) which is less sterically constrained and in which the Ni(III) d _x2-y2 orbital is indeed occupied upon complexation with cyanide. These results thus illustrate the significant effects that the conformations, plasticity or rigidity of Ni porphyrin macrocycles can have on sites of oxidation (metal or porphyrin), spin states (low spin Ni(III) or high spin Ni(II)), and orbital occupancies (d _z2 or d _x2-y2 in Ni(III)).     

Alternatives to 1-H-tetrazole in the preparation of phosphonate diesters and phosphonamidates from phosphonyl dichlorides

5-Ethylthio-1-H-tetrazole (SET) and 4,5-dicyanoimidazole (DCI) were examined as alternatives to 1-H-tetrazole to mediate the synthesis of phosphonate diesters and phosphonamidates from phosphonyl dichlorides through a two-step one-pot reaction in various organic solvents. SET and DCI were comparable to 1-H-tetrazole for catalyzing these reactions. SET afforded slightly greater yields than DCI while benzene was universally the best solvent for this reaction.     

Dynamics of molecules in contact with an external medium at equilibrium

When considering the dynamical properties of a molecule in contact with an external medium whose degrees of freedom are at equilibrium, one can adopt either of two approaches to the problem. The equations of motion which govern the coupled molecule-medium system can be solved (or approximately solved), after which the initial values of the medium's coordinates are averaged over (with an equilibrium distribution function as weighting factor) to obtain the desired property of the molecule. In the second approach, which is the subject of the present work, the effects of the medium on the molecule of interest are incorporated into a time-dependent effective potential which contains the equilibrium average molecule-medium interaction as well as terms arising from fluctuations in the medium's coordinates about their equilibrium values. By introducing such an effective potential via the cumulant expansion technique, the problem of studying dynamical properties of the molecule is reduced to the study of a molecule which is in the presence of a time varying external field which no longer depends explicitly upon the coordinates of the medium. In addition to developing the basic method for treating such problems, applications of the method to interesting problems are briefly discussed.