Determination of clindamycin in animal plasma by high-performance liquid chromatography combined with electrospray ionization mass spectrometry

A method for the quantification of clindamycin in animal plasma using high-performance liquid chromatography combined with electrospray ionization mass spectrometry (LC/ESI-MS/MS) is presented. Lincomycin is used as the internal standard. The sample preparation includes a simple deproteinization step with trichloroacetic acid. Chromatographic separation is achieved on an RP-18 Hypersil column using gradient elution with 0.01 M ammonium acetate and acetonitrile as mobile phase. Good linearity was observed in the range 0-10 microg ml(-1). The limit of quantification of the method is 50 ng ml(-1) and the limit of detection is 1.3 ng ml(-1). The method was shown out to be of use for pharmacokinetic studies of clindamycin formulations in dogs.     

Characterization of the early stages of programmed cell death in maize root cells by using comet assay and the combination of cell electrophoresis with annexin binding

An emerging topic in plant biology is whether plant cells display similar elements of programmed cell death (PCD) as animal cells do. We have studied cell death in maize roots exposed to cold stress by using fluorescence microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL), DNA gel electrophoresis, single cell gel electrophoresis (SCGE), cell electrophoresis, and annexin binding techniques. The results showed that cell death in maize root cells triggered by cold stress was accompanied by a subset of features characteristic of animal PCD such as nuclear condensation and fragmentation, and oligonucleosomal DNA fragmentation. In addition to DNA laddering and TUNEL positivity, a "comet" pattern indicative of DNA breakage appeared as short as after one day of treatment. The maize root cell PCD process was also accompanied by an increase in negative surface charge of the dying cells due to exposure of phosphatiolylserine (PS) from inner to outer membrane. After annexin binding, however, the enhanced electrophoretic mobility (EPM) of the dying cells decreased nearly to normal values. This result suggests that the combination between cell electrophoresis and annexin binding provides a quantitative method for monitoring PS exposure during plant PCD.     

Highly stereoselective C-allylation of an enantiopure alpha-sulfinyl thioacetamide

The alkylation of the lithium enolate of enantiopure alpha-cyclohexylsulfinyl thioacetamide 1 with allyl bromides 5 possessing an electron-withdrawing group at the vinylic position does not occur at the sulfur center - as expected in the sulfur series - but at the carbon center through conjugate addition followed by bromide elimination. The modest to excellent 1,2-asymmetric induction achieved by the alkylsulfinyl group (dr up to 100:0) is explained by an electronic model.     

Optimization of catalyst enantioselectivity and activity using achiral and meso ligands

The optimization of asymmetric catalysts for enantioselective synthesis has conventionally revolved around the synthesis and screening of enantiopure ligands. In contrast, we have optimized an asymmetric reaction by modification of a series of achiral ligands. Thus, employing (S)-3,3'-diphenyl BINOL [(S)-Ph(2)-BINOL] and a series of achiral diimine and diamine activators in the asymmetric addition of alkyl groups to benzaldehyde, we have observed enantiomeric excesses between 96% (R) and 75% (S) of 1-phenyl-1-propanol. Some of the ligands examined have low-energy chiral conformations that can contribute to the chiral environment of the catalyst. These include achiral diimine ligands with meso backbones that adopt chiral conformations, achiral diimine ligands with backbones that become axially chiral on coordination to metal centers, achiral diamine ligands that form stereocenters on coordination to metal centers, and achiral diamine ligands with pendant groups that have axially chiral conformations. Additionally, we have structurally characterized (Ph(2)-BINOLate)Zn(diimine) and (Ph(2)-BINOLate)Zn(diamine) complexes and studied their solution behavior.     

Comparison between 2-hydroxypropyl-��-cyclodextrin and 2-hydroxypropyl-��-cyclodextrin for inclusion complex formation with danazol

Complexation in solution between danazol and two different cyclodextrins [2-hydroxypropyl-??-cyclodextrin (HP-??-CD) and 2-hydroxypropyl-??-cyclodextrin (HP-??-CD)] was studied using phase solubility analysis, and one- and two-dimensional ¹H-NMR. The increase of danazol solubility in the aqueous cyclodextrin solutions showed a linear relationship (A_L profile). The apparent stability constant, K _1:1, of each complex was calculated and found to be 51.7 × 10³ and 7.3 × 10³ M^?1 for danazol?CHP-??-CD and danazol?CHP-??-CD, respectively. ¹H-NMR spectroscopic analysis of varying ratios of danazol and the different cyclodextrins in a mixture of EtOD?CD₂O confirmed the 1:1 stoichiometry. Cross-peaks, from 2D ROESY ¹H-NMR spectra, between protons of danazol and H3?? and H5??of cyclodextrins, which stay inside the cyclodextrin cavity, proved the formation of an inclusion complex between danazol and the cyclodextrins. For HP-??-CD, the inclusion complex is formed by entrance of the isooxazole and the A rings of danazol in the cyclodextrin cavity. For HP-??-CD, two different inclusion structures may exist simultaneously in solution: one with the isooxazole and A ring in the cavity and the other with the C and D ring inside the cavity. DLS showed that self-aggregation of the CD??s was absent in the danazol HP-??-CD system up to a CD concentration of 10% and in the danazol HP-??-CD system up to a CD concentration of 5%.     

Ionization energy reductions in small 2,5-dihydroxybenzoic acid-proline clusters

The photoionization of (pro)(n)DHB (pro = proline, DHB = 2,5-dihydroxybenzoic acid, n = 0, 1, 2 or 4) clusters was studied both experimentally and computationally. Experimentally the (pro)(n)DHB clusters are generated in the gas phase by laser desorption and supersonic jet entrainment. The photoionization thresholds are then determined by the mass-selective measurement of both one- and two-color photoionization efficiency curves. These experiments demonstrate that the ionization energies (IEs) of the (pro)(n)DHB clusters are substantially reduced in comparison with the IE of free DHB. Computational studies of the (pro)(n)DHB clusters provide insights into the mechanism of IE reduction. For the (pro)DHB system the IE reduction results from spin delocalization in the ion state of the cluster. In contrast, for the (pro)(2)DHB and (pro)(4)DHB clusters the IE reduction results from an inductive delocalization of electron density from pro to DHB in the ground state of the cluster. This latter effect, which is a result of the specific hydrogen-bonding interactions occurring in the mixed clusters, leads to IE reductions of >1 eV. Finally, determination of the energetics of the (pro)(2)DHB radical cation demonstrate that the DHB-to-proline proton transfer reaction is a barrierless, exoergic process in the ion state and that energetic demands for cluster dissociation to protonated (pro)(2) plus a deprotonated DHB radical are substantially lower than those for cluster dissociation to (pro)(2) plus DHB(+*). Cumulatively, these studies provide new energetic and mechanistic insights into both primary and secondary MALDI ionization processes.     

Two dimensional reversed-phase-reversed-phase separations isomeric separations incorporating C18 and carbon clad zirconia stationary phases

Informational theory and a geometric approach to factor analysis were employed to evaluate the degree of orthogonality of a two-dimensional reversed-phase-reversed-phase chromatographic system. The system incorporated a C18 column as one dimension and a carbon clad zirconia column as the second dimension. In order to study the resolving power of this system, the separation of a sample matrix containing an artificial mix of 32 isomers (structural and diastereoisomers) was evaluated. Using this system, between 25 and 28 of the 32 isomers could be separated, depending on the mobile phase combinations--with resolution that could not possibly be achieved in a single one dimensional separation. The results from this study indicate that in order to fully evaluate the resolving power of a 2D system multiple methods of analysis are most appropriate. This becomes increasingly important when the sample contains components that are very closely related and the retention of solutes is clustered in one quadrant of the 2D space. Ultimately, the usefulness of the 2D separation is determined by the goals of analyst.