Separation, identification and quantification of riboflavin and its photoproducts in blood products using high-performance liquid chromatography with fluorescence detection: a method to support pathogen reduction technology

A medical device using riboflavin (RB) and light is being developed for the reduction of pathogens in platelet concentrates (MIRASOL pathogen reduction technology [PRT]). A high-performance liquid chromatography (HPLC) method for the quantification of RB and its main photoproduct, lumichrome (LC) in blood components has been developed and validated. In addition, the same method has been used to identify and quantify the presence of additional photoproducts-catabolites of RB. Levels of these agents before and after treatment as well as endogenous levels present in normal donor blood are reported using this analytical technique. The method allows for quantitative and qualitative analysis of RB and LC in blood components using HPLC-fluorescence detection, a Zorbax SB-CN (stable bond cyano) column and a methanol-water mobile phase. Quantitation and qualitative analysis of additional photoproducts of RB was also performed, but the method has not been validated for these other components. The method described has passed an 8 day validation and has been found to be adequate for its intended use. The range of the method for RB is 0.016-1.500 microM and for LC is 0.060-1.500 microM. The method detection limit for RB is 0.0006 microM and for LC is 0.012 microM. The acceptance criteria for repeatability were met; the relative standard deviation for RB was 0.64% and for LC was 0.76%. The acceptance criteria for bias were met with a 97% average recovery for RB and a 102% recovery for LC. Samples were centrifuged and diluted 1:50 with 0.9% saline before analysis. No protein precipitation or extraction was required. A mass balance of approximately 93.4-94.4% was achieved after exposure of products to UV light in the intended pathogen reduction treatment method. The method permitted the identification of photoproducts in blood that were both naturally occurring and produced after photolysis of blood samples treated with the PRT process. The identity of these photoproducts has been established using HPLC Tandem Mass Spectrometry (MS/MS) and UV spectroscopic methods and has been correlated with known metabolites and catabolites of RB. HPLC with fluorescence detection using a reverse phase cyano-column allows for accurate separation, identification and quantification of both RB and LC in blood products without the need for solvent extraction or protein precipitation. Additional photoproducts could also be identified and quantified using this method. The presence of these agents in normal, untreated blood suggests that their presence in blood is ubiquitous.     

An operational supramolecular nanovalve

A functioning nanomachine in the form of a supramolecular nanovalve that opens and closes the orifices to molecular-sized pores and releases a small number of molecules on demand is reported. The nanovalve, which is used to open and close the nanocontainer, is a pseudorotaxane composed of two components-a long thread containing a 1,5-dioxnaphthalene donor unit, which is attached to the solid support, and the moving part, the tetracationic cyclophane acceptor/receptor, cyclobis(paraquat-p-phenylene), which controls access to the interior of the nanopore. The nanocontainer is made out of mesoporous silica by using a dip-coating method. Operating the nanovalve involves three steps: (i) filling the container, (ii) closing the valve, and (iii) opening the valve to release the contents of the container on demand. The tubular pores, which are approximately 2 nm wide, are filled with stable luminescent Ir(ppy)3 molecules by allowing them to diffuse into the open pores. The orifices are then closed by pseudorotaxane formation. An external reducing reagent (NaCNBH3) is used to effect dethreading of the pseudorotaxane so as to unlock the tubes and allow the guest molecules to be released. This nanovalve is a supramolecular machine consisting of a solid framework with moving parts capable of doing useful work.     

Determination of Bentazepam by differential pulse polarography and adsorptive stripping voltammetry

Summary A method is described for the determination of Bentazepam using DPP and ADSV with DP. Bentazepam is determined in buffer Britton-Robinson 0.04 mol l^-1 at pH 9 with detection limits of 3.1×10^-9 mol/l and a relative standard deviation of 0.8 DPP was used to determine Bentazepam in Tiadipona, the commercial product. ADSV was used to determine Bentazepam in urine with a detection limit of 2.7 ng ml^-1 (accumulation time 5 min) and a relative standard deviation of 1.5%.     

Chemical reaction dynamics within anisotropic solvents in time-dependent fields

The dynamics of low-dimensional Brownian particles coupled to time-dependent driven anisotropic heavy particles (mesogens) in a uniform bath (solvent) have been described through the use of a variant of the stochastic Langevin equation. The rotational motion of the mesogens is assumed to follow the motion of an external driving field in the linear response limit. Reaction dynamics have also been probed using a two-state model for the Brownian particles. Analytical expressions for diffusion and reaction rates have been developed and are found to be in good agreement with numerical calculations. When the external field driving the mesogens is held at constant rotational frequency, the model for reaction dynamics predicts that the applied field frequency can be used to control the product composition.     

Thermal stability of aluminium hydroxycarbonates with monovalent cations

Dawsonite-type compounds of formula MAl(OH)₂CO₃ (M = Na. K, NH₄) as well as a laminar hydrotalcite-type hydroxycarbonate of composition [Al₂Li(OH)₆]₂CO₃·4H₂O. have been hydrothermally synthesized The thermal decomposition of these compounds was monitored by DTA and TG, and the resulting products have been studied by X-ray and IR techniques. Sodium and potassium dawsonites are destroyed at 335°C. yielding a poorly crystalline compound in which part of the overall carbonate is present; the remaining carbonate is lost between 600 and 700°C, yielding NaAlO₂ and KAlO₂, respectively Ammonium dawsonite and lithium hydrotalcite are less stable, their thermal decomposition occurring at about 240°C. The ammonium dawsonite heated at 680°C shows the presence of A1₂O₃ with a poorly ordered structure, while lithium hydrotalcite yields poorly crystalline γ-Al₂O₃ at 500°C and a mixture of γ-LiAlO₂ and LiAl₅O₈ when the compound is heated at higher temperatures ( ~ 1000°C).     

Folding behavior of model proteins with weak energetic frustration

The native structure of fast-folding proteins, albeit a deep local free-energy minimum, may involve a relatively small energetic penalty due to nonoptimal, though favorable, contacts between amino acid residues. The weak energetic frustration that such contacts represent varies among different proteins and may account for folding behavior not seen in unfrustrated models. Minimalist model proteins with heterogeneous contacts--as represented by lattice heteropolymers consisting of three types of monomers--also give rise to weak energetic frustration in their corresponding native structures, and the present study of their equilibrium and nonequilibrium properties reveals some of the breadth in their behavior. In order to capture this range within a detailed study of only a few proteins, four candidate protein structures (with their cognate sequences) have been selected according to a figure of merit called the winding index--a characteristic of the number of turns the protein winds about an axis. The temperature-dependent heat capacities reveal a high-temperature collapse transition, and an infrequently observed low-temperature rearrangement transition that arises because of the presence of weak energetic frustration. Simulation results motivate the definition of a new measure of folding affinity as a sequence-dependent free energy--a function of both a reduced stability gap and high accessibility to non-native structures--that correlates strongly with folding rates.     

Determination of 1,4-thienodiazepines by liquid chromatography with spectrophotometric,amperometric and coulometric detection

Spectrophotometric, amperometric and coulometric methods of detection for the liquid chromatographic determination of four 1,4-thienodiazepines were compared. The effects of several experimental parameters on the separation and sensitivity of the method was evaluated. A mobile phase consisting of methanol—water (60 + 40) and containing ammonium acetate buffer (pH 7) appeared to be optimal when using a 4-μm Novapak ODS column. The amperometric and coulometric detectors, equipped with glassy carbon electrodes, were operated at +1.05 V vs. Ag/AgCl. The limits of detection obtained ranged from 0.05 to 0.2 μg ml^?1 for spectrophotometric detection, from 0.05 to 0.2 μg ml^?1 for amperometric detection and from 0.006 to 0.02 μg ml^?1 for coulometric detection.     

Method for the evaluation of the reorganization energy of electron transfer reactions produced under restricted geometry conditions

The kinetics of the electron transfer reactions between S(2)O(8)(2-) and the complexes (Ru(NH(3))(5)L)(2+) (L = pyridine, pyrazine, and 4-cyanopyridine) have been studied in micellar (SDS) solutions. A method for the evaluation of the reorganization energy of these reactions, based on the comparison of their rate constants, is proposed. From the results obtained, we concluded that the observed rate constants go through a minimum for the surfactant concentration in which the reorganization energy goes through a maximum. The method can be applied to any kind of restricted geometry conditions.     

Developments toward a complete micro-total analysis system for Duchenne muscular dystrophy diagnosis

The diagnosis of Duchenne muscular dystrophy (DMD) has historically utilized either PCR or requires Southern blot analysis, a southern blot analysis, however, is not amenable to incorporation in a microdevice format. A PCR amplification-based method has been developed, and we have previously coupled this amplification with microchip separation of the PCR fragments for DMD diagnosis. Diagnoses of affected patients were performed by comparing exon concentrations to those of control samples amplified at the same time. To accurately identify mutations in patient samples, this work established normal ranges for the concentration of each amplified exon fragment using control samples amplified over successive days. Our studies show that the number of cycles used in the amplification process affects this range. Affected patient samples were analyzed using these normal ranges and the mutations detected by Southern blot analysis were also diagnosed using the microchip separation method.

Employing the microchip separation method decreases the time required for the analysis, but the time required for DNA purification and PCR amplification must also be decreased for faster total analysis of patient samples. Development of microchip methods for these processing steps is one approach for reducing the individual times, while also providing the possibility of integrating these steps in a single device. Here we report on the microchip extraction of genomic DNA from whole blood using a novel sol–gel matrix that is easily formed in microdevices. IR-mediated PCR amplification of a β-globin fragment from genomic DNA followed by electrophoretic analysis on a single integrated microdevice is presented for the first time. Work towards the development of a micro-total analysis device for DMD diagnosis, through integration of all processing steps on a single device, is also discussed.     

Reaction of 5-aminopyrazoles with β-dimethylaminopropiophenones. Synthesis of new pyrazolo[3,4-b]pyridines

Several new pyrazolo[3,4-b]pyridines were obtained from the reaction of 5-amino-1-aryl-3-methylpyrazoles 1 with β-dimemylaminopropiophenones 2 in pyridine. The structure elucidation of 4,5-dihydropyrazolo[3,4-b]pyridines 3 is based on nmr measurements and X-ray diffraction. The treatment of compounds 3 with N-bromosuccinimide led to the formation of pyrazolo[3,4-b]pyridines 4 .