Radiolytic stability of N,N-dialkyl amide: effect on Pu(iv) complexes in solution.

The radiolytic degradation of three N,N-dialkyl amide ligands relevant to nuclear fuel reprocessing was studied. The degradation of these ligands: N,N di-2-ethyhexylbutyramide (DEHBA), N,N di-2-ethyhexylisobutyramide (DEHiBA) and N,N di-2-ethyhexyl-3-dimethylbutanamide (DEHDMBA) was examined to evaluate the effect of the structure on the formation of degradation products as well as to compare alpha induced degradation to gamma induced degradation. In situ alpha radiolysis by introduction of plutonium(iv) as the alpha source in the solution and ex situ gamma radiolysis with 60Co as the gamma source were compared. Upon identification of the main degradation products, a degradation scheme was proposed. The effects of radiation on the stability of Pu-monoamide complexes were discussed. Theoretical calculations were also performed to determine bond dissociation energy and estimate the relative strength of the bond in the molecule. The results show that neither the type of radiation (alpha vs. gamma) nor the structure modification (introduction of branching on the alkyl chain off the carbonyl carbon) of the molecule significantly impact the formation of degradation products under the conditions studied. Moreover, it was observed that the overall stability of the monoamide remains good and that Pu complexation is not greatly affected by either alpha or gamma irradiation.

Fast, sensitive point of care electrochemical molecular system for point mutation and select agent detection.

Point of care molecular diagnostics benefits from a portable battery-operated device capable of performing a fast turnaround using reliable inexpensive cartridges. We describe a prototype device for performing a molecular diagnostics test for clinical and biodefense samples in 16 minutes using a prototype capable of an 8 minute PCR reaction, followed by hybridization and detection on an electrochemical microarray based on the i-STAT® system. We used human buccal swabs for hemochromatosis testing including in-device DNA extraction. Additional clinical and biodefense samples included influenza A and bacterial select agents Bacillus anthracis, Yersinia pestis and Francisella tularensis.

Identification of bacterial plant pathogens using multilocus polymerase chain reaction/electrospray ionization-mass spectrometry.

Polymerase chain reaction/electrospray ionization-mass spectrometry (PCR/ESI-MS, previously known as "TIGER") utilizes PCR with broad-range primers to amplify products from a wide array of organisms within a taxonomic group, followed by analysis of PCR amplicons using mass spectrometry. Computer analysis of precise masses allows for calculations of base compositions for the broad-range PCR products, which can then be compared to a database for identification. PCR/ESI-MS has the benefits of PCR in sensitivity and high-throughput capacity, but also has the distinct advantage of being able to detect and identify organisms with no prior characterization or sequence data. Existing broad range PCR primers, designed with an emphasis on human pathogens, were tested for their ability to amplify DNA of well characterized phytobacterial strains, as well as to populate the existing PCR/ESI-MS bacterial database with base counts. In a blinded panel study, PCR/ESI-MS successfully identified 93% of unknown bacterial DNAs to the genus level and 73% to the species/subspecies level. Additionally, PCR/ESI-MS was capable of detecting and identifying multiple bacteria within the same sample. The sensitivity of PCR/ESI-MS was consistent with other PCR based assays, and the specificity varied depending on the bacterial species. Preliminary tests with real life samples demonstrate a high potential for using PCR/ESI-MS systems for agricultural diagnostic applications.

A receptor-based bioassay for quantitative detection of gallium.

The detection of gallium in biological samples is required due to its role in the diagnosis of tumor and for possible treatment of malignancies. However, the use of purely instrumental techniques is unsuitable for detection of low levels of gallium in biological matrixes. We have synthesized new protein conjugates based on 4-(2-pyridylazo) ligands. The conjugates were successfully employed for the detection of gallium in biological matrixes using a nonantibody-based sandwich assay format. The recovery level obtained was between 97 and 101.3 with a relative standard deviation of less than 5%. The assay resulted in a detection limit of 5 x 10(-8) M and a remarkable selectivity for gallium(III) relative to other metals investigated. The new method provided adequate accuracy for gallium applicable for animal physiology and clinical toxicology.

Fourier transform ion cyclotron resonance mass spectrometry in a 20 T resistive magnet.

We present Fourier transform ion cyclotron resonance (FTICR) mass spectra at a magnetic field of 20 T; more than twice the highest field previously used for FTICR. Our instrument is based on a resistive magnet installed at the National High Magnetic Field Laboratory. The magnet has a 50 mm diameter bore and spatial inhomogeneity of approximately 1000 ppm over a 1 cm diameter spherical volume. However, FTICR mass resolving power far in excess of magnet homogeneity is achieved routinely for ions produced by either electron ionization (EI) or matrix-assisted laser desorption/ionization (MALDI). As examples, we show a MALDI mass spectrum of [M + H] quasimolecular ions of the peptide, human luteinizing hormone-releasing hormone (monoisotopic molecular weight, 1181.6 Da) at mass resolving power, m/delta m > 10,000; and an EI mass spectrum of molecular ions of the platinum cluster compound, Pt4(PF3)8 (average molecular weight, 1484 Da at mass resolving power, m/delta m approximately 20,000. Much better FTICR MS performance is predicted for future NHMFL resistive magnets of higher spatial and temporal homogeneity.

Real-time monitoring of the gas phase reactions of a single ion population using the remeasurement experiment in Fourier transform ion cyclotron resonance mass spectrometry.

A single population of multiply charged protein ions formed by electrospray ionization is held in a trapped ion cell and remeasured continuously by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry while undergoing multiple reactions with diethylamine. In a first example, electrosprayed horse myoglobin with an average of 16 attached protons is reacted with base at a pressure of 1.5 x 10(-8) Torr for a period of 60 s. A total of 31 spectra acquired with a duty cycle of 2 s exhibit the charge state-dependent formation of up to three diethylamine adducts and removal of up to nine protons. A realtime measuring experiment is then conducted over a 1 h period to observe charge stripping of electrosprayed myoglobin ions, leaving as few as seven charges. Realtime monitoring is used to evaluate the effect of reagent gas pressure on adduct formation and is used in conjunction with high mass resolution FTICR detection to resolve the isotopic peaks within individual charge states of adduct spectra. The attractive features of real-time reaction monitoring include a dramatic reduction in experiment time and sample consumed while concurrently observing long-lived intermediates and reaction products as they form.

Simplified application of quadrupolar excitation in Fourier transform ion cyclotron resonance mass spectrometry.

A new method for application of quadrupolar excitation to the trapped ion cell of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer is presented. Quadrupolar excitation is conventionally applied to the two pairs of opposed electrodes that normally perform the excitation and detection functions in the FTICR experiment. Symmetry arguments and numerically calculated isopotential contours within the trapped ion cell lead to the conclusion that quadrupolar excitation can be applied to a single pair of opposed side electrodes. Examples of effective quadrupolar axialization via this method include a sevenfold signal-to-noise enhancement derived from 50 remeasurements of a single population of trapped bovine insulin ions and the selective isolation of a single charge state of horse heart myoglobin after an initial measurement that revealed the presence of 14 charge states.

Ion kinetic energy modulation for improved ion trapping in electrospray ionization fourier transform ion cyclotron resonance mass spectrometry.

A new technique for manipulating the kinetic energy distribution of electrospray ions that arrive at a Fourier transform ion cyclotron resonance trapped-ion cell is presented. Narrow kinetic energy distributions can complicate the selection of appropriate trapping conditions for electrospray ions and introduce charge discrimination in resulting mass spectra. Modulation of the applied skimmer potential controllably broadens the kinetic energy distribution, which improves the reproducibility of acquired spectra and eliminates charge discrimination. Mass spectra of horse heart cytochrome c are presented to demonstrate the utility of the technique. For example, applied static skimmer potentials of 12 and 9 V yield charge state distributions ranging from [M+19H](+19) to [M+12H](+12) and [M+15H](+15) to [M+7H](+7), respectively. A 12 ± 2 V, 100-Hz modulation of the skimmer potential yields an electrospray spectrum with charge states that range from [M+19H](+19) to [M+7H](+7), which is more representative of the source distribution.

Inhibition of calcification of glutaraldehyde pretreated porcine aortic valve cusps with sodium dodecyl sulfate: preincubation and controlled release studies.

Calcification of bioprosthetic heart valves fabricated from glutaraldehyde pretreated bovine pericardium or porcine aortic valves (PAV) is a frequent cause of the failure of these devices. Of all strategies considered thus far, only detergent preincubations using compounds such as sodium dodecyl sulfate (SDS) inhibited PAV bioprosthetic mineralization in circulatory sheep bioprosthetic valve replacements. The present study sought to characterize the mechanism of action of SDS preinicubation. Results of transport and material characterization studies showed that SDS had a relatively high affinity for PAV, with a maximum uptake of 167.1 +/- 6.8 micrograms SDS/mg tissue over 24 h at 37 degrees C with a partition coefficient of 19.3. The PAV diffusion of SDS was 1.95 +/- 0.35 10(-6) cm2/sec. The principal effect of SDS on PAV was phospholipid extraction. The residual organic phosphate in the SDS pretreated tissue was 2.22 +/- 0.72 nmol/mg tissue compared to the control untreated group with 18.52 +/- 2.1 nmol/mg tissue. Incubations of PAV specimens in a 1% SDS solution for 24 h significantly inhibited calcification after 21 days in subdermal implants in 3-week-old male rats (PAV Ca2+ = 18.0 +/- 11.8 micrograms/mg) compared to control (177.8 +/- 6.0 micrograms/mg). In contrast, coimplants of 30% SDS silicone rubber polymers, for regional sustained SDS administration, did not impede PAV calcification in 21 day implants Ca2+ = 166.0 +/- 14.0 micrograms/mg compared to the nondrug silicone matrix controls, Ca2+ = 173.0 +/- 6.6 micrograms/mg). Thus, we conclude that the mechanisms of SDS inhibition of PAV calcification is due to material effects which occur during preincubation, and is not facilitated by sustained SDS administration.

Synergistic inhibition of the calcification of glutaraldehyde pretreated bovine pericardium in a rat subdermal model by FeCl3 and ethanehydroxydiphosphonate: preincubation and polymeric controlled release studies.

Calcification is a frequent cause of the clinical failure of bioprosthetic heart valves fabricated from glutaraldehyde-pretreated porcine aortic valves or glutaraldehyde-pretreated bovine pericardium (GPBP). We investigated the hypothesis that ferric chloride (FeCl3) and sodium-ethanehydroxydiphosphonate (EHDP) may act synergistically to prevent bioprosthetic tissue calcification. Pre-incubations and controlled release systems were studied individually. FeCl3-EHDP polymeric controlled release matrices were formulated using silicone rubber and evaluated for in vitro release kinetics at pH 7.4 and 37 degrees C. The effects of Fe-EHDP synergism on GPBP calcification were investigated with 21 d subdermal implants in 3 wk-old male rats. Results demonstrated that levels of Fe3+ and EHDP uptake, measured in GPBP tissues pre-incubated first in an FeCl3 solution (10(-5) M) followed by an EHDP solution (0.1 M), were higher than in the reverse order of incubation. In the first series of rat implants, GPBP was pre-incubated in either FeCl3 or Na2EHDP solutions, or sequential pre-incubations of first FeCl3 and then Na2EHDP solutions, or the reverse. The inhibition of calcification was greatest when FeCl3 (first pre-incubation, 10(-5) M) was combined with Na2EHDP (second pre-incubation, 0.1 M) (1.78 +/- 0.2 micrograms of Ca2+/mg of dried tissue) compared with the other pre-incubation groups: EHDP (first pre-incubation) combined with FeCl3 (second pre-incubation) (21.7 +/- 6.4), FeCl3 solution alone at 10(-5) M (27.9 +/- 10.7), Na2EHDP solution alone at 0.1 M (52.3 +/- 11.9) and the control group (72.3 +/- 10.2). In a second series of implants, GPBP specimens were co-implanted with individual controlled release systems containing one of the following formulations (weight percentage in silicone rubber): 1% FeCl3, 20% CaEHDP, 20% protamine sulphate, 1% FeCl3-20% CaEHDP, and 1% FeCl3-20% protamine sulphate. The 1% FeCl3-20% CaEHDP silicone-rubber matrices were the most effective for inhibiting GPBP mineralization (13.7 +/- 3.0 micrograms Ca2+/mg of dried tissue) compared with non-drug silicone co-implant controls (74.7 +/- 5.58 micrograms Ca2+/mg of dried tissue) and other polymeric treatment groups (32.3 +/- 2.3-80.0 +/- 19.7). No adverse effects on bone or overall growth of any treatment protocols were noted. Thus, combinations of FeCl3 and EHDP, using either pre-incubations or polymeric controlled release, were synergistic for inhibiting GPBP calcification.