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Aqueous nitrite ion determination by selective reduction and gas phase nitric oxide chemiluminescence

An improved method of flow injection analysis for aqueous nitrite ion exploits the sensitivity and selectivity of the nitric oxide (NO) chemilluminescence detector. Trace analysis of nitrite ion in a small sample (5-160 microL) is accomplished by conversion of nitrite ion to NO by aqueous iodide in acid. The resulting NO is transported to the gas phase through a semipermeable membrane and subsequently detected by monitoring the photoemission of the reaction between NO and ozone (O3). Chemiluminescence detection is selective for measurement of NO, and, since the detection occurs in the gas-phase, neither sample coloration nor turbidity interfere. The detection limit for a 100-microL sample is 0.04 ppb of nitrite ion. The precision at the 10 ppb level is 2% relative standard deviation, and 60-180 samples can be analyzed per hour. Samples of human saliva and food extracts were analyzed; the results from a standard colorimetric measurement are compared with those from the new chemiluminescence method in order to further validate the latter method. A high degree of selectivity is obtained due to the three discriminating steps in the process: (1) the nitrite ion to NO conversion conditions are virtually specific for nitrite ion, (2) only volatile products of the conversion will be swept to the gas phase (avoiding turbidity or color in spectrophotometric methods), and (3) the NO chemiluminescence detector selectively detects the emission from the NO + O3 reaction. The method is free of interferences, offers detection limits of low parts per billion of nitrite ion, and allows the analysis of up to 180 microL-sized samples per hour, with little sample preparation and no chromatographic separation. Much smaller samples can be analyzed by this method than in previously reported batch analysis methods, which typically require 5 mL or more of sample and often need chromatographic separations as well.

NASA Discipline Environmental Health

HPLC/ESI-quadrupole ion trap mass spectrometry for characterization and direct quantification of amphoteric and nonionic surfactants in aqueous samples

An amphoteric (cocamidopropylbetaine, CAPB) and a nonionic (alcohol polyethoxylate, AE) surfactant were characterized by electrospray ionization quadrupole ion trap mass spectrometry (ESI-MS) as to their homologue distribution and ionization/fragmentation chemistry. Quantitative methods involving reversed-phase gradient HPLC and (+)ESI-MSn were developed to directly determine these surfactants in hydroponic plant growth medium that received simulated graywater. The predominant homologues, 12 C alkyl CAPB and 9 EO AE, were monitored to represent the total amount of the respective surfactants. The methods demonstrated dynamic linear ranges of 0.5-250 ng (r2 > 0.996) for CAPB and 8-560 ng (r2 > 0.998) for AE homologue mixture, corresponding to minimum quantification limits of 25 ppb CAPB and 0.4 ppm AE with 20-microL injections. This translated into an even lower limit for individual components due to the polydispersive nature of the surfactants. The procedure was successfully employed for the assessment of CAPB and AE biodegradation in a hydroponic plant growth system used as a graywater bioreactor.

NASA Discipline Life Support Systems

High performance liquid chromatographic assay for the quantitation of total glutathione in plasma

A simple and widely used homocysteine HPLC procedure was applied for the HPLC identification and quantitation of glutathione in plasma. The method, which utilizes SBDF as a derivatizing agent utilizes only 50 microl of sample volume. Linear quantitative response curve was generated for glutathione over a concentration range of 0.3125-62.50 micromol/l. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.999. Limit of detection (LOD) and limit of quantitation (LOQ) values were 5.0 and 15 pmol, respectively. Glutathione recovery using this method was nearly complete (above 96%). Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of glutathione was demonstrated successfully using human and rat plasma samples.

Non-NASA Center

The role of ethylene in the development of constant-light injury of potato and tomato

The role of ethylene in the development of constant-light injury of potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum Mill.) was investigated. In one study, silver thiosulfate (STS) was applied to the foliage of four potato cultivars growing under constant light. Leaf area and shoot dry mass of 'Kennebec' and 'Superior', cultivars normally injured by constant light, were greater (P < 0.05) than those of control plants given foliar applications of distilled water. Examination of STS-treated 'Kennebec' leaflets revealed significantly less injury (necrotic spotting and reduced starch content) than the water-treated controls. 'Norland' and 'Denali', cultivars tolerant of constant light, exhibited no differences in growth between treatments. In a second study, injury (necrotic spotting and reduced starch content) was induced in leaflets of 'Denali' when exposed to spray applications of 0.5 mmol L-1 ethephon or air containing 0.5 to 0.8 microL L-1 ethylene. In a third study, three genotypes of 'Ailsa Craig' tomato were grown under constant light. Leaves of the normal 'Ailsa Craig' exhibited epinasty, reduced chlorophyll concentration, and reduced starch content. Leaves of a mutant 'Ailsa Craig', containing the Never ripe mutation, did not exhibit epinasty but exhibited the same amount of reduced chlorophyll concentration and starch content as normal plants. Leaves of a transgenic 'Ailsa Craig', containing an antisense gene of 1-aminocyclopropane 1-carboxylate (ACC) oxidase, were epinastic, but chlorophyll concentration and starch content were greater than in leaves of normal and mutant plants. These results suggest that transgenic plants were more tolerant of constant light than the other genotypes. Evidence from these studies indicates that ethylene, combined with constant light, has an important role in the development of constant-light injury.

Non-NASA Center

On-line technique for measuring stable oxygen and hydrogen isotopes from microliter quantities of water

Detailed here is a method for extracting and analyzing oxygen and hydrogen isotopes from 10 microL-sized water samples. Based on the traditional CO2-H2O equilibration technique, the oxygen isotope exchange reaction is done exclusively in sealed 6-mm (o.d.) Pyrex tubes at 25 degrees C, with full isotope exchange completed in at least 28 h. Using the same water sample employed in the 18O equilibration, D/H extractions are done in separate sealed 6-mm (o.d.) Pyrex tubes by reaction with Zn at 450 degrees C to form H2(g). Provided that a correction factor is applied to 18O analyses, accuracy and precision for both 18O and D/H are comparable to standard techniques using much larger samples.

NASA Center JSC

Simultaneous quantification of poly-dispersed anionic, amphoteric and nonionic surfactants in simulated wastewater samples using C18 high-performance liquid chromatography-quadrupole ion-trap mass spectrometry

This paper describes the development of a guantitative method for direct and simultaneous determination of three frequently encountered surfactants, amphoteric (cocoamphoacetate, CAA), anionic (sodium laureth sulfate, SLES), and nonionic (alcohol ethoxylate, AE) using a reversed-phase C18 HPLC coupled with an ESI ion-trap mass spectrometer (MS). Chemical composition, ionization characteristics and fragmentation pathways of the surfactants are presented. Positive ESI was effective for all three surfactants in agueous methanol buffered with ammonium acetate. The method enables rapid determinations in small sample volumes containing inorganic salts (up to 3.5 g L(-1)) and multiple classes of surfactants with high specificity by applying surfactant specific tandem mass spectrometric strategies. It has dynamic linear ranges of 2-60, 1.5-40, 0.8-56 mg L(-1) with R2 egual or greater than 0.999, 0.98 and 0.999 (10 microL injection) for CAA, SLES, and AE, respectively.

Chromatography, High Pressure Liquid/methods

A Novel Protocol for Decoating and Permeabilizing Bacterial Spores for Epifluorescent Microscopy

Based on previously reported procedures for permeabilizing vegetative bacterial cells, and numerous trial-and-error attempts with bacterial endospores, a protocol was developed for effectively permeabilizing bacterial spores, which facilitated the applicability of fluorescent in situ hybridization (FISH) microscopy. Bacterial endospores were first purified from overgrown, sporulated suspensions of B. pumilus SAFR-032. Purified spores at a concentration of approx equals 10 million spores/mL then underwent proteinase-K treatment, in a solution of 468.5 μL of 100 mM Tris-HCl, 30 μL of 10% SDS, and 1.5 microL of 20 mg/mL proteinase-K for ten minutes at 35 ºC. Spores were then harvested by centrifugation (15,000 g for 15 minutes) and washed twice with sterile phosphate-buffered saline (PBS) solution. This washing process consisted of resuspending the spore pellets in 0.5 mL of PBS, vortexing momentarily, and harvesting again by centrifugation. Treated and washed spore pellets were then resuspended in 0.5 mL of decoating solution, which consisted of 4.8 g urea, 3 mL Milli-Q water, 1 mL 0.5M Tris, 1 mL 1M dithiothreitol (DTT), and 2 mL 10% sodium-dodecylsulfate (SDS), and were incubated at 65 ºC for 15 minutes while being shaken at 165 rpm. Decoated spores were then, once again, washed twice with sterile PBS, and subjected to lysozyme/mutanolysin treatment (7 mg/mL lysozyme and 7U mutanolysin) for 15 minutes at 35 C. Spores were again washed twice with sterile PBS, and spore pellets were resuspended in 1-mL of 2% SDS. This treatment, facilitating inner membrane permeabilization, lasted for ten minutes at room temperature. Permeabilized spores were washed two final times with PBS, and were resuspended in 200 mkcroL of sterile PBS. At this point, the spores were permeable and ready for downstream processing, such as oligonucleotideprobe infiltration, hybridization, and microscopic evaluation. FISH-microscopic imagery confirmed the effective and efficient (≈50% successful permeabilization and recovery) permeabilization of numerous spore preparations. The novelty of the technology developed here is in its applicability to bacterial endospores. While protocols abound for the effective permeabilization of bacterial, archaeal, and eukaryotic vegetative cells, there are no such reliable methods for decoating and permeabilizing bacterial endospores in a manner that is amenable to downstream FISH microscopic analyses. This innovation enables the direct visualization and enumeration of spores via FISH-based microscopic techniques, circumventing the complications that accompany previously required germination regimes. The synergistic enzymatic weakening of the many spore layers facilitates a structural compromise that is just enough to render the spores permeable without degrading the spore to a level, which precludes it from recognition.

LaDuc, Myron T.