Ultrasonic Degradation of Poly(acrylic acid)

The ultrasonic degradation of poly(acrylic acid), a water-soluble polymer, was studied in the presence of persulfates at different temperatures in binary solvent Mixtures of methanol and water. The degraded samples were analyzed by gel permeation chromatography for the time evolution of the molecular weight distributions. A continuous distribution kinetics model based on midpoint chain scission was developed, and the degradation rate coefficients were determined. The decline in the rate of degradation of poly(acrylic acid) with increasing temperature and with an increment in the methanol content in the binary solvent mixture of methanol and water was attributed to the increased vapor pressure of the solutions. The experimental data showed an augmentation of the degradation rate of the polymer with increasing oxidizing agent (persulfate) concentrations. Different concentrations of three persulfates-potassium persulfate, ammonium persulfate, and sodium persulfate-were used. It was found that the ratio of the polymer degradation rate coefficient to the dissociation rate constant of the persulfate was constant. This implies that the ultrasonic degradation rate of poly(acrylic acid) can be determined a priori in the presence of any initiator.

Novel Mass Spectrometric Analysis Methods for Anabolic Androgenic Steroids in Sports Drug Testing

The feasibility of different modern analytical techniques for the mass spectrometric detection of anabolic androgenic steroids (AAS) in human urine was examined in order to enhance the prevalent analytics and to find reasonable strategies for effective sports drug testing. A comparative study of the sensitivity and specificity between gas chromatography (GC) combined with low (LRMS) and high resolution mass spectrometry (HRMS) in screening of AAS was carried out with four metabolites of methandienone. Measurements were done in selected ion monitoring mode with HRMS using a mass resolution of 5000. With HRMS the detection limits were considerably lower than with LRMS, enabling detection of steroids at low 0.2-0.5 ng/ml levels. However, also with HRMS, the biological background hampered the detection of some steroids. The applicability of liquid-phase microextraction (LPME) was studied with metabolites of fluoxymesterone, 4-chlorodehydromethyltestosterone, stanozolol and danazol. Factors affecting the extraction process were studied and a novel LPME method with in-fiber silylation was developed and validated for GC/MS analysis of the danazol metabolite. The method allowed precise, selective and sensitive analysis of the metabolite and enabled simultaneous filtration, extraction, enrichment and derivatization of the analyte from urine without any other steps in sample preparation. Liquid chromatographic/tandem mass spectrometric (LC/MS/MS) methods utilizing electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) were developed and applied for detection of oxandrolone and metabolites of stanozolol and 4-chlorodehydromethyltestosterone in urine. All methods exhibited high sensitivity and specificity. ESI showed, however, the best applicability, and a LC/ESI-MS/MS method for routine screening of nine 17-alkyl-substituted AAS was thus developed enabling fast and precise measurement of all analytes with detection limits below 2 ng/ml. The potential of chemometrics to resolve complex GC/MS data was demonstrated with samples prepared for AAS screening. Acquired full scan spectral data (m/z 40-700) were processed by the OSCAR algorithm (Optimization by Stepwise Constraints of Alternating Regression). The deconvolution process was able to dig out from a GC/MS run more than the double number of components as compared with the number of visible chromatographic peaks. Severely overlapping components, as well as components hidden in the chromatographic background could be isolated successfully. All studied techniques proved to be useful analytical tools to improve detection of AAS in urine. Superiority of different procedures is, however, compound-dependent and different techniques complement each other.

Interactions of natural products with β-lactoglobulins, members of the lipocalin family

Natural products constitute an important source of new drugs. The bioavailability of the drugs depends on their absorption, distribution, metabolism and elimination. To achieve good bioavailability, the drug must be soluble in water, stable in the gastrointestinal tract and palatable. Binding proteins may improve the solubility of drug compounds, masking unwanted properties, such as bad taste, bitterness or toxicity, transporting or protecting these compounds during processing and storage. The focus of this thesis was to study the interactions, including ligand binding and the effect of pH and temperature, of bovine and reindeer β-lactoglobulin (βLG) with such compounds as retinoids, phenolic compounds as well as with compounds from plant extracts, and to investigate the transport properties of the βLG-ligand complex. To examine the binding interactions of different ligands to βLG, new methods were developed. The fluorescence binding method for the evaluation of ligand binding to βLG was miniaturized from a quartz cell to a 96-well plate. A method of ultrafiltration sampling combined with high-performance liquid chromatography was developed to assess the binding of compounds from extracts. The interactions of phenolic compounds or retinoids and βLG were investigated using the 96-well plate method. The majority of flavones, flavonols, flavanones and isoflavones and all of the retinoids included were shown to bind to bovine and reindeer βLG. Phenolic compounds, contrary to retinol, were not released at acidic pH. Those results suggest that βLG may have more binding sites, probably also on the surface of βLG. An extract from Camellia sinensis (L.) O. Kunze (black tea), Urtica dioica L. (nettle) and Piper nigrum (black pepper) were used to evaluate whether βLG could bind compounds from plant extracts. Piperine from P. nigrum was found to bind tightly and rutin from U. dioica weakly to βLG. No components from C. sinensis bound to βLG in our experiment. The uptake and membrane permeation of bovine and reindeer βLG, free and bound with retinol, palmitic acid and cholesterol, were investigated using Caco-2 cell monolayers. Both bovine and reindeer βLG were able to cross the Caco-2 cell membrane. Free and βLG-bound retinol and palmitic acid were transported equally, whereas cholesterol could not cross the Caco-2 cell monolayer free or bound to βLG. Our results showed that βLG can bind different natural product compounds, but cannot enhance transport of retinol, palmitic acid or cholesterol through Caco-2 cells. Despite this, βLG, as a water-soluble binding protein, may improve the solubility of natural compounds, possibly protecting them from early degradation and transporting some of them through the stomach. Furthermore, it may decrease their bad or bitter taste during oral administration of drugs or in food preparations. βLG can also enhance or decrease the health benefits of herbal teas and food preparations by binding compounds from extracts.

Heated Nebulizer Microchips for Mass Spectrometry

Miniaturized analytical devices, such as heated nebulizer (HN) microchips studied in this work, are of increasing interest owing to benefits like faster operation, better performance, and lower cost relative to conventional systems. HN microchips are microfabricated devices that vaporize liquid and mix it with gas. They are used with low liquid flow rates, typically a few µL/min, and have previously been utilized as ion sources for mass spectrometry (MS). Conventional ion sources are seldom feasible at such low flow rates. In this work HN chips were developed further and new applications were introduced. First, a new method for thermal and fluidic characterization of the HN microchips was developed and used to study the chips. Thermal behavior of the chips was also studied by temperature measurements and infrared imaging. An HN chip was applied to the analysis of crude oil – an extremely complex sample – by microchip atmospheric pressure photoionization (APPI) high resolution mass spectrometry. With the chip, the sample flow rate could be reduced significantly without loss of performance and with greatly reduced contamination of the MS instrument. Thanks to its suitability to high temperature, microchip APPI provided efficient vaporization of nonvolatile compounds in crude oil. The first microchip version of sonic spray ionization (SSI) was presented. Ionization was achieved by applying only high (sonic) speed nebulizer gas to an HN microchip. SSI significantly broadens the range of analytes ionizable with the HN chips, from small stable molecules to labile biomolecules. The analytical performance of the microchip SSI source was confirmed to be acceptable. The HN microchips were also used to connect gas chromatography (GC) and capillary liquid chromatography (LC) to MS, using APPI for ionization. Microchip APPI allows efficient ionization of both polar and nonpolar compounds whereas with the most popular electrospray ionization (ESI) only polar and ionic molecules are ionized efficiently. The combination of GC with MS showed that, with HN microchips, GCs can easily be used with MS instruments designed for LC-MS. The presented analytical methods showed good performance. The first integrated LC–HN microchip was developed and presented. In a single microdevice, there were structures for a packed LC column and a heated nebulizer. Nonpolar and polar analytes were efficiently ionized by APPI. Ionization of nonpolar and polar analytes is not possible with previously presented chips for LC–MS since they rely on ESI. Preliminary quantitative performance of the new chip was evaluated and the chip was also demonstrated with optical detection. A new ambient ionization technique for mass spectrometry, desorption atmospheric pressure photoionization (DAPPI), was presented. The DAPPI technique is based on an HN microchip providing desorption of analytes from a surface. Photons from a photoionization lamp ionize the analytes via gas-phase chemical reactions, and the ions are directed into an MS. Rapid analysis of pharmaceuticals from tablets was successfully demonstrated as an application of DAPPI.

Studies of phosphazenes. Part 15. Chloro(phenoxy)cyclotetraphosphazenes, N4P4Cl8-n(OPh)n

The reaction of N4P4Cl8(1) with sodium phenoxide (or phenol in the presence of triethylamine) has been studied under a variety of experimental conditions. The chloro(phenoxy)-derivatives, N4P4Cl8-n(OPh)n[n= 1 or 2 (mixture of four non-geminal isomers), 3(mixture of non-geminal isomers), 4(mixture of isomers), 5(mixture of isomers), 6(mixture of four non-geminal isomers), or 8], have been isolated by column chromatography over silica gel. Attempts to separate geometric isomers were unsuccessful. Structural elucidation of the products is based on the 31P n.m.r. data for the chloro-precursors and 1H and 31P n.m.r. spectra of the dimethylamino- and/or methoxy-derivatives. The chlorine-replacement pattern is discussed.

Antibodies to guanosine: Fractionation and specificities

Bovine serum albumin conjugates of guanosine prepared by the periodate method was used as immunogen to elicit guanosine antibodies in rabbits. The specificities of the antibodies were studied by the inhibition of their binding to [3H]Gox-red, [32P]DNA and [3H]RNA by related non-radioactive compounds. A population of antibodies is specific to Gox-red with an average association constant of around 107M−1 at 4°C. There are a population of antibodies which bind to [32P]ssDNA and [3H]RNA specifically at guanosine residues. RNA binding antibodies were separated into two populations by affinity chromatography.

Volatile components associated with bacterial spoilage of tropical prawns

Analysis of headspace volatiles by gas chromatography/mass spectrometry from king (Penaeus plebejus), banana (P. merguiensis), tiger (P. esculentus/semisulcatus) and greasy (Metapenaeus bennettae) prawns stored in ice or ice slurry, which is effectively an environment of low oxygen tension, indicated the presence of amines at the early stages of storage (less than 8 days) irrespective of the nature of the storage media. Esters were more prevalent in prawns stored on ice (normal oxygen conditions) at the latter stages of storage (more than 8 days) and were only produced by Pseudomonas fragi, whereas sulphides and amines occurred whether the predominant spoilage organism was Ps.fragi or Shewanella putrefaciens. The free amino acid profiles of banana and king prawns were high in arginine (12–14%) and low in cysteine (0.1–0.17%) and methionine (0.1–0.2%). Filter sterilised raw banana prawn broth inoculated with a total of 15 cultures of Ps. fragi and S. putrefaciens and incubated for two weeks at 5°C, showed the presence of 17 major compounds in the headspace volatiles analysed using gas chromatography/mass spectrometry (GC/MS). These were mainly amines, sulphides, ketones and esters. Principal Component Analysis of the results for the comparative levels of the volatiles produced by pure cultures, inoculated into sterile prawn broth, indicated three subgroupings of the organisms; I, Ps. fragi from a particular geographic location; II, S. putrefaciens from another geographic location; and III, a mixture of Ps. fragi and S. putrefaciens from different geographic locations. The sensory impression created by the cultures was strongly related to the chemical profile as determined by GC/MS. Organisms, even within the same subgrouping classified as identical by the usual tests, produced a different range of volatiles in the same uniform substrate.

Spatial and temporal distribution of tropical phytoplankton species and biomass in the Gulf of Carpenteria, Australia

The biomass and species composition of tropical phytoplankton in Albatross Bay, Gulf of Carpentaria, northern Australia, were examined monthly for 6 yr (1986 to 1992). Chlorophyll a (chl a) concentrations were highest (2 to 5.7 mu g l(-1)) in the wet season at inshore sites, usually coinciding with low salinities (30 to 33 ppt) and high temperatures (29 to 32 degrees C). At the offshore sites chi a concentrations were lower (0.2 to 2 mu g l(-1)) and did not vary seasonally. Nitrate and phosphate concentrations were generally low (0 to 3.68 mu M and 0.09 to 3 mu M for nitrate and phosphate respectively), whereas silicate was present in concentrations in the range 0.19 to 13 mu M. The phytoplankton community was dominated by diatoms, particularly at the inshore sites, as determined by a combination of microscopic and high-performance liquid chromatography (HPLC) pigment analyses. At the offshore sites the proportion of green flagellates increased. The cyanobacterium genus Trichodesmium and the diatom genera Chaetoceros, Rhizosolenia, Bacteriastrum and Thalassionema dominated the phytoplankton caught in 37 mu m mesh nets; however, in contrast to many other coastal areas studied worldwide there was no distinct species succession of the diatoms and only Trichodesmium showed seasonal changes in abundance. This reflects a stable phytoplankton community in waters without pulses of physical and chemical disturbances. These results are discussed in the context of the commercial prawn fishery in the Gulf of Carpentaria and the possible effect of phytoplankton on prawn larval growth and survival.

Identification of the Sex Pheromone of Holotrichia Reynaudi

The male attractant pheromone of the scarab beetle Holotrichia reynaudi, an agricultural pest native to southern India, was extracted from abdominal glands of females with hexane and analyzed by gas chromatography– mass spectrometry. Field testing of the candidate chemicals, indole, phenol, and anisole, both alone and as binary mixtures, led us to conclude that anisole was the major component of the sex pheromone. Neither male nor female beetles were attracted to indole or phenol on their own. Similarly, when indole and anisole were combined, the attractiveness of the solution did not increase over that obtained with anisole alone. However, combination of phenol and anisole did alter the attractiveness of anisole, with fewer male beetles attracted to the binary mixture than to anisole on its own. The behavior of female beetles was not altered by any of the chemicals tested. Anisole is also the sex pheromone of H. consanguinea, making this the first known example of two melolonthine scarabs sharing the same pheromone.

Differentiation of Bordetella avium and Related Species by Cellular Fatty Acid Analysis

The fatty acids of 18 strains of Bordetella avium, 3 strains of Alcaligenes faecalis, 5 strains of Bordetella bronchiseptica, and 12 strains of a B. avium-like organism were examined by gas chromatography-mass spectrometry. The presence of a significant amount of the acid 2-OH C14:0 characterized B. avium and the B. avium-like organism. B. avium and the B. avium-like organism differed in their relative concentrations of C16:1 and 3-OH C14:0 acids. B. bronchiseptica and A. faecalis were distinguishable by comparison of the relative concentrations of C18:0 and C18:1 acids.

Isolation and characterization of binding proteins for retinol from the cytosol, nucleosol and chromatin of the oviduct magnum of laying hens

Protein fractions that bind retinol were isolated from the cytosol, nucleosol and chromatin of the oviduct magnum of laying hens. The proteins isolated from the three sources showed similar elution profiles on chromatography through Sephadex G-75 and G-50 columns, and comparable mobility during electrophoresis on sodium dodecyl sulphate/polyacrylamide gels. Their molecular weights were calculated to be around 14500. When oviducts from vitamin A-depleted and vitamin A-repleted immature chicks given oestrogen injections for 6 consecutive days were incubated with [3H]retinyl acetate, uptake of the radioactivity in the nuclei of the vitamin A-depleted tissue was severalfold higher than that in the nuclei from the vitamin A-repleted tissue.

Improved Approach for Analyzing Bromophenols in Seafood Using Stable Isotope Dilution Analysis in Combination with SPME

An analytical method for the measurement of five naturally occurring bromophenols of sensory relevance in seafood (barramundi and prawns) is presented. The method combines simultaneous distillation−extraction followed by alkaline back extraction of a hexane extract and subsequent acetylation of the bromophenols. Analysis of the bromophenol acetates was accomplished by headspace solid phase microextraction and gas chromatography−mass spectrometry using selected ion monitoring. The addition of 13C6 bromophenol stable isotope internal standards for each of the five congeners studied permitted the accurate quantitation of 2-bromophenol, 4-bromophenol, 2,6-dibromophenol, 2,4-dibromophenol, and 2,4,6-tribromophenol down to a limit of quantification of 0.05 ng/g of fish flesh. The method indicated acceptable precision and repeatability and excellent linearity over the typical concentration range of these compounds in seafood (0.5−50 ng/g). The analytical method was applied to determine the concentration of bromophenols in a range of farmed and wild barramundi and prawns and was also used to monitor bromophenol uptake in a pilot feeding trial.

(Z)-11-Hexadecenal and (3Z,6Z,9Z)-Tricosatriene: Sex Pheromone Components of the Red Banded Mango Caterpillar Deanolis sublimbalis

The sex pheromone of the red banded mango caterpillar, Deanolis sublimbalis (Lepidoptera: Crambidae), a serious pest of the mango Mangifera indica (Anacardiaceae) in India and Southeast Asia and a recent invader into northern Australia, has been identified. Three candidate compounds were identified from pheromone gland extracts of female moths, using gas chromatography (GC), GC-electroantennographic detection and GC-mass spectrometric analyses, in conjunction with dimethyldisulfide derivatization. Field bioassays established that both (Z)-11-hexadecenal (Z11-16:Ald) and (3Z,6Z,9Z)-tricosatriene (3Z,6Z,9Z-23:Hy) were required for attraction of male D. sublimbalis moths, and 1,000 μg of a 1:1 mix of Z11-16:Ald and 3Z,6Z,9Z-23:Hy was more attractive to male moths than caged virgin females. However, the binary blend was only attractive when the isomeric purity of the monounsaturated aldehyde was >99%, suggesting that the (E)-isomer was inhibitory. Although (Z)-11-hexadecen-1-ol (Z11-16:OH) was tentatively identified in gland extracts, the addition of this compound to the binary blend did not increase the numbers of moths captured. The pheromone can now be used in integrated pest management strategies.

New initiators for the ring-opening thermal polymerization of hexachlorocyclotriphosphazene: synthesis of linear poly(dichlorophosphazene) in high yields

Ring-opening thermal polymerization of hexachlorocyclotriphosphazene (N3P3C&h)a s been investigated at 250 "C and at 1.333-Pa pressure using chlorocyclotriphosphazenes N3P3C15(N=PPh3) and N3P3Cl,.,(NMe2), (n = 2-4), salt hydrates, triphenylphosphine, and benzoic acid as initiators. The linear poly (dich1orophosphazene) products are phenoxylated, and the phenoxy polymers are characterized by gel permeation chromatography and dilute solution viscometry. Among the various initiators investigated, CaS04.2H20b rings about a high conversion (>60%) of N3P3C&to the linear [NPC12], polymer which possesses a high molecular weight (>5 X lo6). The rationale for the choice of the initiators and possible mechanism(s) of polymerization is discussed. Several mixed substituent polymers, [NP(OPh),(OC6H4Me-p)2,1, and [NP(OPh),(OCHzCF3)2,]nh, ave been prepared and their thermal properties evaluated.

Carotenoid content and flesh color of selected banana cultivars growing in Australia.

Background: The problems of vitamin A deficiency and chronic diseases have emerged in recent years in some countries in the Micronesian region. These problems are associated with the dietary shift towards imported processed foods and lifestyle changes. Research in the Federated States of Micronesia indicates that yellow- and orange-fleshed banana cultivars contain significant levels of provitamin A carotenoids. Objective: To identify further banana cultivars that may be promoted to alleviate vitamin A deficiency among children and women and chronic disease problems among adults. Methods: Ripe fruit of banana cultivars growing in Australia (sourced mostly from a field research collection) were assessed for carotenoid content and flesh color. Ten cultivars with yellow or yellow/orange flesh color (including common cultivars of Southeast Asia and the Pacific Islands) were selected and compared with two cream-fleshed cultivars, including Williams, of the Cavendish group, the most commonly marketed banana worldwide. Carotenoid content was analyzed by high-performance liquid chromatography (HPLC). Flesh color was analyzed by HunterLab colorimetry. Results: The yellow/orange-fleshed Asupina (a Fe'i banana) contained the highest level (1,412 μg/100 g) of trans β-carotene, the most important provitamin A carotenoid, a level more than 20 times higher than that of Williams. All 10 yellow or yellow/orange-fleshed cultivars (Asupina, Kirkirnan, Pisang Raja, Horn Plantain, Pacific Plantain, Kluai Khai Bonng, Wain, Red Dacca, Lakatan, and Sucrier) had significant carotenoid levels, potentially meeting half or all of the estimated vitamin A requirements for a nonpregnant, nonlactating adult woman within normal consumption patterns. All were acceptable for taste and other attributes. The cream-fleshed cultivars had minimal carotenoid levels. There was a positive significant correlation between carotenoid content and deeper yellow/orange coloration indicators. Conclusions: These yellow- or yellow/orange-fleshed carotenoid-rich banana cultivars should be considered for promotion in order to alleviate vitamin A deficiency and chronic disease in susceptible target communities and to provide variety and enjoyment as exotic fruits in both developing and industrialized countries.