Provisional Identification Research Articles

Optimization strategy and validation of one chromatographic method as approach to determine the phenolic compounds from different sources

We have designed a novel working strategy to optimize a unique chromatographic method consisting of diode array detection for the analysis of the most representative phenolic compounds from different food sources. The simultaneous inclusion of standard phenolic compounds, phenolic compounds isolated from food sources and representative real extracts as an ultimate test in analysis has allowed to establish, for the first time, a unique liquid gradient to serve as an excellent medium for the investigation of phenolics in samples from different food sources. Under the optimized conditions, 21 commercially available phenolic compounds and 25 commercially unavailable phenolic structures were analyzed in less than 30 min. The chromatographic method was designed as an alternative for the provisional identification of these compounds before their full characterization. The optimized chromatographic method was carefully validated for precision and accuracy. A high reproducibility in the retention time (<2%), peak area and calibration slope (<5%) as well as recoveries higher than 95% were obtained in all cases. Consequently, the currently described method was successfully employed to study the phenolic compounds in the most representative food samples.

Evaluation of high-performance liquid chromatography for determination of phenolic compounds in pear horticultural cultivars

An analytical evaluation of an HPLC method with diode array detection to separate and quantify polyphenolic compounds from pears has been made. The method was applied to the quantitative analysis of phenolics from five pear horticultural cultivars (“Agua”, “Blanquilla”, “Conference”, “Pasagrana” and “Decana”) in both peel and pulp matrices and evaluated for precision and accuracy. Precision was taken as the reproducibility in peak area of the polyphenols of interest as well as in the slope of calibration graphs. Values ranged 2–5%. Accuracy was evaluated by recovery of all polyphenolic compounds from both peel and pulp in all pears investigated. Accuracy values ranged 92–102%, and were independent of the polyphenolic structure, horticultural cultivar and matrix. Identification was by comparing retention times and UV spectra with those of standards when commercially available. When not available commercially, provisional identification was according to spectral characteristics as well as from isolation and hydrolysis data. Application of the method revealed differences between peel and pulp in all cases studied; the higher levels of phenolics were found in the peels. “Decana” and “Pasagrana” cultivars showed the highest phenolic content compounds whereas “Conference” showed the lowest.

Determination and Monitoring of Polar Compounds and Acidic Herbicides Using a Modified Samos System

A system for the automated monitoring of organic pollutants in surface waters (SAMOS) has been developed over the last 5 years to monitor for the presence of organic contaminants in surface water. It uses a solid-phase extraction (SPE) trace enrichment step followed by on-line elution and separation by HPLC. Detection and provisional identification is obtained with diode array detection. For pesticide and herbicide analysis, mainly medium volatile, neutral compounds such as carbamates, triazines and phenyl ureas have been reported. Ionic species such as phenoxy acid herbicides are poorly preconcentrated at normal river pH. These ionic/polar compounds frequently show breakthrough from the SPE cartridge prior to complete loading of the sample. Any retained polar analytes are also often obscured by the presence of co-extracted humic substances in river water samples. The paper presents the required changes to the original SAMOS system to allow ionised and polar pollutants to be successfully analysed. These changes involve allowing the ionic/polar compounds to break through from the loading onto the primary cartridge (PLRP-S), allowing all but the last few ml of sample to go to waste. When breakthrough of the relevant analytes is achieved, the remaining sample is switched automatically on-line to a secondary cartridge (again PLRP-S) with acid being added just prior to this to neutralise the compounds This secondary cartridge effectively preconcentrates the ionic/polar compounds. The two cartridges are desorbed in two subsequent gradient elution LC-DAD runs. Analysis of several major classes of compounds is achieved, notably members of the triazine, phenyl urea, phenol and acid herbicide groups. The system has been designed and tested in the laboratory and applied at an installation remote from the laboratory on a river site as part of an intake protection programme. Details of the method performance, experiences of operation and access of the system via telemetry are discussed.

Fast separation of (poly)phenolic compounds from apples and pears by high-performance liquid chromatography with diode-array detection

Polyphenolic compounds in apples and pears were analysed by HPLC on C 18-modified silica. Gradient elution with phosphoric acid–methanol mixtures and phosphoric acid–acetonitrile mixtures gave complete separation of all polyphenolics of interest. The use of methanol as modifier was preferred because it provides a more rapid separation (20 min). Diode-array detection was used for the provisional identification of polyphenolic compounds not available as standards.

Liquid chromatographic separation of intermediates of the catalytic hydrogenation of 2,4-dinitrotoluene

An improved HPLC method was developed for the separation and quantification of 2,4-dinitrotoluene and its reduction products. Gradient elution, with a water–acetonitrile mobile phase, was selected for the separation of all components including the 2,4-nitrohydroxyaminotoluene isomers. Provisional identification of the isomer intermediates has been obtained by their UV–Vis spectra.

The bacteriological screening of donated human milk: Laboratory experience of British Paediatric Association's published guidelines

This study was undertaken to assess the application of the British Paediatric Association's (BPA) published guidelines to the bacteriological screening of breast milk donated to a District General Hospital milk bank. Samples of donated milk were subjected to bacterial counts and provisional identification after both 24 and 48 h incubation on cysteine lactose electrolyte-deficient (CLED) and Columbia blood agar. 21.8% (76 out of 348) donations of milk failed to reach the BPA acceptable criteria. The organisms responsible for the rejection of these samples were all evident within 24 h incubation, and were not significantly confined to one medium. A large percentage of rejected samples originated from a small number of donor mothers; 63.2% came from one donor. In applying BPA guidelines, both CLED and Columbia blood agar were found to be equally effective in screening for unacceptable organisms in prepasteurization donated breast milk. The 24 h period allowed for bacteriological screening, prior to pasteurization of milk samples, was sufficient to allow the growth of all potentially pathogenic bacteria in this study. To prevent the donation of consistently contaminated milk, more active communication between the milk bank staff and the donor is recommended.

Identification of Staphylococcus aureus virulence genes in a murine model of bacteraemia using signature-tagged mutagenesis.

Signature-tagged mutagenesis with transposon Tn917 was used to identify genes of Staphylococcus aureus required for virulence in a murine model of bacteraemia. Screening 1248 mutant strains in pools of 96 resulted in the provisional identification of 50 mutants attenuated in virulence. Subsequent individual analysis of many of these mutants confirmed that they are attenuated in virulence. DNA sequence analysis of regions flanking their transposon insertion points revealed that approximately half of them represent genes with unknown function, while most of the remainder are involved in nutrient biosynthesis and cell surface metabolism. Three mutants were found with transposon insertions in different positions in femA, and one mutant had an insertion in femB. Both femA and femB are involved in the formation of cell wall peptidoglycan pentaglycine cross-bridges. A further mutation occurred in a previously unknown gene that shares significant similarity to femB. Mutations were also obtained in recA and lsp (encoding the S. aureus prolipoprotein signal peptidase). On the basis of sequence similarities to proteins of known function, the products of other genes are probably involved in the synthesis of diaminopimelic acid (a component of peptidoglycan), maintenance of surface adhesins and cell surface membrane transport, showing that many components of the S. aureus cell surface are critical for the survival and replication of this pathogen in blood.

Rapid analysis of organic microcontaminants in environmental water samples by trace enrichment and liquid chromatography on a single short column

On-column trace enrichment and liquid chromatography using a single short (20 mm length) high-pressure packed column was optimized for the rapid simultaneous identification and quantification of a wide range of organic microcontaminants in environmental water samples. The quality of different C 18 bonded silica packing materials in terms of their performance (i.e., peak capacity, repeatability) and enrichment/extraction characteristics (i.e., recovery, selectivity) was tested. With 15-ml samples, limits of detection of 0.1–0.4 μg/1 and 0.5–1.1 μg/1 were achieved for tap and surface water, respectively. The applicability of the system was demonstrated by the screening of real environment samples and the provisional identification of unknowns.

Flavonoids, cinnamic acids and coumarins from the different tissues and medicinal preparations of Taraxacum officinale

Three flavonoid glycosides: luteolin 7-glucoside and two luteolin 7-diglucosides were isolated from dandelion flowers and leaves together with free luteolin and chrysoeriol in the flower tissue. The hydroxycinnamic acids, chicoric acid, monocaffeyltartaric acid and chlorogenic acid were found throughout the plant and the coumarins, cichoriin and aesculin were identified in the leaf extracts. This represents the first report of free chrysoeriol (luteolin 3′-methyl ether) in Taraxacum officinale agg. An earlier provisional identification of chicoric acid, chlorogenic acid, cichoriin and aesculin in a phenolic survey of the tribe Cichorieae is confirmed. Chicoric acid and the related monocaffeyltartaric acid were found to be the major phenolic constitutents in flowers, roots, leaves and involucral bracts and also in the medicinal preparations tested.

The First Records of the Blue RunnerCaranx Crysos(Pisces: Carangidae) in British Waters

In September 1992 a blue runner (Caranx crysos) (37 cm) was caught off Portland Harbour, Dorset, by an angler. Initially the specimen was not recognized as a rarity and it was damaged while being prepared as bait for a lobster pot. Fortunately it was retrieved and sent to the Ministry of Agriculture Fisheries and Food (MAFF) laboratory at Lowestoft where it was identified. In August 1993, a second blue runner (29 cm), (see Figure 1) was hand-lined in St Ives Bay, Cornwall by Mr Astrinsky, of Penzance. It was also taken to a MAFF office, at Newlyn, for identification where one of the authors made a provisional identification before sending it to the British Marine Fishes Database at the Marine Biological Association for final verification. These two fishes are the first records of the blue runner C.crysosfrom British waters, and represent a northerly extension to its range.

Purification, properties and possible gene assignment of an ?1,3-fucosyltransferase expressed in human liver

alpha 1,3-Fucosyltransferase solubilized from human liver has been purified 40,000-fold to apparent homogeneity by a multistage process involving cation exchange chromatography on CM-Sephadex, hydrophobic interaction chromatography on Phenyl Sepharose, affinity chromatography on GDP-hexanolamine Sepharose and HPLC gel exclusion chromatography. The final step gave a major protein peak that co-chromatographed with alpha 1,3-fucosyltransferase activity and had a specific activity of approximately 5-6 mumol min-1 mg-1 and an M(r) approximately 44,000 deduced from SDS-PAGE and HPLC analysis. The purified enzyme readily utilized Gal beta 1-4GlcNAc, NeuAc alpha 2-3Gal beta 1-4GlcNAc and Fuc alpha 1-2Gal beta 1-4GlcNAc, with a preference for sialylated and fucosylated Type 2 acceptors. Fuc alpha 1-2Gal beta 1-4Glc and the Type 1 compound Gal beta 1-3GlcNAc were very poor acceptors and no incorporation was observed with NeuAc alpha 2-6Gal beta 1-4GlcNAc. A polyclonal antibody raised against the liver preparation reacted with the homologous enzyme and also with the blood group Lewis gene-associated alpha 1,3/1,4-fucosyltransferase purified from the human A431 epidermoid carcinoma cell line. No cross reactivity was found with alpha 1,3-fucosyltransferase(s) isolated from myeloid cells. Examination by Northern blot analysis of mRNA from normal liver and from the HepG2 cell line, together with a comparison of the specificity pattern of the purified enzyme with that reported for the enzyme expressed in mammalian cells transfected with the Fuc-TVI cDNA, suggests a provisional identification of Fuc-TVI as the major alpha 1,3-fucosyltransferase gene expressed in human liver.

Quantitative trait loci associated with the behavioral response of B x D recombinant inbred mice to restraint stress: a preliminary communication.

Quantitative trait loci (QTL) analysis was used to make provisional identification of loci containing genes influencing vulnerability to stress. The effect of restraint stress on open-field activity was measured in C57BL/6J and DBA/2J inbred strains of mice and in 22 B x D recombinant inbred strains of mice. QTL analyses were performed by correlating the behavioral delta scores for each group with the strain distribution pattern of 1300 markers for the B x D mice. A significant association was found between postrestraint rearings during min 5 through 8 in the open field and the Lamb2 marker on chromosome 1 (r = .718, p < .0001). Significant associations at the p < .0001 level were also found between baseline open-field rearings of control mice during min 0 through 5 and the Zp3, Ache, and Mr66-1 markers on chromosome 5, baseline open-field rearings of control mice during min 5 through 8 and the Pmv42 marker on chromosome 15, and open-field rearings of experimental mice during min 0 through 5 and the D11Ncvs61 marker on chromosome 11.

Detection and identification of gastrointestinal microsporidia using non-invasive techniques.

To detect enteric microsporidia in faecal specimens from patients with the acquired immunodeficiency syndrome (AIDS), and to identify the spores to species level without using invasive procedures. Formalised faecal preparations were examined using a modification of the strong trichrome staining method to demonstrate microsporidian spores. Six positive specimens were prepared for electron microscopy by emulsification and separation using a 9% Ficoll gradient. The modified staining technique readily identified microsporidian spores. Spores of different species showed variation in size. Identification using electron microscopy was successful for five of the six positive specimens examined. It was unsuccessful for one specimen in which spores were less abundant on initial staining. The modified strong trichrome staining method is a useful way of detecting spores of intestinal microsporidia in faecal specimens. Variation in spore size may permit provisional identification by light microscopy. Electron microscopic examination of faecal preparations is useful for identifying spores to species level.

Determination of phenolic compounds in surface water using on-line liquid chromatographic precolumn-based column-switching techniques

Gradient LC-diode array UV detection, combined on-line with a PLRP-S and an ENVI-Chrom P precolumn in series, enables the on-line trace-level determination and provisional identification of phenol and 13 substituted phenols in surface water. Additional selectivity has been incorporated into the system by properly utilizing the breakthrough properties of phenol over the PLPP-S precolumn. With 50-ml sample volumes, the limits of detection for all analytes are at the low- to sub-μg/1 level.

Rapid Identification of Benzothiazole in River Water With On-Line Solid-Phase Extraction-Gas Chromatography-Mass Selective Detection

Abstract Solid-phase extraction coupled on-line with gas chromatography-mass spectometry and off-line with gas chromatography-atomic emission detection is shown to be a rapid technique for the trace-level determination and provisional identification of (an) organic pollutant(s) in surface water.

International spaceflight crew rescue standards

An initial step towards the goal of establishing capabilities for international space crew rescue is the development of a set of international standards. These initial standards should comprise the minimum set which is necessary to support the capability to perform emergency rendezvous with, and rescue of, the crew of a spacecraft by another crew carrying spacecraft. The paper covers: • - the current international spaceflight environment; • - the possible generic rescue scenarios; • - the identification of design and operational factors relevant to the rescue scenarios; • - provisional identification of the common standards; • - proposed strategy for follow up activities including international aspects.

The benomyl test as a fundamental diagnostic method for medical mycology

The fungicide benomyl has long been known to differentially affect major taxonomic groups of fungi. In the present study 163 species or aggregates of closely similar species of medically important fungi and actinomycetes, as well as species commonly isolated as clinical contaminants, were tested to determine their reactions to three concentrations of benomyl. Fungi of basidiomycetous, endomycetous, and microascaceous affinities were highly resistant, including all common yeasts and Geotrichum, Pseudallescheria, Scedosporium, and Scopulariopsis species. Also resistant were fungi of pleosporalean affinities with poroconidial anamorphs, such as Alternaria, Bipolaris, Curvularia, and Exserohilum species. Most other fungi of ascomycetous affinity were moderately to strongly susceptible. Such fungi included dermatophytes; Coccidioides, Blastomyces, and Histoplasma species; Sporothrix schenckii; medically important aspergilli; and "black yeasts." Benomyl testing aided in the provisional identification of nonsporulating mycelia, including common basidiomycetous isolates obtained as contaminants as well as nonsporulating Aspergillus fumigatus from pulmonary sources.

Provisional identification of Haemophilus influenzae from sputum cultures within 1 h by rapid enzyme tests

Possible Haemophilus influenzae colonies in cultures of sputum samples are currently identified by tests for dependence on X and V factors. This method requires further overnight culture and may give a relatively high number of false negative results. Identification of suspected H. influenzae colonies by a 5-min test for production of indole and beta-galactosidase followed by a 1-h porphyrin test was compared with tests for dependence on X and V factors. A commercially produced form of the rapid tests (Haemstrip, Lab M, Bury, Lancs) was used to test 252 potential haemophilus colonies from cultures of sputum samples on heated blood agar. Colonies that were beta-galactosidase-positive after 5 min were considered to be non-H. influenzae and those that were beta-galactosidase-negative but indole-positive were considered to be H. influenzae. At this stage the test had a sensitivity of 99.4% and a specificity of 90.9%. After 1 h, only colonies that were beta-galactosidase- and porphyrin-negative were considered to be H. influenzae, the sensitivity was then 99.5% and the specificity 100%. Similar results were found with colonies from sputum cultures on selective heated blood agar containing bacitracin. The X and V dependence and Haemstrip results were in 97.6% agreement in a double blind test. Of 100 non-haemophilus colonies tested by Haemstrip, two pseudomonads could have been identified as H. influenzae by this method. The high positive predictive value of Haemstrip results depends partly on the initial recognition of potential haemophilus colonies.

A review of the taxonomy of five Ontario genera of freshwater cyclopoid Copepoda (Crustacea)

Examination of material of five cyclopoid copepod genera found in Ontario fresh waters and a comparison with species of these genera from other parts of the world closely related to Ontario species has given some very interesting results. The widespread Ectocyclops phaleratus was the species previously recorded from Ontario. However, the Ontario material fits the description of E. polyspinosus Harada, 1931, known from Taiwan. There is considerable confusion in the nomenclature of North American species of Eucyclops. In Ontario Eucyclops serrulatus is found. This widespread and variable species needs revision on the basis of worldwide material. Hence, only a provisional identification can be made now. The species previously called E. speratus in Ontario is a hitherto underscribed species, E. neomacruroides, closely related to, but we think distinct from, E. macruroides and E. speratus. The third species is Eucyclops macruroides denticulatus and the fourth is the very distinctive E. prionophorus. In the genus Tropocyclops, besides the widely occurring Tropocyclops prasinus prasinus, T. extensus was found. This latter species has been consistently identified as T. prasinus mexicanus since 1959. Four species of the genus Acanthocyclops occur in Ontario. Acanthocyclops robustus is very common; A. vernalis is rare and so are A. venustoides and A. carolinianus. We are unable to resolve the status of A. venustoides bispinosus, as only late copepodid stages of this species, and no mature adults, are available. Mesocyclops americanus, long called M. leuckarti, is now a well-documented species, much rarer than the somewhat atypical (for the genus) M. edax, well known in North America. Our proposed designations for North American species are summarized. There is a need to collect material year round from all available biotopes to document the species composition of Ontario Copepoda. Our work is also a first step in clarifying the status of North American Copepoda, comparing material from North America and elsewhere.

Inhibition of autoxidation of divicine and isouramil by the combination of superoxide dismutase and reduced glutathione

The effects of GSH on the autoxidation of the fava bean pyrimidine aglycones, divicine and isouramil, and on acid-hydrolyzed vicine (provisional identification 2-amino-4,5,6-trihydroxypyrimidine) have been studied. GSH alone promoted redox cycling of each compound, with concomitant GSH oxidation and H 2O 2 production. In the presence of superoxide dismutase, there is a lag period during which little pyrimidine oxidation occurs, followed by a period of accelerated oxidation. With the three pyrimidines, increasing concentrations of GSH extended this lag period and progressively decreased subsequent rates of both pyrimidine oxidation and O 2 uptake. No GSH oxidation or O 2 uptake occurred during the lag. These results show that the combination of GSH and superoxide dismutase is able to inhibit redox cycling of the pyrimidines. With a 10-fold excess of GSH over isouramil or acid-hydrolyzed vicine (20-fold with divicine) this coupled oxidation of GSH and the pyrimidine is almost completely suppressed. This mechanism may be a means whereby GSH in combination with superoxide dismutase protects against the cytotoxic effects of these reactive pyrimidines.