Development of a novel method for detection of Clostridium difficile using HS-SPME-GC-MS.

AIMS: A novel method has been developed that allows successful differentiation between Clostridium difficile culture-positive and culture-negative stool samples based on volatile organic compound (VOC) evolution and detection by headspace solid-phase microextraction coupled with gas chromatography mass spectrometry (HS-SPME-GC-MS). METHODS AND RESULTS: The method is based on the activation of p-hydroxyphenylacetate decarboxylase produced by Cl. difficile and the detection of a specific VOC, that is 2-fluoro-4-methylphenol from an enzyme substrate. In addition, other VOCs were good indicators for Cl. difficile, that is isocaproic acid and p-cresol, although they could not be used alone for identification purposes. One hundred stool samples were tested, of which 77 were positive by culture. Detection using HS-SPME-GC-MS allowed confirmation of the presence of Cl. difficile within 18 h with a sensitivity and specificity of 83·1 and 100%, respectively. CONCLUSIONS: It is recommended that this new approach could be used alongside conventional methods for Cl. difficile detection, including toxin detection methods, which would allow any false-negative results to be eliminated. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to identify Cl. difficile-positive stool samples by the analysis of VOCs could allow the development of a VOC detection device which could allow rapid diagnosis of disease and hence prompt treatment with appropriate antibiotics.

Potentially harmful elements (PHEs) in scalp hair, soil and metallurgical wastes in Mitrovica, Kosovo: the role of oral bioaccessibility and mineralogy in human PHE exposure.

Internationally publicized impacts upon human health associated with potentially harmful element (PHE) exposure have been reported amongst internally displaced populations (IDPs) in Mitrovica, Kosovo, following the Kosovan War. Particular concern has surrounded the exposure to Pb indicated by the presence of highly elevated concentrations of Pb in blood and hair samples. This study utilizes a physiologically-based in-vitro extraction method to assess the bioaccessibility of PHEs in surface soils and metallurgical waste in Mitrovica and assesses the potential daily intake of soil-bound PHEs. Maximum As (210mgkg(-1)), Cd (38mgkg(-1)), Cu (410mgkg(-1)), Pb (18790mgkg(-1)) and Zn (8500mgkg(-1)) concentrations in surface soils (0-10cm) are elevated above guideline values. Samples with high PHE concentrations (e.g. As >1000mgkg(-1); Pb >1500mgkg(-1)) exhibit a wide range of bioaccessibilities (5.40 - 92.20% in the gastric (G) phase and 10.00 - 55.80% in the gastric-intestinal (G-I) phase). Samples associated with lower bioaccessibilities typically contain a number of XRD-identifiable primary and secondary mineral phases, particularly As- and Pb-bearing arsenian pyrite, beudantite, galena and cerrusite. Quantification of the potential human exposure risk associated with the ingestion of soil-associated PHEs indicates that on average, 0.01µg Cd kg(-1) BW d(-1), 0.16µg Cu kg(-1) BW d(--1), 0.12µg As kg(-1) BW d(-1), 7.81µg Pb kg(-1) BW d(-1), and 2.68µg Zn kg(-1) BW d(-1) could be bioaccessible following ingestion of PHE-rich soils in the Mitrovica region, with Pb, and to a lesser extent As, indicating the likely possibility of local populations exceeding the recommended tolerable daily intake. Lead present within surface soils of the area could indeed have contributed to the human Pb burden due to the high bioaccessibility of Pb present within these soils (13.40 - 92.20% in the gastric phase). Data for Pb levels in scalp hair (≤120µgg(-1)) and blood (≥650µgdL(-1); WHO, 2004) for children that have lived within IDP camps in Mitrovica indicate significant Pb uptake has indeed taken place. The highly bioaccessible nature of soil-associated PHEs in this study highlights the need for appropriate environmental management approaches that limit the exposure of local populations to these contaminated soils.

The potential impact of washing machines on laundry malodour generation.

A multidisciplinary approach has been adopted to investigate and identify the source of malodour in washing machines and the potential for cross-contamination of laundry. Four washing machines were olfactively graded, and the number of colony-forming units (CFUs) bacteria was determined in four specific locations. Then, samples of terry-towel and fleece were washed, without the use of detergent, in the machines, and the occurrence of malodour over a 52-h period was assessed. Analysis of the scrapings from the four locations in the two malodorous machines identified a plethora of volatile organic compounds (VOCs) by either olfactory detection or mass spectral identification post-gas chromatographic separation. In addition, microbiological analysis from the swabs from the four locations within all four washing machines was carried out. Quantitative analysis of VOCs from 66 microbiological isolates from either the washing machines or fabrics was carried out. In total, 10 VOCs were identified: dimethyl disulfide, 3-methyl-1-butanol, 2,4-dithiapentane, dimethyl trisulfide, 2-tridecanone, indole, 2-phenylethanol, isovaleric acid, isobutyric acid and 1-undecene.

Determination of quaternary ammonium compounds in seawater samples by solid-phase extraction and liquid chromatography-mass spectrometry.

A method for the simultaneous determination of two biocidal quaternary ammonium compounds; didecyldimethylammonium chloride (didecyldimethyl quat) and dodecylbenzyldimethylammonium chloride (benzyl quat), in seawater by solid phase extraction (SPE) followed by liquid chromatography-mass spectrometry (LC-MS) was developed. The optimised procedure utilised off-line extraction of the analytes from seawater using polymeric (Strata-X) SPE cartridges. Recoveries ranged from 80 to 105%, with detection limits at the low parts-per-trillion (ng/l) level for both analytes. To demonstrate sensitivity, environmental concentrations were measured at three different locations along the North East coast of England with measured values in the range 120-270ng/l.

Oral bioaccessibility of metals in an urban catchment, Newcastle upon Tyne.

The metal content was determined in soils from a former, historic, contaminated land site and now a 'green' public open space in N.E. England. Using a systematic sampling grid approach, 32 soil samples were taken from locations across the site and analyzed for six potentially toxic elements (Cd, Cr, Cu, Ni, Pb, and Zn). Initially, the pseudo-total metal content of the soils was determined using acid digestion followed by inductively coupled plasma mass spectrometry analysis. This data was evaluated against published soil guideline value (SGV) and generic assessment criteria (GAC) values; it was found that 21% (i.e., 41 samples) exceeded the stated lower values. The data was then compared to the oral bioaccessibility of the soils, which was assessed by an in-vitro gastrointestinal extraction procedure. The results, determined as the % BAF, indicated that overall bioaccessibility was low (<10% BAF) for all the elements studied; the exception was Cd. Given that SGV/GAC values are based on generic land-use categories and not a public open space, as investigated in this work, further work is recommended on developing a qualitative risk assessment at the site to estimate the risks posed to human health via the direct and indirect soil ingestion pathway.

Outcomes in percutaneous stenting of non-hepato-biliary/pancreatic malignant jaundice.

The aim of this study is to review the practice and outcomes at our institution of percutaneous transhepatic placement of metallic biliary stents for non-hepato-biliary/pancreatic (non-HBP) malignant obstructive jaundice. A retrospective review was performed of the records of all patients undergoing transhepatic stenting for non-HBP malignant obstructive jaundice over a 7-year period. A total of 25 patients were successfully stented and linear regression analysis of a variety of demographic, clinical and laboratory markers against survival was performed. Survival after stenting varied from 1 to 1354 days (median 58, mean 152). An initial bilirubin level less than 300 micromol/L (P=0.01) and a reduction of greater than 50% in bilirubin post stenting (P=0.02) were strong predictors of improved survival. Older patients survived longer than younger ones (P<0.01). There was a weak association of survival with an albumin>30 g/L (P=0.06), but no statistically significant correlation with creatinine or haemoglobin levels or active tumour treatment after stenting. There were few major complications from the procedures. Transhepatic metallic biliary stenting for non-HBP malignant biliary obstruction is a safe and effective procedure, and with careful patient selection, significant periods of survival and palliation of jaundice can be achieved.

Evaluation of the physiologically based extraction test as an indicator of metal toxicity in mussel tissue.

In order to estimate the bioaccessibility of metals from mussel tissues, an in vitro physiologically based extraction test was applied to simulate the human gastrointestinal conditions. The samples were subjected to human body temperature, and Ag, Co, Cr, Cu, Mn, Ni, Pb and Sn were sequentially extracted with simulated gastric solution, followed by extraction with a simulated intestinal solution. Both gastric and intestinal extracted solutions and microwave-digested residue were analysed by ICP-MS. The procedure was applied to a certified reference material NIST 2977 (mussel tissue) to prove the accuracy of the method. Some mussel tissue samples from Northern Spain were subjected to this procedure to determine their metal content and their metal oral bioaccessibility.

An assessment of the bioavailability of persistent organic pollutants from contaminated soil.

A procedure to assess the bioavailability of persistent organic pollutants (POPs) from soil samples has been developed. The procedure is based on the use of simulated in vitro gastrointestinal extraction to remove POPs from soil matrices. The level of recovery, using this approach, is assessed following liquid-liquid extraction (LLE) and analysis by gas chromatography-mass selective detection (GC-MSD). The remaining soil residue is then extracted using pressurised fluid extraction (PFE) followed by GC-MSD analysis to assess the residual fraction. The residual fraction is monitored to determine the unavailable fraction i.e. not available for absorption in the gastrointestinal tract of humans. The procedure was applied to four soil samples i.e. an aged, spiked soil and three certified reference materials (CRMs) contaminated with POPs. Recoveries of pesticides (lindane, endosulfan I, endrin, DDE, DDD and endosulfan II), phenols (cresol, TCP and PCP), and base neutral compounds (hexachloroethane, acenaphthene, dibenzofuran, fluorene and hexachlorobenzene) from aged, spiked soil following extraction with gastric fluid ranged from 0.8 to 8.3% while following intestinal extraction ranged from 5.5 to 13.5%, irrespective of POP. Recoveries of pesticides (lindane, endosulfan I, endrin, DDE, DDD and endosulfan II) from CRM 805-050 following extraction with gastric fluid were below the limit of detection while following intestinal extraction ranged from 5.3 to 12.8%. Recoveries of phenols (cresol, TCP and PCP) from CRM 401-225 following extraction with gastric fluid ranged from 1.6 to 2.0% while following intestinal extraction ranged from 4.1 to 5.4%. Recoveries of base neutral acid analytes (hexachloroethane, acenaphthene, dibenzofuran, fluorene and hexachlorobenzene) from CRM 107-100 following extraction with gastric fluid ranged from 1.4 to 4.0% while following intestinal extraction ranged from 6.6 to 12.7%. It has been found that the majority of POPs present i.e. >75%, would be excreted if consumed and not be absorbed in the gastrointestinal tract of humans.

SPE/LC/ESI/MS with phthalic anhydride derivatisation for the determination of alcohol ethoxylate surfactants in sewage influent and effluent samples.

A new method for the analysis of alcohol ethoxylates (AEs) using electrospray ionisation liquid chromatography/mass spectrometry (ESI LC/MS) is described. The procedure incorporates a novel derivatisation step with phthalic anhydride for the analysis of EO0-20 ethoxylates in a single analysis. The derivatives obtained have proved to be very stable and the negative ion spectra show reduced background ions and competing adduct formation as compared to positive ion spectra. An automated solid phase extraction (SPE) step is used to allow both pre-concentration and clean-up of the environmental samples. The method provides more efficient recovery of AEs across the C12-C18 range than previously reported in the literature. Recoveries from final effluent spiked at 100 microg/L total AE, for the 126 species analysed, were found to be in the range 55-117%, with approximately 100 of the individual analytes having recoveries of 90-105%. An LOD of 0.02 microg/L for individual ethoxylate components is reported with the instrument operated in scan mode over the range m/z 300-1300. The method was applied to sewage effluent and influent samples, with AEs determined at approximately 7 and 5000 microg/L, respectively.

Characterisation and analysis of persistent organic pollutants and major, minor and trace elements in Calabash chalk.

Analysis of Calabash chalk has been done using energy dispersive X-ray fluorescence spectroscopy (EDXRF), X-ray diffraction (XRD) and pressurised fluid extraction (PFE) followed by gas chromatography (GC) with mass selective detection (MSD). It was found by XRD that the composition of Calabash chalk was an aluminium silicate hydroxide from the kaolin clay group with the possible formula Al(2)Si(2)O(5)(OH)(4). Multi-elemental analysis by EDXRF was able to quantify 22 elements in Calabash chalk including lead at a mean concentration of approximately 40 mg/kg. A range of persistent organic pollutants were identified and quantified in Calabash chalk including alpha lindane, endrin, endosulphan II and p,p'-DDD using PFE-GC-MSD.

Pressurised fluid extraction of bupirimate and ethirimol from aged soils.

This paper assesses the effect of pressurised fluid extraction (PFE) on the recovery of bupirimate and its degradation product, ethirimol from a range of soil types. The analytes were extracted under standard conditions (pressure, 2000 p.s.i.; temperature, 100 degrees C; and, three static flush cycles of 5 min static extraction time each) using a variety of individual and combined solvents. It was found that the recovery of bupirimate was dependent upon the organic matter content of soil.

The strengthening effect of percutaneous vertebroplasty.

AIM: To examine whether single-needle vertebroplasty is effective in strengthening vertebrae. MATERIALS AND METHODS: Four adjacent intact lumbar vertebrae were removed from each of nine adult post-mortem subjects. One or two vertebra of each set was injected with low viscosity acrylic bone cement using a single-track posterolateral approach. Vertebral strength was measured during constant rate axial compression. RESULTS: Single-needle injection delivered different quantities of cement (1-8 ml) in an asymmetric distribution. Injected vertebrae were stronger than controls in 10 out of 12 cases (P < 0.01), suggesting that cement injection increases the load needed to cause initial vertebral fracture. Compression was continued past the point of initial failure, to simulate progressive vertebral collapse. Injected specimens were more resistant to deformation than control specimens (P < 0.05), most of which initially deformed with little increase in load. CONCLUSION: Single-track injection of bone cement appeared to strengthen vertebrae against both initial failure and subsequent collapse. The magnitude of strength increase from vertebroplasty was variable, and did not correlate with the volume of cement injected. Further investigation is needed to optimize the technique.Gishen, P. (2000). Clinical Radiology55, 471-476.

Extraction of DDT [1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane] and its metabolites DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene] and DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)-ethane]) from aged contaminated soil.

Pressurised liquid extraction (PLE) was used to extract DDT [1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane] and its metabolites, DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane] and DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene] from an aged, contaminated soil. Using three sequential static phases, PLE removed an equivalent quantity of DDT and its metabolites as Soxhlet extraction, in less time and with less solvent. Recovery was almost quantitative, implying appropriate sample work-up and manipulation.

Pressurised liquid extraction of pentachlorophenol from dyestuffs and cotton.

Pressurised liquid extraction (PLE) was used to extract pentachlorophenol from cotton and dyestuffs. Five dyes and one dyed cotton were investigated for pentachlorophenol. The dyed cotton was found to have between 5.3 and 5.9 microg g(-1) pentachlorophenol, while the dyes were found to have between 42 and 2569 microg g(-1) pentachlorophenol present. The latter were found to be dye and manufacturing site dependant. Recovery experiments, in all cases, were found to be quantitative indicating appropriate sample work-up and manipulations.

Influence of solvent and soil type on the pressurised fluid extraction of PAHs.

Pressurised fluid extraction (PFE) of polycyclic aromatic hydrocarbons (PAHs) from a certified reference material (CRM) 524 has been firstly optimised following a central composite design. The instrumental parameters of the PFE (pressure, temperature, extraction time and number of solvent cycles) were studied in order to obtain maximum extraction yields. Neither pressure nor extraction time or temperature seemed to have any significant effect on the extraction yield, therefore one extraction cycle was enough to exhaustively extract all the PAHs from CRM 524. Once the instrumental conditions were established, the extraction yields obtained with eight different solvents or solvent mixtures [acetone, dichloromethane, acetonitrile, acetone-dichloromethane (1 + 1 v/v), acetone-isohexane (1 + 1 v/v), isohexane, methanol and toluene] from the CRM 524 were compared and showed that the best recoveries were obtained with acetone-isohexane (1 + 1 v/v). Finally, the effect of sand, silt, clay and the organic matter content of soil was investigated with respect to recovery of PAHs by PFE with different solvents or solvent mixtures for aged soil samples. In this case, eight soils with different sand, silt, clay and organic matter contents were slurry spiked with PAHs and aged for 19 days. Three aliquots of each slurry spiked soil were extracted with the previously mentioned solvents and the results were studied by means of principal component analysis (PCA) of the whole data set (soil composition, solubility parameter of the solvent and recoveries of all PAHs) and partial least squares (PLS). Clay and organic matter content and the squared solubility parameter have the highest correlation with the recovery of PAHs from soil samples.

Intrapituitary fluid levels following haemorrhage: MRI appearances in 13 cases.

Demonstration of fluid levels on MRI is well recognised in cerebral haematomas, tumours and cysts. The occurrence of fluid levels within haemorrhagic pituitary tumours has not previously been described in detail. Evidence of haemorrhage was identified in 27 of 125 pituitary tumours. Fluid levels occurred in 13 of these haemorrhagic tumours. No association with histological type was identified. Recognised risk factors for haemorrhage were identified in half of the cases.

Extraction of pharmaceuticals using pressurised carbon dioxide.

This paper reviews the applications of super- and sub-critical carbon dioxide for the extraction of pharmaceuticals from various matrices. The matrices covered are divided into the following types: animal feed, formulations, biological and miscellaneous, with various sub-divisions as appropriate. The polar nature of most pharmaceuticals often precludes the use of carbon dioxide only, so it is common to find the addition of a more polar solvent, as modifier. As the majority of sample types covered are solid, little if any pre-treatment is required, with the exception of grinding, prior to insertion in the sample extraction cell. For liquid-type matrices, sample pre-treatment is the normal. Often this may involve adsorption on an inert support e.g. Celite or diatomaceous earth, or immobilisation on a functionalised silica surface, e.g. C18. The later may take the form of a solid phase extraction cartridge or disk. An attempt has also been made to sample from liquid matrices directly using a modified extraction cell. The variety of sample types, matrices and analyte polarity places stringent requirements on the use of pressurised carbon dioxide. Its potential for effective recovery is examined in this review.

Determination of antifungals in rodent diet by supercritical fluid extraction followed by packed column supercritical fluid chromatography with ultraviolet detection.

Supercritical fluid extraction (SFE) followed by packed column supercritical fluid chromatography with ultraviolet detection was evaluated as a quantitative method for determining 4 antifungals (fluconazole, tioconazole, hexaconazole, and UK-47,265) in rodent diet. Chromatography was achieved with a cyano-bonded silica column, UV detection at 210 nm, and methanol-modified supercritical carbon dioxide as mobile phase. The effects of modifier concentration, temperature, and column pressure on antifungal retention time was studied. Off-line SFE was optimized at 2 spike levels, ranging from 0.5 to 10 g/kg, for each of the 4 antifungals. Average recoveries ranged from 79.0% for UK-47,265 to 96.5% for hexaconazole. Overall, the procedure provides a suitable method for analyzing antifungals in spiked rodent diet.

Solid-phase microextraction as a method for estimating the octanol-water partition coefficient.

The determination of octanol-water partition coefficients (log K(ow)) is important for the prediction of the fate of organic pollutants in the environment. Traditionally, log K(ow) values are determined by shake-flask, estimated by, e.g., HPLC retention data, or calculated, e.g., from ClogP. In this paper, an alternative approach is reported that allows log K(ow) to be estimated from solid-phase microextraction (SPME) data. Previously reported attempts to correlate SPME data with log K(ow) are discussed. The results obtained in this work for six phenols, using an 85 µm polyacrylate-coated fiber, indicate that SPME is a viable method for estimating log K(ow) values <3.5.