A quantitative phenytoin GC-MS method and it's validation for samples from human ex situ brain microdialysis, blood and saliva using solid-phase extraction

This study describes the development and validation of a gas chromatography-mass spectrometry (GC-MS) method to identify and quantitate phenytoin in brain microdialysate, saliva and blood from human samples. A solid-phase extraction (SPE) was performed with a nonpolar C8-SCX column. The eluate was evaporated with nitrogen (50°C) and derivatized with trimethylsulfonium hydroxide before GC-MS analysis. As the internal standard, 5-(p-methylphenyl)-5-phenylhydantoin was used. The MS was run in scan mode and the identification was made with three ion fragment masses. All peaks were identified with MassLib. Spiked phenytoin samples showed recovery after SPE of ≥94%. The calibration curve (phenytoin 50 to 1,200 ng/mL, n = 6, at six concentration levels) showed good linearity and correlation (r² > 0.998). The limit of detection was 15 ng/mL; the limit of quantification was 50 ng/mL. Dried extracted samples were stable within a 15% deviation range for ≥4 weeks at room temperature. The method met International Organization for Standardization standards and was able to detect and quantify phenytoin in different biological matrices and patient samples. The GC-MS method with SPE is specific, sensitive, robust and well reproducible, and is therefore an appropriate candidate for the pharmacokinetic assessment of phenytoin concentrations in different human biological samples.

An In Vitro Method to Investigate the Microbicidal Potential of Human Macrophages for Use in Psychosomatic Research

OBJECTIVE: Psychological states relate to changes in circulating immune cells, but associations with immune cells in peripheral tissues such as macrophages have hardly been investigated. Here, we aimed to implement and validate a method for measuring the microbicidal potential of ex vivo isolated human monocyte-derived macrophages (HMDMs) as an indicator of macrophage activation. METHODS: The method was implemented and validated for two blood sampling procedures (short-term cannula insertion versus long-term catheter insertion) in 79 participants (34 women, 45 men) aged between 18 and 75 years. The method principle is based on the reduction of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-dis-ulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) by superoxide anions, the first in a series of pathogen-killing reactive oxygen species produced by phorbol myristate acetate-activated HMDM. Cytochrome c reduction and current generation were measured as reference methods for validation purposes. We further evaluated whether depressive symptom severity (Beck Depression Inventory) and chronic stress (Chronic Stress Screening Scale) were associated with macrophage microbicidal potential. RESULTS: The assay induced superoxide anion responses by HMDM in all participants. Assay results depended on blood sampling procedure (cannula versus catheter insertion). Interassay variability as a measure for assay reliability was 10.92% or less. WST-1 reduction scores correlated strongly with results obtained by reference methods (cytochrome c: r = 0.57, p = .026; current generation: r values ≥ 0.47, p values <.033) and with psychological factors (depressive symptom severity: r = 0.35 [cannula insertion] versus r = -0.54 [catheter insertion]; chronic stress: r = 0.36 [cannula insertion]; p values ≤ .047). CONCLUSIONS: Our findings suggest that the implemented in vitro method investigates microbicidal potential of HMDM in a manner that is valid and sensitive to psychological measures.

Fourier transform infrared spectroscopy, a new method for rapid determination of total organic and inorganic carbon and biogenic silica concentration in lake sediments

Abstract We demonstrate the use of Fourier transform infrared spectroscopy (FTIRS) to make quantitative measures of total organic carbon (TOC), total inorganic carbon (TIC) and biogenic silica (BSi) concentrations in sediment. FTIRS is a fast and costeffective technique and only small sediment samples are needed (0.01 g). Statistically significant models were developed using sediment samples from northern Sweden and were applied to sediment records from Sweden, northeast Siberia and Macedonia. The correlation between FTIRS-inferred values and amounts of biogeochemical constituents assessed conventionally varied between r = 0.84–0.99 for TOC, r = 0.85– 0.99 for TIC, and r = 0.68–0.94 for BSi. Because FTIR spectra contain information on a large number of both inorganic and organic components, there is great potential for FTIRS to become an important tool in paleolimnology.

DEVELOPING A METHOD FOR IDENTIFYING AND REDUCING FUNCTIONAL DISCREPANCIES OF INFORMATION SYSTEMS

Currently more than half of Electronic Health Record (EHR) projects fail. Most of these failures are not due to flawed technology, but rather due to the lack of systematic considerations of human issues. Among the barriers for EHR adoption, function mismatching among users, activities, and systems is a major area that has not been systematically addressed from a human-centered perspective. A theoretical framework called Functional Framework was developed for identifying and reducing functional discrepancies among users, activities, and systems. The Functional Framework is composed of three models – the User Model, the Designer Model, and the Activity Model. The User Model was developed by conducting a survey (N = 32) that identified the functions needed and desired from the user’s perspective. The Designer Model was developed by conducting a systemic review of an Electronic Dental Record (EDR) and its functions. The Activity Model was developed using an ethnographic method called shadowing where EDR users (5 dentists, 5 dental assistants, 5 administrative personnel) were followed quietly and observed for their activities. These three models were combined to form a unified model. From the unified model the work domain ontology was developed by asking users to rate the functions (a total of 190 functions) in the unified model along the dimensions of frequency and criticality in a survey. The functional discrepancies, as indicated by the regions of the Venn diagrams formed by the three models, were consistent with the survey results, especially with user satisfaction. The survey for the Functional Framework indicated the preference of one system over the other (R=0.895). The results of this project showed that the Functional Framework provides a systematic method for identifying, evaluating, and reducing functional discrepancies among users, systems, and activities. Limitations and generalizability of the Functional Framework were discussed.

Towards a Hybrid Method to Categorize Interruptions and Activities in Healthcare

Objective Interruptions are known to have a negative impact on activity performance. Understanding how an interruption contributes to human error is limited because there is not a standard method for analyzing and classifying interruptions. Qualitative data are typically analyzed by either a deductive or an inductive method. Both methods have limitations. In this paper a hybrid method was developed that integrates deductive and inductive methods for the categorization of activities and interruptions recorded during an ethnographic study of physicians and registered nurses in a Level One Trauma Center. Understanding the effects of interruptions is important for designing and evaluating informatics tools in particular and for improving healthcare quality and patient safety in general. Method The hybrid method was developed using a deductive a priori classification framework with the provision of adding new categories discovered inductively in the data. The inductive process utilized line-by-line coding and constant comparison as stated in Grounded Theory. Results The categories of activities and interruptions were organized into a three-tiered hierarchy of activity. Validity and reliability of the categories were tested by categorizing a medical error case external to the study. No new categories of interruptions were identified during analysis of the medical error case. Conclusions Findings from this study provide evidence that the hybrid model of categorization is more complete than either a deductive or an inductive method alone. The hybrid method developed in this study provides the methodical support for understanding, analyzing, and managing interruptions and workflow.

Towards a hybrid method to categorize interruptions and activities in healthcare.

OBJECTIVE: Interruptions are known to have a negative impact on activity performance. Understanding how an interruption contributes to human error is limited because there is not a standard method for analyzing and classifying interruptions. Qualitative data are typically analyzed by either a deductive or an inductive method. Both methods have limitations. In this paper, a hybrid method was developed that integrates deductive and inductive methods for the categorization of activities and interruptions recorded during an ethnographic study of physicians and registered nurses in a Level One Trauma Center. Understanding the effects of interruptions is important for designing and evaluating informatics tools in particular as well as improving healthcare quality and patient safety in general. METHOD: The hybrid method was developed using a deductive a priori classification framework with the provision of adding new categories discovered inductively in the data. The inductive process utilized line-by-line coding and constant comparison as stated in Grounded Theory. RESULTS: The categories of activities and interruptions were organized into a three-tiered hierarchy of activity. Validity and reliability of the categories were tested by categorizing a medical error case external to the study. No new categories of interruptions were identified during analysis of the medical error case. CONCLUSIONS: Findings from this study provide evidence that the hybrid model of categorization is more complete than either a deductive or an inductive method alone. The hybrid method developed in this study provides the methodical support for understanding, analyzing, and managing interruptions and workflow.

IMILAST: A community effort to intercompare extratropical cyclone detection and tracking algorithms: assessing method-related uncertainties

The variability of results from different automated methods of detection and tracking of extratropical cyclones is assessed in order to identify uncertainties related to the choice of method. Fifteen international teams applied their own algorithms to the same dataset - the period 1989-2009 of interim European Centre for Medium-Range Weather Forecasts (ECMWF) Re-Analysis (ERAInterim) data. This experiment is part of the community project Intercomparison of Mid Latitude Storm Diagnostics (IMILAST; see www.proclim.ch/imilast/index.html). The spread of results for cyclone frequency, intensity, life cycle, and track location is presented to illustrate the impact of using different methods. Globally, methods agree well for geographical distribution in large oceanic regions, interannual variability of cyclone numbers, geographical patterns of strong trends, and distribution shape for many life cycle characteristics. In contrast, the largest disparities exist for the total numbers of cyclones, the detection of weak cyclones, and distribution in some densely populated regions. Consistency between methods is better for strong cyclones than for shallow ones. Two case studies of relatively large, intense cyclones reveal that the identification of the most intense part of the life cycle of these events is robust between methods, but considerable differences exist during the development and the dissolution phases.

An Aquaculture-Based Method for Calibrated Bivalve Isotope Paleothermometry

To quantify species- specific relationships between bivalve carbonate isotope geochemistry ( delta O-18(c)) and water conditions ( temperature and salinity, related to water isotopic composition [delta O-18(w)]), an aquaculture-based methodology was developed and applied to Mytilus edulis ( blue mussel). The four- by- three factorial design consisted of four circulating temperature baths ( 7, 11, 15, and 19 degrees C) and three salinity ranges ( 23, 28, and 32 parts per thousand ( ppt); monitored for delta O-18(w) weekly). In mid- July of 2003, 4800 juvenile mussels were collected in Salt Bay, Damariscotta, Maine, and were placed in each configuration. The size distribution of harvested mussels, based on 105 specimens, ranged from 10.9 mm to 29.5 mm with a mean size of 19.8 mm. The mussels were grown in controlled conditions for up to 8.5 months, and a paleotemperature relationship based on juvenile M. edulis from Maine was developed from animals harvested at months 4, 5, and 8.5. This relationship [ T degrees C = 16.19 (+/- 0.14) - 4.69 (+/- 0.21) {delta O-18(c) VPBD - delta O-18(w) VSMOW} + 0.17 (+/- 0.13) {delta O-18(c) VPBD - delta O-18(w) VSMOW}(2); r(2) = 0.99; N = 105; P < 0.0001] is nearly identical to the Kim and O'Neil ( 1997) abiogenic calcite equation over the entire temperature range ( 7 - 19 degrees C), and it closely resembles the commonly used paleotemperature equations of Epstein et al. ( 1953) and Horibe and Oba ( 1972). Further, the comparison of the M. edulis paleotemperature equation with the Kim and O'Neil ( 1997) equilibrium- based equation indicates that M. edulis specimens used in this study precipitated their shell in isotopic equilibrium with ambient water within the experimental uncertainties of both studies. The aquaculture- based methodology described here allows similar species- specific isotope paleothermometer calibrations to be performed with other bivalve species and thus provides improved quantitative paleoenvironmental reconstructions.

A Modification of the Response Method of Tidal Analysis

An easily implemented extension of the standard response method of tidal analysis is outlined. The modification improves the extraction of both the steady and the tidal components from problematic time series by calculating tidal response weights uncontaminated by missing or anomalous data. Examples of time series containing data gaps and anomalous events are analyzed to demonstrate the applicability and advantage of the proposed method.

Quantitative morphometric analysis of lakes using GIS: rectangularity R , ellipticity E , orientation O , and the rectangularity vs. ellipticity index, REi

Quantitative measures of polygon shapes and orientation are important elements of geospatial analysis. These kinds of measures are particularly valuable in the case of lakes, where shape and orientation patterns can help identifying the geomorphological agents behind lake formation and evolution. However, the lack of built-in tools in commercial geographic information system (GIS) software packages designed for this kind of analysis has meant that many researchers often must rely on tools and workarounds that are not always accurate. Here, an easy-to-use method to measure rectangularity R, ellipticity E, and orientation O is developed. In addition, a new rectangularity vs. ellipticity index, REi, is defined. Following a step-by-step process, it is shown how these measures and index can be easily calculated using a combination of GIS built-in functions. The identification of shapes and estimation of orientations performed by this method is applied to the case study of the geometric and oriented lakes of the Llanos de Moxos, in the Bolivian Amazon, where shape and orientation have been the two most important elements studied to infer possible formation mechanisms. It is shown that, thanks to these new indexes, shape and orientation patterns are unveiled, which would have been hard to identify otherwise.

New method for porewater extraction from claystone and determination of transport properties with results for Opalinus Clay (Switzerland)

A new technique to porewater extraction from claystone employs advective displacement of the in situ porewater by traced artificial porewater. Monitoring of tracer breakthrough yields species-specific transport properties. Results for Opalinus Clay from the Mont Terri Research Laboratory indicate that the chemical disturbances due to the method are minimal, and the observed significant differences in transport properties for Br– and 2H are in agreement with existing data. Sampling times are 2–4 months, and observation of tracer breakthrough takes 12–24 months at hydraulic conductivity of ∼10-13 m/s.

Continuous dynamic mapping of the corticospinal tract during surgery of motor eloquent brain tumors: evaluation of a new method

OBJECT The authors developed a new mapping technique to overcome the temporal and spatial limitations of classic subcortical mapping of the corticospinal tract (CST). The feasibility and safety of continuous (0.4-2 Hz) and dynamic (at the site of and synchronized with tissue resection) subcortical motor mapping was evaluated. METHODS The authors prospectively studied 69 patients who underwent tumor surgery adjacent to the CST (< 1 cm using diffusion tensor imaging and fiber tracking) with simultaneous subcortical monopolar motor mapping (short train, interstimulus interval 4 msec, pulse duration 500 μsec) and a new acoustic motor evoked potential alarm. Continuous (temporal coverage) and dynamic (spatial coverage) mapping was technically realized by integrating the mapping probe at the tip of a new suction device, with the concept that this device will be in contact with the tissue where the resection is performed. Motor function was assessed 1 day after surgery, at discharge, and at 3 months. RESULTS All procedures were technically successful. There was a 1:1 correlation of motor thresholds for stimulation sites simultaneously mapped with the new suction mapping device and the classic fingerstick probe (24 patients, 74 stimulation points; r(2) = 0.98, p < 0.001). The lowest individual motor thresholds were as follows: > 20 mA, 7 patients; 11-20 mA, 13 patients; 6-10 mA, 8 patients; 4-5 mA, 17 patients; and 1-3 mA, 24 patients. At 3 months, 2 patients (3%) had a persistent postoperative motor deficit, both of which were caused by a vascular injury. No patient had a permanent motor deficit caused by a mechanical injury of the CST. CONCLUSIONS Continuous dynamic mapping was found to be a feasible and ergonomic technique for localizing the exact site of the CST and distance to the motor fibers. The acoustic feedback and the ability to stimulate the tissue continuously and exactly at the site of tissue removal improves the accuracy of mapping, especially at low (< 5 mA) stimulation intensities. This new technique may increase the safety of motor eloquent tumor surgery.

A quantitiative LC-MS/MS method for the measurement of arachidonic acid, prostanoids, endocannabinoids, N-acylethanolamines and steroids in human plasma.

Free arachidonic acid is functionally interlinked with different lipid signaling networks including those involving prostanoid pathways, the endocannabinoid system, N-acylethanolamines, as well as steroids. A sensitive and specific LC-MS/MS method for the quantification of arachidonic acid, prostaglandin E2, thromboxane B2, anandamide, 2-arachidonoylglycerol, noladin ether, lineoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, steroyl ethanolamide, aldosterone, cortisol, dehydroepiandrosterone, progesterone, and testosterone in human plasma was developed and validated. Analytes were extracted using acetonitrile precipitation followed by solid phase extraction. Separations were performed by UFLC using a C18 column and analyzed on a triple quadrupole MS with electron spray ionization. Analytes were run first in negative mode and, subsequently, in positive mode in two independent LC-MS/MS runs. For each analyte, two MRM transitions were collected in order to confirm identity. All analytes showed good linearity over the investigated concentration range (r>0.98). Validated LLOQs ranged from 0.1 to 190ng/mL and LODs ranged from 0.04 to 12.3ng/mL. Our data show that this LC-MS/MS method is suitable for the quantification of a diverse set of bioactive lipids in plasma from human donors (n=32). The determined plasma levels are in agreement with the literature, thus providing a versatile method to explore pathophysiological processes in which changes of these lipids are implicated.