A Novel Dried Blood Spot-LCMS Method for the Quantification of Methotrexate Polyglutamates as a Potential Marker for Methotrexate Use in Children

Objective: Development and validation of a selective and sensitive LCMS method for the determination of methotrexate polyglutamates in dried blood spots (DBS).

Methods: DBS samples [spiked or patient samples] were prepared by applying blood to Guthrie cards which was then dried at room temperature. The method utilised 6-mm disks punched from the DBS samples (equivalent to approximately 12 μl of whole blood). The simple treatment procedure was based on protein precipitation using perchloric acid followed by solid phase extraction using MAX cartridges. The extracted sample was chromatographed using a reversed phase system involving an Atlantis T3-C18 column (3 μm, 2.1x150 mm) preceded by Atlantis guard column of matching chemistry. Analytes were subjected to LCMS analysis using positive electrospray ionization.

Key Results: The method was linear over the range 5-400 nmol/L. The limits of detection and quantification were 1.6 and 5 nmol/L for individual polyglutamates and 1.5 and 4.5 nmol/L for total polyglutamates, respectively. The method has been applied successfully to the determination of DBS finger-prick samples from 47 paediatric patients and results confirmed with concentrations measured in matched RBC samples using conventional HPLC-UV technique.

Conclusions and Clinical Relevance: The methodology has a potential for application in a range of clinical studies (e.g. pharmacokinetic evaluations or medication adherence assessment) since it is minimally invasive and easy to perform, potentially allowing parents to take blood samples at home. The feasibility of using DBS sampling can be of major value for future clinical trials or clinical care in paediatric rheumatology. © 2014 Hawwa et al.

Dried blood spot liquid chromatography assay for therapeutic drug monitoring of metformin

The use of blood spot collection cards is a simple way to obtain specimens for analysis of drugs for the purpose of therapeutic drug monitoring, assessing adherence to medications and preventing toxicity in routine clinical setting. We describe the development and validation of a microanalytical technique for the determination of metformin from dried blood spots. The method is based on reversed phase high-performance liquid chromatography with ultraviolet detection. Drug recovery in the developed method was found to be more than 84%. The limits of detection and quantification were calculated to be to be 90 and 150 ng/ml, respectively. The intraday and interday precision (measured by CV%) was always less than 9%. The accuracy (measured by relative error, %) was always less than 12%. Stability analysis showed that metformin is stable for at least 2 months when stored at -70 degrees C. The small volume of blood required (10 mu L), combined with the simplicity of the analytical technique makes this a useful procedure for monitoring metformin concentrations in routine clinical settings. The method is currently being applied to the analysis of blood spots taken from diabetic patients to assess adherence to medications and relationship between metformin level and metabolic control of diabetes. (c) 2006 Elsevier B.V. All rights reserved.

The manufacture and characterisation of hot-melt extruded enteric tablets

The aim of this highly novel study was to use hot-melt extrusion technology as an alternative process to enteric coating. In so doing, oral dosage forms displaying enteric properties may be produced in a continuous, rapid process, providing significant advantages over traditional pharmaceutical coating technology. Eudragit (R) L100-55, an enteric polymer, was pre-plasticized with triethyl citrate (TEC) and citric acid and subsequently dry-mixed with 5-aminosalicylic acid, a model active pharmaceutical ingredient (API), and an optional gelling agent (PVP (R) K30 or Carbopol (R) 971P). Powder blends were hot-melt extruded as cylinders, cut into tablets and characterised using powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC) and dissolution testing conducted in both pH 1.2 and pH 6.8 buffers. Increasing the concentration of TEC significantly lowered the glass transition temperature (T,) of Eudragit (R) L100-55 and reduced temperatures necessary for extrusion as well as the die pressure. Moreover, citric acid (17% w/w) was shown to act as a solid-state plasticizer. HME tablets showed excellent gastro-resistance, whereas milled extrudates compressed into tablets released more than 10% w/w of the API in acidic media. Drug release from HME tablets was dependent upon the concentration of TEC, the presence of citric acid, PVP K30, and Carbopol (R) 971P in the matrix, and pH of the dissolution media. The inclusion of an optional gelling agent significantly reduced the erosion of the matrix and drug release rate at pH 6.8; however, the enteric properties of the matrix were lost due to the formation of channels within the tablet. Consequently this work is both timely and highly innovative and identifies for the first time a method of producing an enteric matrix tablet using a continuous hot-melt extrusion process.

Development and validation of a dried blood spot-LC-APCI-MS assay for estimation of canrenone in paediatric samples

A selective and sensitive liquid chromatography (LC)-atmospheric pressure chemical ionisation (APCI)-mass spectroscopic (MS) assay of canrenone has been developed and validated employing Dried Blood Spots (DBS) as the sample collection medium. DBS samples were prepared by applying 30 mu l of spiked whole blood onto Guthrie cards. A 6 mm disc was punched from the each DBS and extracted with 2 ml of methanolic solution of 17 alpha-methyltestosterone (Internal Standard). The methanolic extract was evaporated to dryness and reconstituted in acetonitrile:water (1:9, v/v). The reconstituted solution was further subjected to solid phase extraction using HLB cartridges. Chromatographic separation was achieved using Waters Sunfire C18 reversed-phase column using isocratic elution, followed by a high organic wash to clear late eluting/highly retained components. The mobile phase consisted of methanol:water (60:40, v/v) pumped at a flow rate of 0.3 ml/min. LC-APCI-MS detection was performed in the selected-ion monitoring (SIM) mode using target ions at m/z 341.1 and 303.3 for canrenone and internal standard respectively. The selectivity of the method was established by analysing DBS samples from 6 different sources (individuals). The calibration curve for canrenone was found to be linear over 25-1000 ng/ml (r >0.994). Accuracy (% RE) and precision (% CV) values for within and between day were

Spin-orbit angle measurements for six southern transiting planets. New insights into the dynamical origins of hot Jupiters

Context. Several competing scenarios for planetary-system formation and evolution seek to explain how hot Jupiters came to be so close to their parent stars. Most planetary parameters evolve with time, making it hard to distinguish between models. The obliquity of an orbit with respect to the stellar rotation axis is thought to be more stable than other parameters such as eccentricity. Most planets, to date, appear aligned with the stellar rotation axis; the few misaligned planets so far detected are massive (> 2 MJ). Aims: Our goal is to measure the degree of alignment between planetary orbits and stellar spin axes, to search for potential correlations with eccentricity or other planetary parameters and to measure long term radial velocity variability indicating the presence of other bodies in the system. Methods: For transiting planets, the Rossiter-McLaughlin effect allows the measurement of the sky-projected angle ß between the stellar rotation axis and a planet's orbital axis. Using the HARPS spectrograph, we observed the Rossiter-McLaughlin effect for six transiting hot Jupiters found by the WASP consortium. We combine these with long term radial velocity measurements obtained with CORALIE. We used a combined analysis of photometry and radial velocities, fitting model parameters with the Markov Chain Monte Carlo method. After obtaining ß we attempt to statistically determine the distribution of the real spin-orbit angle ?. Results: We found that three of our targets have ß above 90°: WASP-2b: ß = 153°+11-15, WASP-15b: ß = 139.6°+5.2-4.3 and WASP-17b: ß = 148.5°+5.1-4.2; the other three (WASP-4b, WASP-5b and WASP-18b) have angles compatible with 0°. We find no dependence between the misaligned angle and planet mass nor with any other planetary parameter. All six orbits are close to circular, with only one firm detection of eccentricity e = 0.00848+0.00085-0.00095 in WASP-18b. No long-term radial acceleration was detected for any of the targets. Combining all previous 20 measurements of ß and our six and transforming them into a distribution of ? we find that between about 45 and 85% of hot Jupiters have ? > 30°. Conclusions: Most hot Jupiters are misaligned, with a large variety of spin-orbit angles. We find observations and predictions using the Kozai mechanism match well. If these observational facts are confirmed in the future, we may then conclude that most hot Jupiters are formed from a dynamical and tidal origin without the necessity to use type I or II migration. At present, standard disc migration cannot explain the observations without invoking at least another additional process.

Development and validation of a dried blood spot LC-MS/MS assay to quantify ranitidine in paediatric samples.

A novel approach has been developed to determine ranitidine in paediatric samples using dried blood spots (DBS) on Guthrie cards (Whatman 903). A selective and sensitive HPLC-MS/MS assay has been developed and validated using small volumes of blood (30µl). A 6mm disc was punched from each DBS and extracted with methanolic solution of the internal standard (IS) nizatidine. This was further subjected to solid phase extraction (SPE), followed by reversed phase HPLC separation, using a XBridge™ C18 column and mobile phase 10mM ammonium acetate/methanol (98:2 v/v) with a flow rate of 0.3mL/min. This was combined with multiple reaction monitoring (MRM) mass detection using electrospray ionisation (ESI). The calibration curve for ranitidine was found linear over the range 10-500ng/mL (r=0.996). The limit of quantification (LOQ) of the method was validated at 10ng/mL. Accuracy and precision values for within and between days were

WASP-41b: A Transiting Hot Jupiter Planet Orbiting a Magnetically Active G8V Star

We report the discovery of a transiting planet with an orbital period of 3.05 days orbiting the star TYC 7247-587-1. The star, WASP-41, is a moderately bright G8 V star (V=11.6) with a metallicity close to solar ([Fe/H]=-0.08±0.09). The star shows evidence of moderate chromospheric activity, both from emission in the cores of the Ca ii H and K ines and photometric variability with a period of 18.4 days and an amplitude of about 1%. We use a new method to show quantitatively that this periodic signal has a low false-alarm probability. The rotation period of the star implies a gyrochronological age for WASP-41 of 1.8 Gyr with an error of about 15%. We have used a combined analysis of the available photometric and spectroscopic data to derive the mass and radius of the planet (0.92±0.06 M, 1.20±0.06 R). Further observations of WASP-41 can be used to explore the connections between the properties of hot Jupiter planets and the level of chromospheric activity in their host stars.

WASP-23b: a transiting hot Jupiter around a K dwarf and its Rossiter-McLaughlin effect

We report the discovery of a new transiting planet in the southern hemisphere. It was found by the WASP-south transit survey and confirmed photometrically and spectroscopically by the 1.2 m Swiss Euler telescope, LCOGT 2m Faulkes South Telescope, the 60 cm TRAPPIST telescope, and the ESO 3.6 m telescope. The orbital period of the planet is 2.94 days. We find that it is a gas giant with a mass of 0.88 ± 0.10 MJ and an estimated radius of 0.96 ± 0.05 RJ. We obtained spectra during transit with the HARPS spectrograph and detect the Rossiter-McLaughlin effect despite its small amplitude. Because of the low signal-to-noise ratio of the effect and a small impact parameter, we cannot place a strong constraint on the projected spin-orbit angle. We find two conflicting values for the stellar rotation. We find, via spectral line broadening, that v sin I = 2.2 ± 0.3 km s-1, while applying another method, based on the activity level using the index log R'_HK, gives an equatorial rotation velocity of only v = 1.35 ± 0.20 km s-1. Using these as priors in our analysis, the planet might be either misaligned or aligned. This result raises doubts about the use of such priors. There is evidence of neither eccentricity nor any radial velocity drift with time. Using WASP-South photometric observations confirmed with LCOGT Faulkes South Telescope, the 60 cm TRAPPIST telescope, the CORALIE spectrograph and the camera from the Swiss 1.2 m Euler Telescope placed at La Silla, Chile, as well as with the HARPS spectrograph, mounted on the ESO 3.6 m, also at La Silla, under proposal 084.C-0185. The data is publicly available at the CDS Strasbourg and on demand to the main author.RV data is only available at the CDS via anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/531/A24Appendix is available in electronic form at http://www.aanda.org

A comprehensive aerosol spray method for the rapid photocatalytic grid area analysis of semiconductor photocatalyst thin films

Indicator inks, previously shown to be capable of rapidly assessing photocatalytic activity via a novel photo-reductive mechanism, were simply applied via an aerosol spray onto commercially available pieces of Activ (TM) self-cleaning glass. Ink layers could be applied with high evenness of spread, with as little deviation as 5% upon UV-visible spectroscopic assessment of 25 equally distributed positions over a 10 cm x 10 cm glass cut. The inks were comprised of either a resazurin (Rz) or dichloroindophenol (DCIP) redox dye with a glycerol sacrificial electron donor in an aqueous hydroxyethyl cellulose (HEC) polymer media. The photo-reduction reaction under UVA light of a single spot was monitored by UV-vis spectroscopy and digital images attained from a flat-bed scanner in tandem for both inks. The photo-reduction of Rz ink underwent a two-step kinetic process, whereby the blue redox dye was initially reduced to a pink intermediate resorufin (Rf) and subsequently reduced to a bleached form of the dye. In contrast, a simple one-step kinetic process was observed for the reduction of the light blue redox dye DCIP to its bleached intermediates. Changes in red-green-blue colour extracted from digital images of the inks were inversely proportional to the changes seen at corresponding wavelengths via UV-visible absorption spectroscopy and wholly indicative of the reaction kinetics. The photocatalytic activity areas of cuts of Activ (TM) glass, 10 cm x 10 cm in size, were assessed using both Rz and DCIP indicator inks evenly sprayed over the films: firstly using UVA lamp light to activate the underlying Activ (TM) film (1.75 mW cm(-2)) and secondly under solar conditions (2.06 +/- 0.14 mW cm(-2)). The photo-reduction reactions were monitored solely by flat-bed digital scanning. Red-green-blue values of a generated 14 x 14 grid (196 positions) that covered the entire area of each film image were extracted using a Custom-built program entitled RGB Extractor(C). A homogenous degradation over the 196 positions analysed for both Rz (Red colour deviation = 19% UVA, 8% Solar: Green colour deviation = 17% UVA, 12% Solar) and DCIP (Red colour deviation = 22% UVA, 16% Solar) inks was seen in both UVA and solar experiments, demonstrating the consistency of the self-cleaning titania layer on Activ (TM). The method presented provides a good solution for the high-throughput photocatalytic screening of a number of homogenous photocatalytically active materials simultaneously or numerous positions on a single film; both useful in assessing the homogeneity of a film or determining the best combination of reaction components to produce the optimum performance photocatalytic film. (C) 2010 Elsevier B.V. All rights reserved.

The VLT-FLAMES survey of massive stars: wind properties and evolution of hot massive stars in the Large Magellanic Cloud

We have studied the optical spectra of a sample of 28 O- and early B-type stars in the Large Magellanic Cloud, 22 of which are associated with the young star forming region N11. Our observations sample the central associations of LH9 and LH10, and the surrounding regions. Stellar parameters are determined using an automated fitting method ( Mokiem et al. 2005), which combines the stellar atmosphere code fastwind ( Puls et al. 2005) with the genetic algorithm based optimisation routine PIKAIA ( Charbonneau 1995). We derive an age of 7.0 +/- 1.0 and 3.0 +/- 1.0 Myr for LH9 and LH10, respectively. The age difference and relative distance of the associations are consistent with a sequential star formation scenario in which stellar activity in LH9 triggered the formation of LH10. Our sample contains four stars of spectral type O2. From helium and hydrogen line fitting we find the hottest three of these stars to be similar to 49- 54 kK ( compared to similar to 45- 46 kK for O3 stars). Detailed determination of the helium mass fraction reveals that the masses of helium enriched dwarfs and giants derived in our spectroscopic analysis are systematically lower than those implied by non-rotating evolutionary tracks. We interpret this as evidence for efficient rotationally enhanced mixing leading to the surfacing of primary helium and to an increase of the stellar luminosity. This result is consistent with findings for SMC stars by Mokiem et al. ( 2006). For bright giants and supergiants no such mass discrepancy is found; these stars therefore appear to follow tracks of modestly or non-rotating objects. The set of programme stars was sufficiently large to establish the mass loss rates of OB stars in this Z similar to 1/2 Z(circle dot) environment sufficiently accurate to allow for a quantitative comparison with similar objects in the Galaxy and the SMC. The mass loss properties are found to be intermediate to massive stars in the Galaxy and SMC. Comparing the derived modified wind momenta D-mom as a function of luminosity with predictions for LMC metallicities by Vink et al. ( 2001) yields good agreement in the entire luminosity range that was investigated, i.e. 5.0

A simple bioanalytical method for the quantification of antiepileptic drugs in dried blood spots

An increasing number of publications on the dried blood spot (DBS) sampling approach for the quantification of drugs and metabolites have been spurred on by the inherent advantages of this sampling technique. In the present research, a selective and sensitive high-performance liquid chromatography method for the concurrent determination of multiple antiepileptic drugs (AEDs) [levetiracetam (LVT), lamotrigine (LTG), phenobarbital (PHB)], carbamazepine (CBZ) and its active metabolite carbamazepine-10,11 epoxide (CBZE)] in a single DBS has been developed and validated. Whole blood was spotted onto Guthrie cards and dried. Using a standard punch (6. mm diameter), a circular disc was punched from the card and extracted with methanol: acetonitrile (3:1, v/v) containing hexobarbital (Internal Standard) and sonicated prior to evaporation. The extract was then dissolved in water and vortex mixed before undergoing solid phase extraction using HLB cartridges. Chromatographic separation of the AEDs was achieved using Waters XBridge™ C18 column with a gradient system. The developed method was linear over the concentration ranges studied with r=0.995 for all compounds. The lower limits of quantification (LLOQs) were 2, 1, 2, 0.5 and 1. µg/mL for LVT, LTG, PHB, CBZE and CBZ, respectively. Accuracy (%RE) and precision (%CV) values for within and between day were

Finding exoplanets orbiting young active stars - I. Technique

Stellar activity, such as starspots, can induce radial velocity (RV) variations that can mask or even mimic the RV signature of orbiting exoplanets. For this reason RV exoplanet surveys have been unsuccessful when searching for planets around young, active stars and are therefore failing to explore an important regime which can help to reveal how planets form and migrate. This paper describes a new technique to remove spot signatures from the stellar line-profiles of moderately rotating, active stars (v sin i ranging from 10 to 50 km s(-1)). By doing so it allows planetary RV signals to be uncovered. We used simulated models of a G5V type star with differing dark spots on its surface along with archive data of the known active star HD 49933 to validate our method. The results showed that starspots could be effectively cleaned from the line-profiles so that the stellar RV jitter was reduced by more than 80 per cent. Applying this procedure to the same models and HD 49933 data, but with fake planets injected, enabled the effective removal of starspots so that Jupiter mass planets on short orbital periods were successfully recovered. These results show that this approach can be useful in the search for hot-Jupiter planets that orbit around young, active stars with a v sin i of similar to 10-50 km/s.

A theoretical assessment of surface defect machining and hot machining of nanocrystalline silicon carbide

In this paper, a newly proposed machining method named “surface defect machining” (SDM) [Wear, 302, 2013 (1124-1135)] was explored for machining of nanocrystalline beta silicon carbide (3C-SiC) at 300K using MD simulation. The results were compared with isothermal high temperature machining at 1200K under the same machining parameters, emulating ductile mode micro laser assisted machining (µ-LAM) and with conventional cutting at 300 K. In the MD simulation, surface defects were generated on the top of the (010) surface of the 3C-SiC work piece prior to cutting, and the workpiece was then cut along the <100> direction using a single point diamond tool at a cutting speed of 10 m/sec. Cutting forces, sub-surface deformation layer depth, temperature in the shear zone, shear plane angle and friction coefficient were used to characterize the response of the workpiece. Simulation results showed that SDM provides a unique advantage of decreased shear plane angle which eases the shearing action. This in turn causes an increased value of average coefficient of friction in contrast to the isothermal cutting (carried at 1200 K) and normal cutting (carried at 300K). The increase of friction coefficient however was found to aid the cutting action of the tool due to an intermittent dropping in the cutting forces, lowering stresses on the cutting tool and reducing operational temperature. Analysis shows that the introduction of surface defects prior to conventional machining can be a viable choice for machining a wide range of ceramics, hard steels and composites compared to hot machining.

A high resolution study of complex organic molecules in hot cores

We present the results of a line identification analysis using data from the IRAM Plateau de Bure Inferferometer, focusing on six massive star-forming hot cores: G31.41+0.31, G29.96-0.02, G19.61-0.23, G10.62-0.38, G24.78+0.08A1 and G24.78+0.08A2. We identify several transitions of vibrationally excited methyl formate (HCOOCH$_3$) for the first time in these objects as well as transitions of other complex molecules, including ethyl cyanide (C$_2$H$_5$CN), and isocyanic acid (HNCO). We also postulate a detection of one transition of glycolaldehyde (CH$_2$(OH)CHO) in two new hot cores. We find G29.96-0.02, G19.61-0.23, G24.78+0.08A1 and 24.78+0.08A2 to be chemically very similar. G31.41+0.31, however, is chemically different: it manifests a larger chemical inventory and has significantly larger column densities. We suggest that it may represent a different evolutionary stage to the other hot cores in the sample, or it may surround a star with a higher mass. We derive column densities for methyl formate in G31.41+0.31, using the rotation diagram method, of $\times$10$^{17}$ cm$^{-2}$ and a T$_{rot}$ of $\sim$170 K. For G29.96-0.02, G24.78+0.08A1 and G24.78+0.08A2, glycolaldehyde, methyl formate and methyl cyanide all seem to trace the same material and peak at roughly the same position towards the dust emission peak. For G31.41+0.31, however, glycolaldehyde shows a different distribution to methyl formate and methyl cyanide and seems to trace the densest, most compact inner part of hot cores.

Gastroretentive Extended-Release Floating Granules Prepared Using a Novel Fluidized Hot Melt Granulation (FHMG) Technique

The objective of this work was to investigate the feasibility of using a novel granulation technique, namely, fluidized hot melt granulation (FHMG), to prepare gastroretentive extended-release floating granules. In this study we have utilized FHMG, a solvent free process in which granulation is achieved with the aid of low melting point materials, using Compritol 888 ATO and Gelucire 50/13 as meltable binders, in place of conventional liquid binders. The physicochemical properties, morphology, floating properties, and drug release of the manufactured granules were investigated. Granules prepared by this method were spherical in shape and showed good flowability. The floating granules exhibited sustained release exceeding 10 h. Granule buoyancy (floating time and strength) and drug release properties were significantly influenced by formulation variables such as excipient type and concentration, and the physical characteristics (particle size, hydrophilicity) of the excipients. Drug release rate was increased by increasing the concentration of hydroxypropyl cellulose (HPC) and Gelucire 50/13, or by decreasing the particle size of HPC. Floating strength was improved through the incorporation of sodium bicarbonate and citric acid. Furthermore, floating strength was influenced by the concentration of HPC within the formulation. Granules prepared in this way show good physical characteristics, floating ability, and drug release properties when placed in simulated gastric fluid. Moreover, the drug release and floating properties can be controlled by modification of the ratio or physical characteristics of the excipients used in the formulation.