Considerations for quantification of lipids in nerve tissue using matrix-assisted laser desorption/ionization mass spectrometric imaging

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging is a technique that provides the ability to identify and characterize endogenous and exogenous compounds spatially within tissue with relatively little sample preparation. While it is a proven methodology for qualitative analysis, little has been reported for its utility in quantitative measurements. In the current work, inherent challenges in MALDI quantification are addressed. Signal response is monitored over successive analyses of a single tissue section to minimize error due to variability in the laser, matrix application, and sample inhomogeneity. Methods for the application of an internal standard to tissue sections are evaluated and used to quantify endogenous lipids in nerve tissue. A precision of 5% or less standard error was achieved, illustrating that MALDI imaging offers a reliable means of in situ quantification for microgram-sized samples and requires minimal sample preparation.     

Determination of chemically reduced pyrrolobenzodiazepine SJG-136 in human plasma by HPLC-MS/MS: application to an anticancer phase I dose escalation study

SJG-136 1,1'-[[(propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-methylidene-1,2,3,11a-tetrahydro-5H-pyr- rolo[2,1-c][1,4]benzodiazepin-5-one]] (NSC 694501), is a bifunctional pyrrolobenzodiazepine (PBD) dimer that forms selective, irreversible, interstrand DNA cross-links via exocyclic N2 atoms of two guanine bases, with a preference for 5'PuGATCPy binding sites. SJG-136 is highly cytotoxic in human tumor cells in vitro and in human tumor xenograft models in vivo at subnanomolar concentrations and is currently in anticancer phase I clinical trials in the United Kingdom and United States. To support correlative pharmacokinetics studies, a highly sensitive HPLC-MS/MS assay was developed and validated for the reliable quantitation of SJG-136 in human plasma, using the structurally similar PBD dimer DSB-120 as an internal standard. Chemical reduction of SJG-136 to its corresponding amine (SJG-136-H(4), [M + H](+)m/z 561) improved HPLC peak resolution and sensitivity by minimizing complications that arose from the reactivity of the labile imine moieties. Plasma samples were processed by protein precipitation and centrifugal membrane dialysis; components were separated by HPLC using an Agilent Rapid Resolution HT 1.8 mm (2.1 mm x 50 mm) analytical column. The total analysis time from injection to injection was 11 min. Electrospray MS/MS detection of SJG-136-H(4) was based on the selected reaction monitoring (SRM) transition [M + H](+)m/z 561 --> 301. The analytical response ratio was linearly proportional to the plasma concentration of SJG-136 over the nominal concentration range of 25 pg/ml to 250 ng/ml, with a coefficient of determination of r > or = 0.999. The intrarun absolute %RE was < or =19.6, 14.2, and 14.0% at 0.056, 2.83, and 56.3 ng/ml, respectively. The corresponding %RSD was < or =14.9%, 9.01, and 4.59%. The interday %RSD was < or =2.72, 3.46, and 5.20%. The lower and upper limits of quantitation were 0.056 and 56 ng/ml, respectively; recovery of SJG-136 from plasma was > or = 62% across the validated concentration range. The sensitivity of the validated assay was sufficient to detect SJG-136 in human subjects for up to 6 h after intravenous administration of 6 microg/m(2), the starting dose of an NCI-sponsored dose escalation study.     

Selective Homogeneous Palladium(0)-Catalyzed Hydrogenation of Alkynes to (Z)-Alkenes

Zero-valent palladium precatalysts containing rigid bidentate bis(arylimino)acenaphthene ligands (shown schematically) facilitate the highly stereoselective homogeneous catalytic hydrogenation of alkynes to (Z)-alkenes. Internal, terminal, aryl-substituted, and cyclic alkynes are suitable substrates, as are some enynes, which are chemoselectively hydrogenated to dienes. E=CO(2)Me; R(1), R(2)=4-OCH(3), 4-CH(3), 2,6-(CH(3))(2).     

A critical investigation of the effects of the radio frequency potential on the trapping of externally injected ions in ion trap mass spectrometry

The sensitivity of all ion trap mass spectrometry (ITMS) methods is dependent on the trapping efficiency of the instrument. For ITMS instruments utilizing external ion sources, such as laser desorption, trapping efficiency is known to depend on the phase and amplitude of the radio frequency (RF) potential applied to the ring electrode at the time of ion introduction. It is remarkable that, in a considerable body of literature, no consensus exists regarding the effects of these parameters on the efficacy of trapping externally generated ions. In this paper, a summary of the literature is presented in order to highlight significant discrepancies. New laser desorption ion trap mass spectrometry (LD-ITMS) data are also presented, from which conclusions are drawn in our effort to clarify some of the confusion. Copyright 1999 John Wiley & Sons, Ltd.     

Photoelastic determination of stresses in multiple-pin connectors

The design, fabrication, and testing of photoelastic models of double-lap, multiple-pin connectors are discussed. Interest is in the stresses in the inner laps. These stresses are determined by constructing models with photoelastic inner laps and transparent-acrylic outer laps. The connectors have two pins, in tandem, parallel to the load direction. A photoelastic-isotropic point is shown to permit the evaluation of load sharing between the two pins. A numerical scheme, utilizing the isochromatic- and isoclinic-photoelastic data and a finite-difference representation of the planestress equilibrium equations, is used to compute the stresses around the two pins. Representative stress distributions and stress-concentration factors are shown.     

Quantitation of melatonin and n-acetylserotonin in human plasma by nanoflow LC-MS/MS and electrospray LC-MS/MS

Melatonin (MEL) and its chemical precursor N-acetylserotonin (NAS) are believed to be potential biomarkers for sleep-related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC-MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies, and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays, plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1?×?100 mm, 3.5?μm) or on a polyimide-coated, fused-silica capillary self-packed with 17?cm AquaC18 (3?μm, 125??). Quantitation was done using the SRM transitions m/z 233?→?174 and m/z 219?→?160 for MEL and NAS, respectively. The analytical response ratio versus concentration curves were linear for MEL (nanoflow LC: 11.7-1165 pg/mL, LC: 1165-116,500 pg/mL) and for NAS (nanoflow LC: 11.0-1095 pg/mL).     

Quantitation of melatonin and n‐acetylserotonin in human plasma by nanoflow LC‐MS/MS and electrospray LC‐MS/MS

Melatonin (MEL) and its chemical precursor N‐acetylserotonin (NAS) are believed to be potential biomarkers for sleep‐related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC‐MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies, and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays, plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1 × 100 mm, 3.5 µm) or on a polyimide‐coated, fused‐silica capillary self‐packed with 17 cm AquaC18 (3 µm, 125 Å). Quantitation was done using the SRM transitions m/z 233 → 174 and m/z 219 → 160 for MEL and NAS, respectively. The analytical response ratio versus concentration curves were linear for MEL (nanoflow LC: 11.7–1165 pg/mL, LC: 1165–116500 pg/mL) and for NAS (nanoflow LC: 11.0–1095 pg/mL). Copyright © 2012 John Wiley & Sons, Ltd.     

The NEMESIS planetary atmosphere radiative transfer and retrieval tool

With the exception of in situ atmospheric probes, the most useful way to study the atmospheres of other planets is to observe their electromagnetic spectra through remote observations, either from ground-based telescopes or from spacecraft. Atmospheric properties most consistent with these observed spectra are then derived with retrieval models. All retrieval models attempt to extract the maximum amount of atmospheric information from finite sets of data, but while the problem to be solved is fundamentally the same for any planetary atmosphere, until now all such models have been assembled ad hoc to address data from individual missions.In this paper, we describe a new general-purpose retrieval model, Non-linear Optimal Estimator for MultivariatE Spectral analySIS (NEMESIS), which was originally developed to interpret observations of Saturn and Titan from the composite infrared spectrometer on board the NASA Cassini spacecraft. NEMESIS has been constructed to be generally applicable to any planetary atmosphere and can be applied from the visible/near-infrared right out to microwave wavelengths, modelling both reflected sunlight and thermal emission in either scattering or non-scattering conditions. NEMESIS has now been successfully applied to the analysis of data from many planetary missions and also ground-based observations.     

Glutathionylated γG and γA subunits of hemoglobin F: a novel post‐translational modification found in extremely premature infants by LC‐MS and nanoLC‐MS/MS

Oxidative stress plays an important role in the development of various disease processes and is a putative mechanism in the development of bronchopulmonary dysplasia, the most common complication of extreme preterm birth. Glutathione, a major endogenous antioxidant and redox buffer, also mediates cellular functions through protein thiolation. We sought to determine if post‐translational thiol modification of hemoglobin F occurs in neonates by examining erythrocyte samples obtained during the first month of life from premature infants, born at 23 0/7 – 28 6/7 weeks gestational age, who were enrolled at our center in the Prematurity and Respiratory Outcomes Program (PROP). Using liquid chromatography‐mass spectrometry (LC‐MS), we report the novel finding of in vivo and in vitro glutathionylation of γG and γA subunits of Hgb F. Through tandem mass spectrometry (nanoLC‐MS/MS), we confirmed the adduction site as the Cys‐γ94 residue and through high‐resolution mass spectrometry determined that the modification occurs in both γ subunits. We also identified glutathionylation of the β subunit of Hgb A in our patient samples; we did not find modified α subunits of Hgb A or F. In conclusion, we are the first to report that glutathionylation of γG and γA of Hgb F occurs in premature infants. Additional studies of this post‐translational modification are needed to determine its physiologic impact on Hgb F function and if sG‐Hgb is a biomarker for clinical morbidities associated with oxidative stress in premature infants. Copyright © 2014 John Wiley & Sons, Ltd.     

Investigation of new band parameters with temperature dependence for self-broadened methane gas in the range 9000 to 14,000 cm−1 (0.71 to 1.1 μm)

This paper describes new measurements and modelling of the absorption of methane gas, one of the most important gases observed in the atmospheres of the outer planets and Titan, between 9000 and 14,000 cm^?1 (0.7 to 1.1 μm) and compares them with current best available spectral models.A series of methane spectra were measured at the UK's Natural Environment Research Council (NERC) Molecular Spectroscopy Facility (based at the Rutherford Appleton Laboratory, Oxfordshire, UK) using a Brüker 125HR Fourier transform spectrometer. To approximate the conditions found in outer planet atmospheres, the spectra were measured over a wide range of pressures (5 bar to 38 mbar) and temperatures (290–100 K) with path lengths of 19.3, 17.6, 16.0 and 14.4 m. The spectra were recorded at a moderate resolution of 0.12 cm^?1 and then averaged to 10 cm^?1 resolution prior to fitting a series of increasingly complex band-models including temperature dependence. Using the most complex model, a Goody line distribution with a Voigt line shape and two lower energy state levels, the typical rms residual error in the fit is between 0.01 and 0.02 in the wings of the main absorption bands.The new spectral parameters were then compared with the measured spectra and spectra calculated using existing data and shown to be able to accurately reproduce the measured absorption. The improvement in the temperature dependence included in the model is demonstrated by comparison with existing cold methane spectral data for a typical Jovian path.