Bioanalysis of acetylcarnitine in cerebrospinal fluid by HILIC–mass spectrometry

Acetyl‐l ‐carnitine (ALCAR) is a potential biomarker for the modulation of brain neurotransmitter activity, but is also present in cerebrospinal fluid (CSF). Recent studies have utilized hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC‐MS/MS) based assays to detect and quantify ALCAR within biofluids such as urine, plasma and serum, using various sample pretreatment procedures. In order to address the need to quantify ALCAR in CSF on a high‐throughput scale, a new and simple HILIC‐MS/MS assay has been successfully developed and validated. For rapid analysis, CSF sample pretreatment was performed via ‘dilute and shoot’ directly onto an advanced HILIC column prior to MS/MS detection. This newly developed HILIC‐MS/MS assay shows good recoveries of ALCAR without the need for chemical derivatization and multistep sample extraction procedures. The employment of this assay is suitable for the high‐throughput bioanalysis and quantification of ALCAR within the CSF of various animal models and human clinical studies. Copyright © 2015 John Wiley & Sons, Ltd.     

Energy and momentum in chiral theories

We calculate the energy momentum tensor to orderE ⁴ in chiral perturbation theory. New terms not present in previous work enter the effective Lagrangian. We describe these and estimate the values of the new coupling constants, using the results of a disperisve analysis of the π andK energy momentum tensors and relying on tensor meson dominance for the spin two component. In addition, we compare our findings with the predictions of known scalar meson dominance models of the conformal anomaly.     

Tuning the singlet oxygen quantum yield of near-IR-absorbing porphyrazines

We report the quantum yields for singlet oxygen production by a series of porphyrazines (pz) of the form M[pz(An;B4-n)] (Scheme 1), where the peripheral substituent A is [S-R]2 with R = (CH2CH2O)3H, B is a fused alpha,alpha'-dialkoxybenzo group and M = 2H, Mg or Zn. These compounds show intense near-IR absorbance/emission (longest wavelength emission, approximately 830 nm). Their solubilities vary with R, whereas their optical properties do not. We show that singlet oxygen sensitization by these luminescent compounds can be "tuned" from essentially off to on by varying n and selection among M = 2H, Mg or Zn. The quantum yields vary ca 60-fold within the set of compounds studied, from phidelta = 0.007 for compound 3 to phidelta = approximately 0.4 for compound 11.     

Gallium cluster "magic melters"

Calorimetry measurements (using a method based on multicollision induced dissociation) have been performed for unsupported gallium clusters, Gan+ (n = 30-50 and 55). Melting transitions have been identified from spikes in the heat capacities recorded as a function of temperature. There are enormous fluctuations in the melting temperatures and the heats of fusion with cluster size. Clusters with n = 31, 33, 37, and 45-47 are "magic melters" with particularly well-defined melting transitions. There is a strong correlation between the heats of fusion, entropies of fusion, and the stabilities of the clusters. However, these quantities are not strongly correlated with the melting temperatures.     

Detection of single nucleotide polymorphisms using electrospray ionization mass spectrometry: validation of a one-well assay and quantitative pooling studies

Single nucleotide polymorphisms (SNPs) are currently being mapped and databased at a remarkable pace, providing a viable means for understanding disease susceptibility, differential drug response and human evolution. Consequently, there is an increasing demand for SNP genotyping technologies that are simple, rapid, cost effective and readily amenable to automation for high-throughput analyses. In this study, we improved the Survivor Assay, a SNP detection method based on electrospray ionization mass spectrometry (ESI-MS), with several developments. One improvement is the development of a one-well assay, requiring no off-line purification of the polymerase chain reaction product, achieved by simple addition of reagent solution into a single well. Another is the on-line separation of magnesium and dideoxynucleotides using an in-house made monolithic metal chelating column, eliminating any off-line sample preparation prior to mass spectrometric analysis. Here the Survivor Assay is extended from a proof-of-principle concept to a validated method by genotyping six SNPs from five different regions of human genomic DNA in 55 individual samples with 100% accuracy. This improved Survivor Assay eliminates the tedious and time-consuming steps of sample preparation, minimizes sample handing and offers a high-throughput analysis of SNPs by ESI-MS. The current combined preparation and analysis time is 2 min per sample. The simplicity of this method has potential for full automation and parallel chromatography and, thus, reduced analysis time. In addition, we have adapted the Survivor Assay for quantitative SNP analysis in pooled DNA samples. The capabilities and sensitivity of this approach were evaluated. We demonstrate that an allele occurring at a frequency of 2% can consistently be quantitated.     

A fully automated nanoelectrospray tandem mass spectrometric method for analysis of Caco-2 samples

Caco-2 cells offer a means to rapidly screen permeability of drug candidates, allowing pharmaceutical companies to eliminate candidates unable to cross the intestinal barrier early in the discovery process. This screening process is typically performed by conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS), which can require time-consuming method development. An alternative to LC/MS/MS, automated nanoelectrospray tandem mass spectrometry (nanoESI-MS/MS), is introduced. This novel approach requires an off-line ZipTip desalting step followed by automated nanoESI-MS/MS, using the NanoMate 100 and ESI Chip. In addition to reduced method development time, automated nanoESI-MS/MS also offers no carry-over between samples, low sample consumption, and ease-of-use as compared with conventional pulled-capillary nanoelectrospray. Furthermore, the infusion system described has the potential to be high-throughput. A comparison of Caco-2 samples analyzed both by LC/MS/MS and by automated nanoESI-MS/MS is presented. The permeability and recovery data of the two compounds analyzed in this study obtained from conventional LC/MS/MS and by automated nanoESI-MS/MS were in excellent agreement.     

Lens alpha-crystallin and hypericin: a photophysical mechanism explains observed lens

Determining whether alpha-crystallin (the major lens protein) affects the photophysics of hypericin, a photosensitizing agent found in various plants, such as St. John's Wort, is important. Hypericin shows promise in cancer and human immunodeficiency virus therapy but may harm individuals taking St. John's Wort extracts (for mild to moderate depression). Hypericin causes hypericism, which is characterized by cellular damage in light-exposed areas. Ocular tissues are at risk for photosensitized damage; thus, we investigated the effects on hypericin photophysics by alpha-crystallin. We measured the transient absorption spectra and the 1270 nm luminescence of singlet (1Deltag) oxygen produced from hypericin in the presence of alpha-crystallin. alpha-Crystallin complexes hypericin, extending the lifetime of its triplet excited state; the Stern-Volmer slope is negative, but not linear, after a saturation curve. Damage to the lens protein by hypericin is known to occur via singlet oxygen, which oxidizes methionine, tryptophan and histidine residues. Binding to alpha-crystallin does not inhibit singlet oxygen formation by hypericin. alpha-Crystallin reacts with singlet oxygen with a rate constant of 1.3 x 10(8) M(-1) s(-1). Thus, we anticipate that hypericin will be an effective photosensitizer in the lens.