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.     

Middle-ear response in the chinchilla and its relationship to mechanics at the base of the cochlea

The responses of the malleus and the stapes to sinusoidal acoustic stimulation have been measured in the middle ears of anesthetized chinchillas using the M?ssbauer technique. With "intact" bullas (i.e., closed except for venting via capillary tubing), the vibrations of the tip of the malleus reach a maximal peak velocity of about 2 mm/s in responses to 100-dB SPL tones in the frequency range 500-6000 Hz; vibration velocity diminishes toward lower frequencies with a slope of about 6 dB/oct. Opening the bulla widely increases the responses to low-frequency stimuli by as much as 16 dB. At low frequencies, malleus response sensitivity with either open or intact bullas far exceeds all previous measurements in cats and matches or exceeds such measurements in guinea pigs. Whether measured in open or intact bullas, phase-versus-frequency curves closely approximate those predicted from the magnitude-versus-frequency curves by minimum phase theory. The stapes responses are similar to those of the malleus, except that stapes response magnitude is lower, on the average, by 7.5 dB at frequencies below 2 kHz and 10.7 dB at 2 kHz and above. Comparison of the responses of the middle ear with those of the basilar membrane at a site 3.5 mm from the stapes indicates that, at frequencies below 150 Hz, the basilar membrane displacement is proportional to stapes acceleration. At frequencies between 150 and 2000 Hz, basilar membrane displacement is proportional to stapes velocity.     

Cochlear microphonics and the initiation of spikes in the auditory nerve: correlation of single-unit data with neural and receptor potentials recorded from the round window

On the basis of comparisons of responses of guinea pig ganglion cells and inner hair cells to intense low-frequency tones, Sellick et al. [Hear. Res. 7, 199-221 (1982)] have proposed that basal inner hair cells can be depolarized (and thus, VIII-N. spikes generated) by the extracellular microphonic generated during hyperpolarization of outer hair cells. VIII-N. data for the chinchilla have been presented that, to a first approximation, support such a hypothesis [Ruggero and Rich, J. Acoust. Soc. Am. 73, 2096-2108 (1983)]. However, an apparent discrepancy exists in our results, vis à vis Sellick et al.'s hypothesis, in that basal fiber near-threshold responses precede maximal negativity of the round window microphonic (i.e., maximal hyperpolarization of outer hair cells) by up to 90 degrees (but generally less than 45 degrees), depending on frequency. It is shown here that the discrepancy is resolved if certain nonlinear phase changes and overall distortion of the microphonic waveshapes, both of which occur at intense stimulus levels, are taken into account. It is also shown that compound action potentials (AP's), superimposed on the round window microphonics, can be identified at multiple times within each stimulus cycle, closely matching the near-threshold response phases of single-unit excitation. AP1 is nearly synchronous with the negative-to-positive transition of round window microphonics and with the excitation of fibers innervating apical-to-middle cochlear regions. AP2 is synchronous with the positive-to-negative transition of the microphonics and with the excitation of basal fibers. One or two other AP's probably reflect "peak splitting" in the responses of both basal and apical fibers.     

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.     

Convenient synthesis of nucleoside and isonucleoside analogues

[reaction: see text]. A very simple methodology to stereoselectively achieve tricyclic isonucleosides (nucleobase = thymine, uracil, and 5-fluoruracil) and 3'-C-branched nucleosides (nucleobase = theophylline) was performed by means of a DBU-mediated addition process using a readily available 2-bromo sugar. The mechanism for these transformations implies the loss of both substituents at C-2 and C-3 on the sugar moiety, and although it seems that DBU is probably involved, its involvement has not yet been ascertained. Cytosine did not react under these conditions.     

A highly diastereoselective synthesis of 4-octulose and 2-deoxy-4-octulose from a -fructose derivative

Bishydroxylation of methyl

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1 with osmium tetraoxide proceeded with extremed high diastereoselectivity to give only methyl

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2. Configurations of the new stereogenic centers (C-2,3) in 2 were determined by degradation of the C-5,6,7,8 fragment to the well-known methyl

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7. Transformation of 2 into the required

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10, was achieved by a methodology that implied, protection to 8, reduction of the ester group in 8 to a hydroxymethyl group in 9, and finally deprotection to the free

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10. On the other hand, epoxidation reaction on

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11 afforded only the corresponding 2,3-anhydro derivative 12 with configuration, as could be demonstrated by degradation to (S)-1,2,4-trimetoxybutane 16, which synthesis is reported herein.     

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.     

Determination of cadmium in biological and environmental samples by slurry electrothermal atomic absorption spectrometry

The retention of cadmium by the bacteria Escherichia coli and Pseudomonas putida was optimized in order to develop a rapid and selective preconcentration method for cadmium from biological and environmental samples prior to determination by electrothermal atomic absorption spectrometry. Living and lyophilized cells for both bacteria were used, but the method using dead cells shows better analytical capabilities. Cadmium from aqueous solutions is easily retained on the outer membrane of both bacteria in the pH range 4-10, although the selected working pHs for E. coli and P. putida were 5 and 9, respectively. Cadmium retained by the bacteria was dispersed in 3.5 M nitric acid and the slurry was introduced directly into the graphite tube. The best sensitivity and detection limit were obtained for E. coli (0.03 ng ml(-1) and 0.04 ng ml(-1) respectively, in the absence of any chemical modifier). A strong spectral interference from nickel chloride was found and methods to overcome it were developed. The proposed extraction procedure was tested by the determination of cadmium in different standard biological and environmental samples.