Absolute quantification of nicotinamide mononucleotide in biological samples by double isotope-mediated liquid chromatography-tandem mass spectrometry (dimeLC-MS/MS).

Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite for fundamental biological phenomena, including aging. Nicotinamide mononucleotide (NMN) is a key NAD+ intermediate that has been extensively tested as an effective NAD+-boosting compound in mice and humans. However, the accurate measurement of NMN in biological samples has long been a challenge in the field. Here, we have established an accurate, quantitative methodology for measuring NMN by using liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) with double isotopic NMN standards. In this new methodology, the matrix effects of biological samples were properly adjusted, and the fate of NMN could be traced during sample processing. We have demonstrated that this methodology can accurately quantitate NMN levels in mouse plasma and confirmed quick, direct NMN uptake into blood circulation and cells. This double isotope-mediated LC-MS/MS (dimeLC-MS/MS) can easily be expanded to other NAD+-related metabolites as a reliable standard methodology for NAD+ biology.

Solid-phase microextraction- probe electrospray ionization devices for screening and quantitating drugs of abuse in small amounts of biofluids.

Probe electrospray ionization (PESI) is an ambient ionization mass spectrometry technique (AIMS) that is primarily used in qualitative studies, though researchers have recently combined it with sample preparation for the quantitative analysis of various analytes in biological matrices. This study presents a method that integrates solid-phase microextraction with PESI for direct coupling to a triple quadrupole mass spectrometer, and examines its ability to quantitate drugs of abuse. Intra- and inter-probe reproducibility experiments were conducted to assess the stability and reproducibility of the extraction-phase-coated PESI probes (coating length: 2 mm; coating thickness: 6.5 µm). This research is the first documented instance wherein highly sensitive determinations were successfully attained using these microextraction and micro-desorption techniques in conjunction with small volumes of sample and extraction phase. A mixture consisting of IPA/H2O (1/1 v/v) + 0.1% FA was determined to be the optimal desorption solvent for SPME-PESI-MS/MS, as it facilitated high analyte enrichment in a picolitre of the solvent, which acted at the same time as efficient electrospray media. Furthermore, a method of quantifying drugs of abuse in 30 µL of plasma without matrix modification was also developed. This method had an intra-day accuracy within the 80-120% range for all eight drugs of abuse at concentrations of 3, 30, and 90 pg µL-1; the exception to this result was lorazepam at 30 pg µL-1, which had an intra-day accuracy of 122%. The lower limit of quantification (LLOQ) for fentanyl and nordiazepam was pg µL-1; the LLOQ for buprenorphine, codeine, diazepam, lorazepam, and propranolol was 5 pg µL-1; and the LLOQ of oxazepam was 10 pg µL-1.

Multivariate approach to on-line supercritical fluid extraction - supercritical fluid chromatography - mass spectrometry method development.

Coupling supercritical fluid extraction (SFE) on-line with supercritical fluid chromatography (SFC) - tandem mass spectrometry (MS/MS) provides a single platform for efficient extraction, separation, and detection in a chemical analysis. SFE-SFC-MS/MS requires consideration of many extraction and chromatographic variables to not only provide the most efficient extraction, but also to analytically transfer the extracted analytes to the column for separation. There is a fundamental lack of understanding of how the variables in SFE affect those in SFC. Typically, a univariate approach is taken in on-line SFE-SFC-MS/MS method development, but this provides little insight into the relative importance of variables and their potential interactions. Here, a multivariate approach was used to develop a better understanding of the synergistic relationship between the extraction and separation processes by focusing on the optimization of extraction parameters for target analytes with a wide range of physicochemical properties in matrices of variable retentivity. The methodology used a set of optimal on-line SFE-SFC-MS/MS extraction parameters for 18 analytes of variable physicochemical properties in three different silica gel-based sample matrices are presented.

Photoinduced Ligand Release from a Silicon Phthalocyanine Dye Conjugated with Monoclonal Antibodies: A Mechanism of Cancer Cell Cytotoxicity after Near-Infrared Photoimmunotherapy.

Photochemical reactions can dramatically alter physical characteristics of reacted molecules. In this study, we demonstrate that near-infrared (NIR) light induces an axial ligand-releasing reaction, which dramatically alters hydrophilicity of a silicon phthalocyanine derivative (IR700) dye leading to a change in the shape of the conjugate and its propensity to aggregate in aqueous solution. This photochemical reaction is proposed as a major mechanism of cell death induced by NIR photoimmunotherapy (NIR-PIT), which was recently developed as a molecularly targeted cancer therapy. Once the antibody-IR700 conjugate is bound to its target, activation by NIR light causes physical changes in the shape of antibody antigen complexes that are thought to induce physical stress within the cellular membrane leading to increases in transmembrane water flow that eventually lead to cell bursting and necrotic cell death.

On-line supercritical fluid extraction-supercritical fluid chromatography-mass spectrometry of polycyclic aromatic hydrocarbons in soil.

On-line supercritical fluid extraction - supercritical fluid chromatography - mass spectrometry (SFE-SFC-MS) has been applied for the determination of polycyclic aromatic hydrocarbons (PAHs) in soil. The purpose of this study was to develop and validate the first on-line SFE-SFC-MS method for the quantification of PAHs in various types of soil. By coupling the sample extraction on-line with chromatography and detection, sample preparation is minimized, diminishing sample loss and contamination, and significantly decreasing the required extraction time. Parameters for on-line extraction coupled to chromatographic analysis were optimized. The method was validated for concentrations of 10-1500 ng of PAHs per gram of soil in Certified Reference Material (CRM) sediment, clay, and sand with R2 ≥ 0.99. Limits of detection (LOD) were found in the range of 0.001-5 ng/g, and limits of quantification (LOQ) in the range of 5-15 ng/g. The method developed in this study can be effectively applied to the study of PAHs in the environment, and may lay the foundation for further applications of on-line SFE-SFC-MS.

[Case of Rh (-) patient's right lobectomy of the liver with massive hemorrhage evading allogeneic blood transfusion by hemodilutional autologous blood transfusion].

A 44-year-old man (ASA-PS 1) underwent right lobectomy of the liver under total intravenous anesthesia with propofol, remifentanil, ketamine and rocuronium. In order to evade allogeneic blood transfusion, 1,200 g of the patient's blood was taken and hemodilution was induced for autologous blood transfusion (HAT) after the induction of anesthesia. As intraoperative blood loss amounted to about 4,000 g, Hb level decreased from 13.6 to 6.2 g x dl(-1). However, as intraoperative hemodynamics was relatively stable with crystalloidal and colloidal transfusion with no ischemic change on ECG and no metabolic acidosis, autologous blood transfusion was withheld. After returning the autologous blood, Hb increased to 9.8 g x dl(-1). Any postoperative complications related to the low Hb level were not recognized. HAT is a useful method to evade or at least decrease the amount of allogeneic blood transfusion by anesthesiologists.

Atf1 is a target of the mitogen-activated protein kinase Pmk1 and regulates cell integrity in fission yeast.

In fission yeast, knockout of the calcineurin gene resulted in hypersensitivity to Cl(-), and the overexpression of pmp1(+) encoding a dual-specificity phosphatase for Pmk1 mitogen-activated protein kinase (MAPK) or the knockout of the components of the Pmk1 pathway complemented the Cl(-) hypersensitivity of calcineurin deletion. Here, we showed that the overexpression of ptc1(+) and ptc3(+), both encoding type 2C protein phosphatase (PP2C), previously known to inactivate the Wis1-Spc1-Atf1 stress-activated MAPK signaling pathway, suppressed the Cl(-) hypersensitivity of calcineurin deletion. We also demonstrated that the mRNA levels of these two PP2Cs and pyp2(+), another negative regulator of Spc1, are dependent on Pmk1. Notably, the deletion of Atf1, but not that of Spc1, displayed hypersensitivity to the cell wall-damaging agents and also suppressed the Cl(-) hypersensitivity of calcineurin deletion, both of which are characteristic phenotypes shared by the mutation of the components of the Pmk1 MAPK pathway. Moreover, micafungin treatment induced Pmk1 hyperactivation that resulted in Atf1 hyperphosphorylation. Together, our results suggest that PP2C is involved in a negative feedback loop of the Pmk1 signaling, and results also demonstrate that Atf1 is a key component of the cell integrity signaling downstream of Pmk1 MAPK.

[Overview of cardiac contrast examination using 64-row multislice CT].

ECG-gated multislice CT(MSCT)now supports not only morphological but also functional diagnosis. In order to improve accuracy in the functional analysis of cardiac motion, it is important to depict the endocardial and epicardial contours. The present study was conducted to evaluate a method in which contrast medium and physiological saline solution(saline)are injected simultaneously, and the iodinated contrast medium concentration is varied in a stepwise manner(multi-stage injection)or continuously(cross injection). The results showed that the cross-injection method exhibited small differences in myocardial CT values between the right and left heart and was thought to be effective for automatic region extraction of the cardiac chambers and myocardium. However, cross-injection requires saline and contrast medium to be injected in equal volumes, necessitating a higher injection speed. Therefore, a newly developed trapezoidal cross-injection method was included in the study as an improved method. The results showed that trapezoidal cross injection provided high CT values. The relationship between the patient's body weight and the amount of iodinated contrast medium used also was investigated, and a negative correlation was observed for all three injection methods: multi-stage injection(r=0.63), cross injection(r=0.54), and trapezoidal cross injection(r=0.8). The trapezoidal cross-injection method showed the strongest correlation.

Giant macrocycles composed of thiophene, acetylene, and ethylene building blocks.

Fully conjugated giant macrocyclic oligothiophenes with 60pi, 90pi,120pi, 150pi, and 180pi frames (1, 2, 3, 4 and 5) have been designed, and their butyl-substituted derivatives (1a, 2a, 3a, 4a, and 5a) have been synthesized using modified Sonogashira and McMurry coupling reactions as key steps. The 60-180pi systems 1-5 are circular with 1.8-6 nm inner cavities and 3.3-7.5 nm outside molecular diameters. Compound 1a containing ten 3,4-dibutyl-2,5-thienylene, eight ethynylene, and two vinylene units has been converted into macrocyclic oligo(3,4-dibutyl-2,5-thienylene-ethynylene) 6a using bromination/dehydrobromination procedure. Giant macrocycles 1a-6a exhibit a red shift of their absorption spectra and a fairly strong fluorescence with a large Stokes shift as compared to a linear conjugated counterpart having five thiophene rings. Compounds 1a-6a exhibit multistep reversible redox behaviors with fairly low first oxidation potentials, reflecting their cyclic conjugation. Furthermore, chemical oxidation of 1a-6a with FeCl3 shows drastic changes of spectroscopic properties due to intramolecular and intermolecular pi-pi interactions. Doping of 1a-3a with iodine forms semiconductor due to its pi-donor properties and pi-pi stacking ability. X-ray analysis of 1a confirmed a round, planar structure with nanoscale inner cavity, and revealed host ability for alkanes and unique packing structure. Interestingly, 2a and 3a self-aggregate in the solid state to form "molecular wires," which are about 200 nm thick and more than 1 mm long. The internal structures of fibrous aggregates have been investigated by optical microscope, scanning electron microscopy, atomic force microscopy, and X-ray diffraction analyses.

Fully automated micro- and nanoscale one- or two-dimensional high-performance liquid chromatography system for liquid chromatography-mass spectrometry compatible with non-volatile salts for ion exchange chromatography.

A one- or two-dimensional high performance liquid chromatography system for electrospray ionization mass spectrometers has been developed that is optimized for ion exchange and reversed phase separations. A unique and simple valve configuration permits the use of a variety of non-volatile salts; ammonium sulfate was used in an example of strong cation exchange separations. The system was designed and evaluated for both micro- and nanoflow chromatography. The peptide detection limit was approximately 100 fmol for micro- and 20 fmol for nanoflow, demonstrating the concentration and mass sensitivity improvements expected with nanoelectrospray ionization. The 1D/2D-HPLC MS system is fully automated for routine peptide analyses, compatible with direct injection of proteolytic digests, and exhibits chromatographic reproducibility and sensitivity. Software permits operator selection of either a 1D or 2D configuration with corresponding system parameters as required for individual samples. The hardware elements and resulting performance are described in this paper.