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.

Optimization of microflow LC-MS/MS and its utility in quantitative discovery bioanalysis.

Aim: The sensitivity advantage of microflow LC (µFLC)-MS/MS is potentially impactful for challenging compounds not detectable by conventional flow LC-MS/MS in drug discovery bioanalysis. Relatively new to µFLC technology, discovery bioanalytical scientists would benefit from an effective strategy for method development and optimization. Results: A systematic µFLC-MS/MS method optimization approach was developed in this study. With optimized conditions, µFLC-MS/MS demonstrated an improved sensitivity compared with conventional LC-MS/MS analysis, ranging from 6× to 49× (by peak area) depending on the compounds, with acceptable analytical performance and robustness. The optimized conditions demonstrated universal applicability to various compounds of diverse properties. Conclusion: The systematic method optimization strategy, and the general applicability of the optimized conditions could facilitate the routine utilization of µFLC in quantitative discovery bioanalysis.

Simultaneous Detection of 13 Allergens in Thermally Processed Food Using Targeted LC-MS/MS Approach.

Food allergy is a major concern for public health and food industries. Because of the large numbers of food ingredients to be tested, MS is considered an alternative to existing techniques in terms of high selectivity, sensitivity, and capability to analyze multiple allergens simultaneously. In this study, we developed the method for monitoring significant peptides derived from 13 food allergens (milk, eggs, cod, shrimp, lobster, almonds, brazil nuts, cashew nuts, hazelnuts, walnuts, peanuts, wheat, and soybeans) and evaluated it in thermally processed foods (bread, cookie, fried fish, and frozen pasta). To select significant peptides to monitor, we used a bioinformatics-based approach and experimental confirmatory analysis. It was demonstrated that the developed method could detect target food ingredients from thermally processed foods successfully.

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.

Method for the Compound Annotation of Conjugates in Nontargeted Metabolomics Using Accurate Mass Spectrometry, Multistage Product Ion Spectra and Compound Database Searching.

Owing to biotransformation, xenobiotics are often found in conjugated form in biological samples such as urine and plasma. Liquid chromatography coupled with accurate mass spectrometry with multistage collision-induced dissociation provides spectral information concerning these metabolites in complex materials. Unfortunately, compound databases typically do not contain a sufficient number of records for such conjugates. We report here on the development of a novel protocol, referred to as ChemProphet, to annotate compounds, including conjugates, using compound databases such as PubChem and ChemSpider. The annotation of conjugates involves three steps: 1. Recognition of the type and number of conjugates in the sample; 2. Compound search and annotation of the deconjugated form; and 3. In silico evaluation of the candidate conjugate. ChemProphet assigns a spectrum to each candidate by automatically exploring the substructures corresponding to the observed product ion spectrum. When finished, it annotates the candidates assigning a rank for each candidate based on the calculated score that ranks its relative likelihood. We assessed our protocol by annotating a benchmark dataset by including the product ion spectra for 102 compounds, annotating the commercially available standard for quercetin 3-glucuronide, and by conducting a model experiment using urine from mice that had been administered a green tea extract. The results show that by using the ChemProphet approach, it is possible to annotate not only the deconjugated molecules but also the conjugated molecules using an automatic interpretation method based on deconjugation that involves multistage collision-induced dissociation and in silico calculated conjugation.

Nano-scale liquid chromatography coupled to tandem mass spectrometry using the multiple reaction monitoring mode based quantitative platform for analyzing multiple enzymes associated with central metabolic pathways of Saccharomyces cerevisiae using ultra fast mass spectrometry.

A widely targeted quantitative proteome analysis of Saccharomyces cerevisiae enzymes was performed employing an ultra fast mass spectrometry (UFMS). Nano-liquid chromatography-UFMS analysis of trypsin digested peptide samples derived from yeast strains successfully determined relative abundances of 303 peptides of 137 proteins.

Development of a practical metabolite identification technique for non-targeted metabolomics.

Metabolite identification is one of the major challenges of non-targeted metabolomics involving liquid chromatography coupled with mass spectrometry (LC-MS). Compound databases contain enormous numbers of records, which makes compound identification difficult in practice because each search will return a large number of candidates. We therefore developed a practical compound identification system using LC-MS with high mass accuracy and MS(n) capability, combined with a compound database. A large number of candidates were evaluated by score calculation based on a combination of formulae and spectral assignments. Here, we demonstrate this method using green tea extract as a model sample. We applied our approach to predict the structures of compounds of interest, and the correct identification of several candidates was confirmed by comparisons to analysis of chemical standards.