Application of graphite carbon black assisted-laser desorption ionization-mass spectrometry for soy sauce product discrimination.

In a previous study, we developed a novel analytical method to directly and simultaneously detect taste- and odor-active compounds using graphite carbon black (GCB)-assisted laser desorption ionization mass spectrometry (LDI-MS). In this study, we aimed to evaluate food quality using a variety of soy sauces using the method to discriminate each product. Graphite carbon black-laser desorption ionization-mass spectrometry allowed the provision of hundreds of MS peaks derived from soy sauces in both positive and negative modes without any tedious sample pretreatments. Principal component analysis using the obtained MS peaks clearly distinguished three soy sauce products based on the manufacturing countries (Japan, China, and India). Moreover, this method identified distinct MS peaks for discrimination, which significantly correlated with their quantitative amounts in the products. Thus, GCB-LDI-MS analysis was established as a simple and rapid technique for food analysis, illustrating the chemical patterns of food products.

Pharmacokinetic profiles of 3-(4-hydroxy-3-methoxyphenyl) propionic acid and its conjugates in Sprague-Dawley rats.

3-(4-hydroxy-3-methoxyphenyl)propionic acid (HMPA) is one of the end-products from gut microbiota from dietary polyphenols, which might contribute to their health benefits. This study aims to investigate the absorption, metabolism, and tissue accumulation of HMPA in Sprague-Dawley (SD) rats. After HMPA (10 mg/kg body weight) was orally administered, intact and conjugated HMPAs in the bloodstream were detected and reached the maximum concentration in 15 min (HMPA, 2.6 ± 0.4 nmol/mL; sulfated HMPA, 3.6 ± 0.9 nmol/mL; glucuronidated HMPA, 0.55 ± 0.09 nmol/mL). HMPA and its conjugates were also detected in the target organs 6 h postadministration, indicating that HMPA undergoes rapid conversion into conjugates, and they broadly distribute to organs with similar profiles (kidneys > liver > thoracic aorta > heart > soleus muscle > lungs). This study demonstrated that orally administered HMPA (10 mg/kg) in SD rats undergoes rapid metabolism and wide tissue distribution with ≥1.2% absorption ratio.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging of Tissues via the Formation of Reproducible Matrix Crystals by the Fluorescence-Assisted Spraying Method: A Quantification Approach.

The application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging to quantitative analyses is restricted by the variability of MS intensity of the analytes in nonreproducible matrix crystals of tissues. To overcome this challenge, a fluorescence-assisted spraying method was developed for a constant matrix amount employing an MS-detectable fluorescent reagent, rhodamine 6G (R6G), which was sprayed with the matrix. To form a homogeneous matrix crystal on the tissue section, a matrix solution, 1,5-diaminonaphthalene (10 mg/mL), containing R6G (40 µg/mL) and O-dinitrobenzene (O-DNB, 10 mg/mL) was sprayed until the desired constant fluorescence intensity was achieved. Compared with that obtained via conventional cycle-number-fixed spraying [relative standard deviation (RSD) = 31.1%], the reproducibility of the relative MS intensity of the analyte [ferulic acid (FA), RSD = 3.1%] to R6G was significantly improved by the fluorescence-assisted matrix spraying. This result indicated that R6G could be employed as an index of the matrix amount and an MS normalizing standard. The proposed matrix spraying successfully quantified nifedipine (0.5-40 pmol/mm2 in the positive mode, R2 = 0.965) and FA (0.5-75 pmol/mm2 in the negative mode, R2 = 0.9972) in the kidney section of a rat. Employing the quantitative MALDI-MS imaging assay, FA, which accumulated in the kidney of the rat after 50 mg/kg was orally administered, was visually determined to be 3.5, 3.0, and 0.2 µmol/g tissue at 15, 30, and 60 min, respectively.

Accumulation of Plasma-Derived Lipids in the Lipid Core and Necrotic Core of Human Atheroma: Imaging Mass Spectrometry and Histopathological Analyses.

Objective: To clarify the pathogenesis of human atheroma, the origin of deposited lipids, the developmental mechanism of liponecrotic tissue, and the significance of the oxidation of phospholipids were investigated using mass spectrometry-aided imaging and immunohistochemistry.Atherosclerotic lesions in human coronary arteries were divided into 3 groups: pathologic intimal thickening with lipid pool, atheroma with lipid core, and atheroma with necrotic core. The lipid pool and lipid core were characterized by the deposition of extracellular lipids. The necrotic core comprised extracellular lipids and liponecrotic tissue. The proportion of cholesteryl linoleate in cholesteryl linoleate+cholesteryl oleate fraction in the extracellular lipid and liponecrotic regions differed significantly from that of the macrophage foam cell-dominant region, and the plasma-derived components (apolipoprotein B and fibrinogen) were localized in the regions. The liponecrotic region was devoid of elastic and collagen fibers and accompanied by macrophage infiltration in the surrounding tissue. Non-oxidized phospholipid (Non-OxPL), OxPL, and Mox macrophages were detected in the three lesions. In the atheroma with lipid core and atheroma with necrotic core, non-OxPL tended to localize in the superficial layer, whereas OxPL was distributed evenly. Mox macrophages were colocalized with OxPL epitopes.In human atherosclerosis, plasma-derived lipids accumulate to form the lipid pool of pathologic intimal thickening, lipid core of atheroma with lipid core, and necrotic core of atheroma with necrotic core. The liponecrotic tissue in the necrotic core appears to be developed by the loss of elastic and collagen fibers. Non-OxPL in the accumulated lipids is oxidized to form OxPL, which may contribute to the lesion development through Mox macrophages.

Identification of the Principle of Taste Sensors to Detect Non-Charged Bitter Substances by 1H-NMR Measurement.

A taste sensor with lipid/polymer membranes is attracting attention as a method to evaluate taste objectively. However, due to the characteristic of detecting taste by changes in membrane potential, taste sensors cannot measure non-charged bitter substances. Many foods and medicines contain non-charged bitter substances, and it is necessary to quantify these tastes with sensors. Therefore, we have been developing taste sensors to detect bitter tastes caused by non-charged substances such as caffeine. In previous studies, a sensor for detecting bitterness caused by caffeine and theobromine, theophylline, was developed, using a membrane modified with hydroxybenzoic acid (HBA) as the sensing part. The sensor was designed to form intramolecular hydrogen bonds (H-bonds) between the hydroxy group and carboxy group of HBA and to successively cause the intermolecular H-bonds between HBA and caffeine molecules to be measured. However, whether this sensing principle is correct or not cannot be confirmed from the results of taste sensor measurements. Therefore, in this study, we explored the interaction between HBA and caffeine by 1H-nuclear magnetic resonance spectroscopy (NMR). By the 1H NMR detection, we confirmed that both the substances interact with each other. Furthermore, the nuclear Overhauser effect (NOE) of intermolecular spatial conformation in solution was measured, by which 2,6-dihydroxybenzoic acid (2,6-DHBA) preferably interacted with caffeine via the H-bonding and stacking configuration between aromatic rings. Identifying the binding form of 2,6-DHBA to caffeine was estimated to predict how the two substances interact.

The cooperative induction of macrophage activation by fucoidan derived from Cladosiphon okamuranus and ß-glucan derived from Saccharomyces cerevisiae.

The present study investigated immune stimulatory effects of Cladosiphon okamuranus-derived fucoidan to activate murine macrophage-like cell line RAW264, and the functional relationship with zymosan, a Saccharomyces cerevisiae-derived ß-glucan. The production of nitric oxide (NO) and tumor necrosis factor-α (TNF-α) in RAW264 cells were remarkably enhanced in the presence of 10 µg/mL fucoidan, and the stimulatory effects of fucoidan were maximally augmented in combinational treatment with 500 ng/mL zymosan, whereas any TLR ligands had no those effects. Confocal microscopic analyses suggested that fucoidan bound on plasma membrane, and it was estimated that some cell surface molecules acted as receptor for fucoidan because cytochalasin D, an inhibitor of phagocytosis, did not affect the immune enhancing activities, whereas methyl-ß-cyclodextrin (MßCD), a general agent for disruption of lipid rafts, diminished that. Furthermore, it was revealed that the additive effects of zymosan on the immune activation with fucoidan was thought to be mediated by dectin-1 based on the results with dectin-1-knockdown RAW264 cells. All of results suggested that fucoidan and some kinds of ß-glucan would cooperatively reinforce the activity of innate immune cells via interactive receptor crosstalk.

Activation of Nod1 Signaling Induces Fetal Growth Restriction and Death through Fetal and Maternal Vasculopathy.

Intrauterine fetal growth restriction (IUGR) and death (IUFD) are both serious problems in the perinatal medicine. Fetal vasculopathy is currently considered to account for a pathogenic mechanism of IUGR and IUFD. We previously demonstrated that an innate immune receptor, the nucleotide-binding oligomerization domain-1 (Nod1), contributed to the development of vascular inflammations in mice at postnatal stages. However, little is known about the deleterious effects of activated Nod1 signaling on embryonic growth and development. We report that administration of FK565, one of the Nod1 ligands, to pregnant C57BL/6 mice induced IUGR and IUFD. Mass spectrometry analysis revealed that maternally injected FK565 was distributed to the fetal tissues across placenta. In addition, maternal injection of FK565 induced robust increases in the amounts of CCL2, IL-6, and TNF proteins as well as NO in maternal, placental and fetal tissues. Nod1 was highly expressed in fetal vascular tissues, where significantly higher levels of CCL2 and IL-6 mRNAs were induced with maternal injection of FK565 than those in other tissues. Using Nod1-knockout mice, we verified that both maternal and fetal tissues were involved in the development of IUGR and IUFD. Furthermore, FK565 induced upregulation of genes associated with immune response, inflammation, and apoptosis in fetal vascular tissues. Our data thus provided new evidence for the pathogenic role of Nod1 in the development of IUGR and IUFD at the maternal-fetal interface.

[Research of the Cancer-Odor].

Early detection and resection of cancer is the most effective in the treatment of solid cancer. Development of a new cancer detection method is expected to become a breakthrough to solve various problems for early detection. It has been reported that there is the specific odors of cancer by using bio olfaction such as dogs, and it has been recognized that there is the odors of cancer. Cancer cells acquire malignant traits as a result of metabolic changes originating from genetic mutation. The cancer specific odorous substances may be considered to be the end products of their metabolic changes. Omics researches such as genomics, proteomics, and metabolomics have been extensively performed to comprehensively analyze changes in DNA, RNA, protein, metabolism and its products specific to cancer for the purpose of developing a new cancer detection marker. It is thought that the research on the odor of cancer is also on the line of omics research. It is difficult to identify cancer-specific odorants buried in various environmental substances. However, it is expected that human will be able to acquire the technology, from the fact that they can be recognized by biological olfaction. We are continuing the research with the dream that identification of the odorous substances as a new cancer detection marker and sensor development for it will lead to the happiness of colleagues in the world.

Anti-prediabetic effect of rose hip (Rosa canina) extract in spontaneously diabetic Torii rats.

BACKGROUND: Prediabetes, a high-risk state for developing diabetes showing impaired glucose tolerance but a normal fasting blood glucose level, has an increasing prevalence worldwide. However, no study investigating the prevention of impaired glucose tolerance at the prediabetic stage by anti-diabetic functional foods has been reported. Thus, the present study aimed to evaluate the anti-prediabetic effect of rose hip in a prediabetic rat model. RESULTS: Spontaneously diabetic Torii (SDT) rats were supplemented with hot-water extract of rose hip at a dose of 100 mg kg-1 body weight day-1 for 12 weeks. The results obtained showed that the supplementation of rose hip extract improved impaired glucose tolerance, promoted insulin secretion, preserved pancreatic beta-cell function and suppressed plasma advanced glycation end-products formation of methylglyoxal-derived hydroimidazolone (MG-H1) residue and Nϵ -carboxymethyl-lysine residues (e.g. MG-H1, control: 465.5 ± 43.8 versus rose hip: 59.1 ± 13.0 pmol mg protein-1 , P < 0.05) in SDT rats at the prediabetic stage (12-20 weeks old). CONCLUSION: The present study provides the first evidence showing that a hot-water extract of rose hip could exert an anti-prediabetic effect in a rat model. © 2017 Society of Chemical Industry.

An extract from pork bones containing osteocalcin improves glucose metabolism in mice by oral administration.

Osteocalcin (OC) is a bone-derived hormone that regulates energy metabolism. OC exists in two forms, carboxylated (GlaOC) and uncaboxylated (GluOC), but only the latter appears to have an endocrine function. In this study, we prepared an extract containing both Gla- and GluOC from boiled pork bone using 0.2 M carbonate buffer at pH 9.5, and tested whether the extract had beneficial effects on improving metabolic parameters in obese mice. The extract equivalent of 1.2 µg of GluOC/mouse was orally administrated to C57BL/6 female mice fed a high-fat, high-sucrose diet. Daily oral administration of the extract for four weeks decreased blood glucose levels and promoted glucose tolerance as well as insulin sensitivity. Our study shows for the first time that boiled pork bones are a source material for osteocalcin in the large-scale production of supplements designed to improve glucose metabolism.

Inhibition of calcium-calmodulin complex formation by vasorelaxant basic dipeptides demonstrated by in vitro and in silico analyses.

BACKGROUND: Tryptophan-histidine (Trp-His) was found to suppress the activity of the Ca²âº/calmodulin (CaM)-dependent protein kinases II (CaMKII), which requires the Ca²âº-CaM complex for an initial activation. In this study, we attempted to clarify whether Trp-His inhibits Ca²âº-CaM complex formation, a CaMKII activator. METHODS: The ability of Trp-His and other peptides to inhibit Ca²âº-CaM complex formation was investigated by a Ca²âº-encapsulation fluorescence assay. The peptide-CaM interactions were illustrated by molecular dynamic simulation. RESULTS: We showed that Trp-His inhibited Ca²âº-CaM complex formation with a 1:1 binding stoichiometry of the peptide to CaM, considering that Trp-His reduced Hill coefficient of Ca²âº-CaM binding from 2.81 to 1.92. His-Trp also showed inhibitory activity, whereas Trp+His, 3-methyl His-Trp, and Phe-His did not show significant inhibitory activity, suggesting that the inhibitory activity was due to a peptide skeleton (irrespective of the sequence), a basic amino acid, a His residue, the N hydrogen atom of its imidazole ring, and Trp residue. In silico studies suggested the possibility that Trp-His and His-Trp interacted with the Ca²âº-binding site of CaM by forming hydrogen bonds with key Ca²âº-binding residues of CaM, with a binding free energy of -49.1 and -68.0 kJ/mol, respectively. CONCLUSIONS: This is the first study demonstrating that the vasoactive dipeptide Trp-His possesses inhibitory activity against Ca²âº-CaM complex formation, which may elucidate how Trp-His inhibited CaMKII in a previous study. GENERAL SIGNIFICANCE: The results provide a basic idea that could lead to the development of small peptides binding with high affinity to CaM and inhibiting Ca²âº-CaM complex formation in the future.

NMR spectroscopic and quantum mechanical analyses of enhanced solubilization of hesperidin by theasinensin a.

PURPOSE: The use of hesperidin in the pharmaceutical field is limited by its aqueous insolubility. The effects of natural compounds in tea on the solubility of hesperidin were evaluated and the underlying mechanism was investigated by nuclear-magnetic resonance (NMR) and quantum mechanical calculations. METHODS: The solubility of hesperidin was measured by liquid chromatography time-of-flight mass spectrometry; the structure of the hesperidin/theasinensin A complex was characterized by (1)H-NMR, diffusion-ordered NMR spectroscopy, and rotating frame NOE spectroscopy, as well as theoretically by quantum mechanical calculations. RESULTS: Among the natural compounds in tea, theasinensin A was the most effective in improving hesperidin solubility. The complexation of hesperidin with theasinensin A led to changes in the chemical shift of protons in hesperidin (Δδ: 0.01-0.27 ppm) and diffusion coefficient (ΔD: 0.66-1.32 × 10(-10) m(2)/s) of hesperidin. ROE correlation signals between hesperidin and theasinensin A and quantum mechanical calculations revealed that two hesperidin molecules formed a stable complex with theasinensin A (2:1 complex) with a ΔG energy of -23.5 kJ/mol. CONCLUSIONS: This is the first study that provides insight into the enhanced solubility of hesperidin through interactions with theasinensin A via a 2:1 complex formation between hesperidin and theasinensin A.

Quantitative analysis of methylglyoxal, glyoxal and free advanced glycation end-products in the plasma of Wistar rats during the oral glucose tolerance test.

The purpose of this study was to gain insight into the production behavior of free adducts of advanced glycation end-products (AGEs) in Wistar rats under acute hyperglycemic conditions. Five AGE-free adducts as well as their precursors (i.e., highly reactive carbonyl intermediates of methylglyoxal and glyoxal) in rat plasma were quantitatively determined at greater than nanomolar levels using the liquid chromatography/tandem mass spectrometry method coupled with 2,4,6-trinitrobenzene sulfonate and 2,3-diaminonaphthalene derivatization techniques. An oral glucose (2 g/kg dose) tolerance test to 10-week-old Wistar rats provided evidence that the plasma levels of diabetes-related metabolites did not change acutely within 120 min, irrespective of increasing blood glucose levels.

Theaflavins enhance intestinal barrier of Caco-2 Cell monolayers through the expression of AMP-activated protein kinase-mediated Occludin, Claudin-1, and ZO-1.

We investigated the effect of theaflavins (TFs) on membrane barrier of Caco-2 cells. For fluorescein-transport experiments, the apparent permeability (Papp) of fluorescein in Caco-2 cells pretreated with 20 µM TFs were significantly decreased compared with that in untreated cells. Although the respective monomeric catechins did not show any Papp reduction, purpurogallin pretreatment resulted in a significant Papp reduction similar to that of TF-3'-O-gallate (TF3'G) pretreatment. This indicates that the benzotropolone moiety may play a crucial role in the Papp reduction or tight junction (TJ)-closing effect induced by TFs. In TF-3'-O-gallate-pretreated Caco-2 cells, fluorescein transport was completely restored by compound C (AMPK inhibitor). In addition, TF3'G significantly increased both the mRNA and protein expression of TJ-related proteins (occludin, claudin-1, and ZO-1) as well as the phosphorylation of AMPK. It was, thus, concluded that TFs could enhance intestinal barrier function by increasing the expression of TJ-related proteins through the activation of AMPK in Caco-2 cells.

Orally administrated dipeptide Ser-Tyr efficiently stimulates noradrenergic turnover in the mouse brain.

In this study, we examined the effect of orally administrated dipeptides containing Tyr (Y) on the metabolism of catecholamines in mouse brains. We found that among eight synthetic dipeptides whose sequences are present frequently in soy proteins, Ser-Tyr (SY), Ile-Tyr, and Tyr-Pro had the highest apparent permeability coefficients in monolayers of human intestinal epithelial Caco-2 cells. When administrated orally, SY markedly increased tyrosine content in the cerebral cortex compared to the vehicle control, Ile-Tyr, Tyr-Pro, and Y alone. The oral administration of SY more effectively increased 3-methoxy-4-hydroxyphenylethyleneglycol, the principal metabolite of noradrenaline, in the cerebral cortex and hippocampus than did Ile-Tyr, Tyr-Pro, or Y alone. Central noradrenergic turnover was also markedly stimulated by SY administration. These in vivo observations strongly suggest that SY is more potent in boosting central catecholamine transmission, particularly the noradrenergic system, than Y alone or other dipeptides that include Y.

Adiponectin-Receptor Agonistic Dipeptide Tyr-Pro Stimulates the Acetylcholine Nervous System in NE-4C Cells.

The dipeptide Tyr-Pro has physiological potential for intact transportability into the brain parenchyma, prevention of cognitive impairment, and an adiponectin receptor 1 (AdipoR1) agonistic effect. The present study aimed to understand the effect of Tyr-Pro on the acetylcholine (ACh) nervous system and its underlying mechanism in NE-4C nerve cells. Concentration-dependent ACh production was induced by stimulation with Tyr-Pro and AdipoRon (an AdipoR1 agonist), along with the expression of AdipoR1 and choline acetyltransferase (ChAT) in NE-4C cells. By knocking down AdipoR1 in the cells, Tyr-Pro promoted ChAT expression, along with the activations of AMPK and ERK 1/2. Tyr-Pro did not alter acetylcholinesterase or ACh receptors, indicating that the dipeptide might operate as an ACh accelerator in nerve cells. This study provides the first evidence that the AdipoR1 agonistic Tyr-Pro is a promising dipeptide responsible for the stimulation of the ACh nervous system by AdipoR1-induced ChAT activation.

Graphite Sheet-Assisted Laser Desorption Ionization-Mass Spectrometry for Small Organic Compound Analysis.

Carbon-based nanopowders have been used as ionization materials for laser desorption ionization-mass spectrometry (LDI-MS) and are very efficient at detection in low m/z regions. In this study, we aimed to develop a new sheet-type graphite material that possessed a randomly grooved nanostructured surface consisting of developed sp2-conjugated atomic carbon to facilitate the desorption/ionization of small compounds in LDI-MS. The graphite sheet exhibited higher UV absorption and provided higher ionization efficiency and survival yield in the LDI-MS detection of a thermometer ion, 4-chloro-benzopyridinium, than those of highly oriented graphite plates. These properties demonstrate that the present graphite sheet is suited for use as an LDI-MS material. Graphite sheet-assisted LDI-MS successfully detected various substances, including amino acids, peptides, and polyethylene glycol polymers, with higher ion intensities and less noise than those associated with conventional organic matrix-assisted LDI-MS (MALDI-MS). Furthermore, graphite sheet-assisted LDI-MS analysis provided more peaks (252 peaks) derived from soy sauce than those obtained by MALDI-MS (36 peaks) and required fewer preparation processes (dilution and air-dried) compared with previously established graphite carbon black-assisted LDI-MS (171 peaks) in the positive mode. This study demonstrates that graphite sheet-assisted LDI-MS has the potential for small organic compound analyses in the biomedical and food science fields.

The PepT1-transportable soy tripeptide VPY reduces intestinal inflammation.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammation of the gastrointestinal tract. The peptide transporter PepT1 is responsible for the intestinal uptake of dietary peptides, and its expression in the gastrointestinal tract is up-regulated during intestinal inflammation, indicating that PepT1 may be a promising target for IBD therapeutics. METHODS: The transport of soy-derived di- and tripeptides across Caco-2 intestinal epithelial cells was examined, and the anti-inflammatory effects of the transported peptide VPY were evaluated in vitro in Caco-2 and THP-1 macrophages, and in vivo in a mouse model of DSS-induced colitis. RESULTS: VPY inhibited the secretion of IL-8 and TNF-α, respectively, from Caco-2 and THP-1 cells. VPY transport and anti-inflammatory activity in Caco-2 cells was reduced in the presence of Gly-Sar, indicating this activity was mediated by PepT1. In mice, VPY treatment reduced DSS-induced colitis symptoms and weight loss, improved colon histology, reduced MPO activity, and decreased gene expression of the pro-inflammatory cytokines TNF-α, IL-6, IL-1ß, IFN-γ and IL-17 in the colon. CONCLUSIONS AND GENERAL SIGNIFICANCE: VPY is a novel PepT1 substrate that can inhibit the production of pro-inflammatory mediators in vitro in intestinal epithelial and immune cells, and reduce the severity of colitis in mice by down-regulating the expression of pro-inflammatory cytokines in the colon, suggesting that VPY may be promising for the treatment of IBD.

Enhanced visualization of small peptides absorbed in rat small intestine by phytic-acid-aided matrix-assisted laser desorption/ionization-imaging mass spectrometry.

Enhanced visualization of small peptides absorbed through a rat intestinal membrane was achieved by matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-IMS) with the aid of phytic acid as a matrix additive. Penetrants through intestinal peptide transporter 1, i.e., glycyl-sarcosine (Gly-Sar, 147.1 m/z) and antihypertensive dipeptide, Val-Tyr (281.2 m/z), were chosen for MALDI-IMS. The signal-to-noise (S/N) ratios of dipeptides Gly-Sar and Val-Tyr were seen to increase by 2.4- and 8.0-fold, respectively, when using a 2',4',6'-trihydroxyacetophenone (THAP) matrix containing 5.0 mM phytic acid, instead of the THAP matrix alone. Owing to the phytic-acid-aided MALDI-IMS method, Gly-Sar and Val-Tyr absorbed in the rat intestinal membrane were successfully visualized. The proposed imaging method also provided useful information on intestinal peptide absorption; to some extent, Val-Tyr was rapidly hydrolyzed to Tyr by peptidases located at the intestinal microvillus during the absorption process. In conclusion, the strongly acidic additive, phytic acid, is beneficial for enhancing the visualization of small peptides using MALDI-IMS, owing to the suppression of ionization-interfering salts in the tissue.

Highly-sensitive detection of free advanced glycation end-products by liquid chromatography-electrospray ionization-tandem mass spectrometry with 2,4,6-trinitrobenzene sulfonate derivatization.

The common derivatization method of primary amino groups by 2,4,6-trinitrobenzene sulfonate (TNBS) was applied to the detection of free adducts of advanced glycation end-products (AGEs), in combination with liquid chromatography-tandem mass spectrometry-multiple reaction monitoring (LC-MS/MS-MRM). The proposed TNBS-MS method provided a surprisingly significant improvement of the detection of AGE-free adducts (e.g., by a factor of >1000 for methylglyoxal-derived hydroimidazolone) with a detection limit of 1.0 nM (10 fmol/injection volume), which was due to the high ionization efficiency of the derived trinitrophenol moiety and its hydrophobicity. With the aid of stable-isotope-labeled internal standard (MG-H1-d3), the convenient TNBS method that allowed the derivation of AGE-free adducts bearing amino groups under mild reaction conditions (30 mM, pH 8.5, 30 min, 30 °C) also permitted successive detection and quantification of five typical AGE-free adducts at nanomolar levels in rat plasma (50 µL) with high reproducibility (2-9% of RSD) and recovery (93-113%), using LC-MS/MS-MRM.