Combination Of High-performance Liquid Chromatography Research Articles

Generation of Enantiomeric Amino Acids during Acid Hydrolysis of Peptides Detected by the Liquid Chromatography/Tandem Mass Spectroscopy

The number of reports indicating the occurrence of D-amino acids in various proteins and natural peptides is increasing. For a usual detection of peptidyl D-amino acids, proteins or peptides are subjected to acid hydrolysis, and the products obtained are analyzed after cancellation of the effect of amino acid racemization during the hydrolysis. However, this method does not seem reliable enough to determine the absence or presence of a small amount of innate D-amino acids. We introduce a modification of an alternative way to distinguish true innate D-amino acids from those artificially generated during hydrolysis incubation. When model peptides (L-Ala)(3), D-Ala-(L-Ala)(2) are hydrolyzed in deuterated hydrochloric acid (DCl), only newly generated D-amino acids are deuterated at the alpha-H-atom. Both innate D-amino acids and artificially generated ones are identified by the combination of high-performance liquid chromatography and liquid chromatography/tandem mass spectrometry equipped with a chiral column. When a peptide containing D-Phe residues was analyzed by this method, the hydrolysis-induced conversion to L-Phe was similarly identified.

Structural and Functional Characterization of Diffusible Signal Factor Family Quorum-Sensing Signals Produced by Members of the Burkholderia cepacia Complex

Previous work has shown that Burkholderia cenocepacia produces the diffusible signal factor (DSF) family signal cis-2-dodecenoic acid (C(12):Delta(2), also known as BDSF), which is involved in the regulation of virulence. In this study, we determined whether C(12):Delta(2) production is conserved in other members of the Burkholderia cepacia complex (Bcc) by using a combination of high-performance liquid chromatography, mass spectrometry, and bioassays. Our results show that five Bcc species are capable of producing C(12):Delta(2) as a sole DSF family signal, while four species produce not only C(12):Delta(2) but also a new DSF family signal, which was identified as cis,cis-11-methyldodeca-2,5-dienoic acid (11-Me-C(12):Delta(2,5)). In addition, we demonstrate that the quorum-sensing signal cis-11-methyl-2-dodecenoic acid (11-Me-C(12):Delta(2)), which was originally identified in Xanthomonas campestris supernatants, is produced by Burkholderia multivorans. It is shown that, similar to 11-Me-C(12):Delta(2) and C(12):Delta(2), the newly identified molecule 11-Me-C(12):Delta(2,5) is a potent signal in the regulation of biofilm formation, the production of virulence factors, and the morphological transition of Candida albicans. These data provide evidence that DSF family molecules are highly conserved bacterial cell-cell communication signals that play key roles in the ecology of the organisms that produce them.

Immunological Characterization and Distribution of Three GnRH Forms in the Brain and Pituitary Gland of Chub Mackerel (Scomber japonicus)

The presence of three gonadotropin-releasing hormone (GnRH) forms in the brain of the chub mackerel, Scomber japonicus, namely, salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and seabream GnRH (sbGnRH), was confirmed by combined high performance liquid chromatography (HPLC) and time-resolved fluoroimmunoassay (TR-FIA). Immunocytochemical localization of the three GnRH forms in the brain was Investigated by using specific antisera, to elucidate possible roles of each GnRH form in reproduction in this species, and double immunolabeling was used to localize GnRH-ir (immunoreactive) fibers Innervating the pituitary. sGnRH-ir neurons were localized in the ventral olfactory bulb and terminal nerve ganglion region. Further, sGnRH-ir fibers were found in different regions of the brain, with prominent fibers running in parallel in the preoptic area (POA) without entering the pituitary. cGnRH-II-ir cell bodies were observed only in the midbrain tegmentum region, with a wide distribution of fibers, which were dense in the midbrain tegmentum and spinal cord. SbGnRH-ir cell bodies were localized in the nucleus preopticus of the POA, with fibers in the olfactory bulb, POA, and hypothalamus. Among the three GnRH forms, only SbGnRH-ir fibers innervated the pituitary gland from the preoptic-hypothalamic region, targeting follicle stimulating hormone (FSH) and luteinizing hormone (LH)-producing cells in the proximal pars distalis, as demonstrated by double immunocytochemistry. The localization of the GnRH-ir system was similar in male and female fish. These results demonstrate that multiple GnRH forms exist in the brain of the chub mackerel and suggest that they serve different functions, with SbGnRH having a significant role in reproduction in stimulating FSH- and LH-producing cells, and sGnRH and cGnRH-II serving as neurotransmitters or neuromodulators.

Arsenic accumulation and thiol status in lichens exposed to As(V) in controlled conditions

Thalli of epiphytic lichen Hypogymnia physodes (L.) Nyl. and terricolous Cladonia furcata (Huds.) Schrad., collected from an area with background arsenic concentrations, were exposed to 0, 0.1, 1 and 10 microg mL(-1) arsenate (As(V)) solutions for 24 h. After exposure they were kept in the metabolically active state for 0, 24 and 48 h in a growth chamber. In the freeze dried samples glutathione (GSH), glutathione disulphide (GSSG), cysteine (Cys) and cystine were analysed and induction of phytochelatin (PC) synthesis measured by reversed-phase high-performance liquid chromatography in combination with fluorescence detection or UV spectrometry. Total arsenic content in thalli was measured by instrumental neutron activation analysis (INAA). In H. physodes, which contained higher amounts of arsenic compared to C. furcata, total glutathione content significantly decreased in samples exposed to 10 microg mL(-1) As(V), whereas in C. furcata a significant increase was observed. In both species PC synthesis was induced in thalli exposed to 10 microg mL(-1).

N- and O-linked oligosaccharides completely lack galactose residues in the gms1och1 mutant of Schizosaccharomyces pombe

Unlike their counterparts in budding yeast Saccharomyces cerevisiae, the glycoproteins of Schizosaccharomyces pombe contain, in addition to alpha-D-mannose (Man), a large number of alpha-D-galactose (Gal) residues. In both yeasts, large outer chains are attached to the oligosaccharide cores of glycoproteins during export via Golgi. Formation of the yeast-specific large outer chain is initiated by alpha-1,6-mannosylatransferase encoded by the och1+ gene, the disruption of which blocked outer chain elongation. We previously reported that N-linked oligosaccharide structures of S. pombe och1Delta mutant consisted of Gal(2-6)Man(9)GlcNAc(2) with alpha-linked Gal residues attached to the core oligosaccharide moiety. The disruption of gms1+, a gene encoding the UDP-galactose transporter required for the synthesis of galactomannan, abolished cell surface galactosylation in S. pombe. In this study, we constructed a gms1Deltaoch1Delta double mutant and determined the N- and O-linked oligosaccharide structures present on the cell surface. Oligosaccharides were liberated from glycoproteins by hydrazinolysis and labeled with the fluorophore, 2-aminopyridine. The pyridylaminated N-linked oligosaccharides were analyzed by high-performance liquid chromatography in combination with alpha1,2-mannosidase digestion and partial acetolysis. These analyses revealed that the N-linked oligosaccharides of gms1Deltaoch1Delta cells consisted of alpha1,2-linked Man-extended core oligosaccharides (Man(8-12)GlcNAc2) from which the fission yeast-specific alpha-linked Gal residues were completely absent.

Incorporation and/or adduction of formic acid with DNA in vivo studied by HPLC–AMS

The contribution of incorporation and/or adduction of formic acid with liver DNA in mouse was investigated using accelerator mass spectrometry (AMS) associated with high performance liquid chromatography (HPLC). Four kinds of 5′-formylated adducts, which were prepared by the reaction of formic acid and deoxyribonucleosides in vitro, were used as references for the HPLC–AMS analysis of in vivo adduction. After the administration of sodium 14 C-formate to mice, the liver DNA pellets were isolated and enzymatically digested to deoxyribonucleosides. A precise analysis of the hydrolysate by HPLC–AMS indicates that a majority of formic acid incorporates directly into DNA, whereas less than 1.5% might form instable formylated DNA adducts in vivo . The results greatly support the important perspective that formic acid is not carcinogenic. Moreover, this study demonstrates that a combination of HPLC with AMS is an essential means for the evaluation of DNA adduction.

Cholesterol overloading leads to hepatic L02 cell damage through activation of the unfolded protein response

Reported data indicate that cholesterol loading in the liver can cause hepatic injury. To explore the possible mechanisms of cell damage resulting from cholesterol overloading in hepatocytes, cell apoptosis, the unfolded protein response (UPR) and the correlation between them were assessed in the cholesterol-overloaded normal human hepatic cell line L02. L02 cells were incubated with 200 microg/ ml of low density lipoprotein (LDL) for 24 h with or without 20 microg/ml 58035, an inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT). In the LDL+58035 group, the intracellular cholesterol level was dramatically increased, which was measured by an enzymatic combined high performance liquid chromatography assay. Expression of immunoglobulin-binding protein, X-box binding protein 1, activating transcription factor 6, activating transcription factor 4, CCAAT/enhancer-binding protein homologous protein-10, markers of endoplasmic reticulum stress (ERS)/ UPR, were up-regulated as determined using reverse transcription-polymerase chain reaction (RT-PCR) or Western blot analysis. The rate of cell apoptic death increased 21.3+/-2.4%. Meanwhile, the active caspase-3 protein expression was increased 8.4-fold compared to the active caspase-3 protein expression in the controls. Furthermore, 4-phenylbutyric acid, an inhibitor of UPR, partly reduced cell apoptosis and activation of caspase-3. This study suggests that cholesterol overloading in hepatic L02 cells induces ERS and activates the UPR which, in part, leads to the apoptotic damage of cells.

The Meibomian puzzle: combining pieces together.

The purpose of this review was to summarize the available information on lipidomic analysis of human meibum and tear film, and critically evaluate the pertinent past and present analytical procedures and results obtained in various laboratories. Human meibum was shown to be a very complex mixture of lipids of various classes. For decades, their exact structures have remained elusive. Because of the limitations of the then-current techniques, most of the complex lipids that constitute meibum could not be analyzed as whole molecules and required prior hydrolysis and/or transesterification of the entire lipid pool. These procedures effectively made it very difficult, and often impossible, to reconstruct the complete structures of the original intact compounds, which prompted us to call this The Meibomian Puzzle. Modern techniques such as high-performance liquid chromatography in combination with mass spectrometry help in solving this puzzle by allowing a researcher to detect and analyze intact molecules of complex lipid compounds, even if present in extremely low concentrations. This current de-facto standard procedure in lipidomic analysis of natural lipids and their mixtures is compared with other experimental techniques such as nuclear magnetic resonance spectroscopy, infrared spectroscopy, gas chromatography, and thin layer chromatography, among the others. The results obtained by older techniques, and their limitations and deficiencies are discussed. It appears that some of the earlier findings did not withstand a scrupulous re-evaluation and need to be modified and/or corrected. The most intriguing development is the virtual absence in meibum of typical phospholipids - an important group of amphiphilic compounds whose role in the human tear film was thought to be to stabilize the entire tear film structure. Instead, another group of previously unidentified compounds, very long chain (O-acyl)-omega-hydroxy fatty acids, appears to be a stabilizing factor which might be related to tear film stability and deterioration. Thus, these compounds may become an important target in biochemistry and (patho)physiology of ocular surface and dry eye research.

Uthethane dimethacrylate: A molecule that may cause confusion in dental research

In recent years, the elution of monomers from dental materials has been a cause for public concern. Urethane dimethacrylate, commonly abbreviated to UDMA, is one of the monomers that are most often tested with regard to elution from and cytotoxicity of resin-based materials. Although each chemical name represents the chemical type, chemical structure, and molecular weight of a molecule, it does not seem to be the same with UDMA. In the present paper, the different forms of UDMA are presented. These include those used by dental manufacturers to produce composite materials and the different types of urethane dimethacrylate used in studies concerning the elution of monomers from composite materials. High-performance liquid chromatography (HPLC) is usually used to detect the eluted monomers, but it does not appear to be adequate in determining the different forms of UDMA. The combination of HPLC with mass spectrometry is shown to be able to specifically identify the compounds eluted in addition to those compounds used as standards in the various studies. The fact that the same name is given to different molecules causes confusion about the results of studies testing the elusion of monomers from composite materials and their possible toxicity.

Interactions of Lipopolysaccharide and Polymyxin Studied by NMR Spectroscopy

In the light of occurrence of bacterial strains with multiple resistances against most antibiotics, antimicrobial peptides that interact with the outer layer of Gram-negative bacteria, such as polymyxin (PMX), have recently received increased attention. Here we present a study of the interactions of PMX-B, -E, and -M with lipopolysaccharide (LPS) from a deep rough mutant strain of Escherichia coli. A method for efficient purification of biosynthetically produced LPS using reversed-phase high-performance liquid chromatography in combination with ternary solvent mixtures was developed. LPS was incorporated into a membrane model, dodecylphosphocholine micelles, and its interaction with polymyxins was studied by heteronuclear NMR spectroscopy. Data from chemical shift mapping using isotope-labeled LPS or labeled polymyxin, as well as from isotope-filtered nuclear Overhauser effect spectroscopy experiments, reveal the mode of interaction of LPS with polymyxins. Using molecular dynamics calculations the complex of LPS with PMX-B in the presence of dodecylphosphocholine micelles was modeled using restraints derived from chemical shift mapping data and intermolecular nuclear Overhauser effects. In the modeled complex the macrocycle of PMX is centered around the phosphate group at GlcN-B, and additional contacts from polar side chains are formed to GlcN-A and Kdo-C, whereas hydrophobic side chains penetrate the acyl-chain region.

Comparison of two methods for the quantification and identification of hemoglobin variants

Background The Sebia Capillarys 2, a capillary electrophoresis method, was compared to a high performance liquid chromatography (HPLC) and electrophoresis at alkaline and acid pH for the presumptive identification and quantitation of hemoglobin variants. Methods The concordance of hemoglobin variant identification on the Sebia Capillarys 2 with the combination of HPLC and electrophoresis at alkaline and acid pH was evaluated by analyzing samples on both systems. The quantification, expressed as % of total hemoglobin, of the common hemoglobin variants on the Capillarys 2 and the Bio Rad VARIANT II β thalassemia methods were compared. Results The % hemoglobin variant results for the two methods were similar for HbS and slightly different for HbC and D Punjab and significantly different for HbE. The Sebia Capillarys 2 correctly identified a number of hemoglobin variants. Conclusion The Sebia Capillarys 2 is suitable for the presumptive identification and quantification of hemoglobin variants producing results comparable with existing HPLC and electrophoresis methods.

Elucidation of O-Glycosylation Structures of the β-Amyloid Precursor Protein by Liquid Chromatography−Mass Spectrometry Using Electron Transfer Dissociation and Collision Induced Dissociation

Accumulation and deposition of beta-amyloid peptide, a major constituent in neuritic plaques are hallmarks of Alzheimer's disease (AD) and AD-related neurodegenerative diseases. beta-Amyloid (Abeta) is derived from the proteolytic cleavage of amyloid precursor protein (APP), a transmembrane protein present in three major isoforms in brain comprising 695, 751 and 770 amino acids, respectively. Among other post-translational modifications, APP is modified during maturation by N- and O-glycosylation, which are thought to be responsible for its expression and secretion. Unlike N-glycosylation, no sites of O-glycosylation of APP have previously been reported. We report here the identification of three specific O-glycosylation sites of the secreted APP695 (sAPP695) produced in CHO cells, using a combination of high-performance liquid chromatography and electrospray-tandem mass spectrometry. With the use of electron transfer dissociation and collision induced dissociation (ETD and CID), we identified type, composition and structures of the Core 1 type O-linked glycans attached at the residues Thr 291, Thr 292 and Thr 576 of the full-length APP695. The glycosylations comprise multiple short glycans, containing N-acetyl galactosamine (GalNAc), Gal-GalNAc and sialic acid terminated structures. The presence of the glycopeptides in the tryptic mixture was identified using the CID-generated sugar oxonium ions. ETD proved to be valuable for the unambiguous identification of the modified sites as ETD fragmentation occurred along the peptide backbone with little or no cleavage of the glycans. Thus, the combination of the CID and ETD techniques in LC-MS is shown here, as a powerful tool for de novo identification of O-glycosylations at unknown modification sites in proteins.

Preconcentration and determination of high leachable pesticides residues in water using solid-phase extraction coupled with high-performance liquid chromatography

A multi-residue procedure was developed for analysis of four common pesticides in water using solid-phase extraction and high-performance liquid chromatography in combination with UV detection at 230 nm. The investigated pesticides (atrazine, dicloran, metazachlor and simazine) are highly leachable and easily migrate within the soil. Multi-walled carbon nanotubes (MWCNTs) adsorbent outperformed C18 bonded silica and graphitized carbon black for preconcentration of the pesticides from solution. The optimum experimental conditions for pesticides extraction were carefully studied and optimised. At the optimum preconcentration conditions (sample volume: 700 mL; adsorbent mass: 300 mg; solution pH: 5.0; flow rate: 3.0 mL min−1; and elution medium: methanol), a quantitiative recovery for pesticides was reported and a high preconcentration factor (1400) was attained. The detection limits of the proposed method were found in the range: 5–15 ng L−1. The dynamic range for simazine and atrazine determination was extended from 15 to 1000 ng L−1, while for metazachlor and dicloran the range was within 60–1000 ng L−1. Five replicate determinations of 70 ng of pesticides mixture present in 700 mL solution gave good results with RSD values within the range 2.5 to 4.6%. The present method gave recoveries from 92.7 to 95.3% for determination of 100 ngL−1 of pesticides in tap water and recoveries from 85.3 to 87.0 in well water with satisfactory RSD values (≤6%).

Postcolumn Renewal of Sensor Surfaces for High-Performance Liquid Chromatography−Surface Plasmon Resonance Detection

The combination of high-performance liquid chromatography (HPLC) with surface plasmon resonance (SPR) for continuous separation and label-free detection of protein samples is described. The detection was realized by electrostatic adsorption of proteins bearing positive and negative charges onto chemically modified SPR sensors in two separate SPR channels. One SPR channel is coated with carboxymethylated dextran which facilitates the detection of positively charged proteins, whereas the other, devoted to the monitoring of negatively charged proteins, is covered with ethylenediamine molecules attached onto a dextran surface. Renewal of the sensor surface in the channels can be accomplished by introducing regeneration solutions through two six-port valves. The coupled technique (HPLC-SPR) was assessed for its analytical figures of merit and applied to the quantification of lysozyme in human milk samples. Unlike the SPR detection of bulk solution refractive index changes during chromatographic peak elutions, the highest sensitivity of SPR is retained in this work since the measurement is performed at the SPR sensor surface where the evanescent field is the strongest. The renewable SPR detection of continuous separations is reproducible and versatile and does not require the separated proteins to contain chromophores or to be prelabeled with a tag (e.g., a redox-active or fluorescent molecule). Such generality makes SPR complementary to other types of chromatographic detectors.

Development of techniques for the analysis of isoflavones in soy foods and nutraceuticals

For over 20 years, soy isoflavones have been investigated for their ability to prevent a wide range of cancers and cardiovascular problems, and numerous other disease states. This research is underpinned by the ability of researchers to analyse isoflavones in various forms in a range of raw materials and biological fluids. This review summarizes the techniques recently used in their analysis. The speed of high performance liquid chromatography analysis has been improved, allowing analysis of more samples, and increasing the sensitivity of detection techniques allows quantification of isoflavones down to nanomoles per litre levels in biological fluids. The combination of high-performance liquid chromatography with immunoassay has allowed identification and estimation of low-level soy isoflavones. The use of soy isoflavone supplements has shown an increase in their circulating levels in plasma and urine, aiding investigation of their biological effects. The significance of the metabolite equol has spurned research into new areas, and recently the specific enantiomers have been studied. High-performance liquid chromatography, capillary electrophoresis and gas chromatography are widely used with a range of detection systems. Increasingly, immunoassay is being used because of its high sensitivity and low cost.

Identification of major alkaloids and steroidal saponins in rat serum by HPLC‐diode array detection‐MS/MS following oral administration of Huangbai‐Zhimu herb‐pair Extract

Huangbai-Zhimu herb-pair (HBZMHP) is a widely used Chinese traditional medicine formula in treating various diseases; however, its active components have remained unknown. In this paper, serum chemistry and combined high-performance liquid chromatography (HPLC), diode-array detection and mass-spectrometry (MS) techniques were used to study the constituents of HBZMHP extract absorbed into rat serum after oral administration. A total of nine characteristic HPLC peaks in the TIC chromatograms were identified as magnoflorine (1), menisperine (2), palmatine (3), berberine (4), timosaponin N or timosaponin E1 (5), timosaponin D (6), timosaponin BIII, anemarsaponin C or xilingsaponin B (7) timosaponin BII (8) and timosaponin AIII (9). All of the identified peaks were constituents of HBZMHP extract. The results narrow the range of active compounds to be found in HBZMHP extract, and pave the way for the follow-up action mechanism research.

Phosphate-Modified TiO2 Nanoparticles for Selective Detection of Dopamine, Levodopa, Adrenaline, and Catechol Based on Fluorescence Quenching

For the first time, an aqueous solution, comprising 6-nm phosphate-modified titanium dioxide (P-TiO2) nanoparticles (NPs) and fluorescein, has been used for sensing dopamine (DA), levodopa (L-DOPA), adrenaline, and catechol. The complexes obtained by means of chelation of surface Ti(IV) ions with an enediol group exhibit strong absorption at 428 nm; thus, they can be designed as efficient quenchers for fluorescein. The fluorescence of a fluorescein solution containing 1.4 mM P-TiO2 NPs at pH 8.0 decreases if the solution comprises DA, L-DOPA, adrenaline, and catechol, but not noradrenaline, ascorbic acid, and salicylic acid. We consider that P-TiO2 NPs have a number of advantages over bare TiO2 NPs, such as ease of preparation, high selectivity, and high stability. By measuring fluorescence quenching, the limits of detection at a signal-to-noise ratio of 3 are calculated as 33.5, 81.8, 20.3, and 92.1 nM for DA, L-DOPA, adrenaline, and catechol, respectively. In contrast, UV-vis absorption reveals the relatively poor sensitivity of these compounds. We have validated the applicability of our method by means of analyses of DA in urine samples. High-performance liquid chromatography in combination with an electrochemical cell has been used to further confirm our results. We believe that this approach has great potential for diagnostic purposes.

Lipopolysaccharide modulation of thyrotropin-releasing hormone (TRH) and TRH-like peptide levels in rat brain and endocrine organs

Lipopolysaccharide (LPS) is a proinflammatory and depressogenic agent whereas thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH2) is an endogenous antidepressant and neuroprotective peptide. LPS and TRH also have opposing effects on K+ channel conductivity. We hypothesized that LPS can modulate the expression and release of not only TRH but also TRH-like peptides with the general structure pGlu-X-Pro-NH2, where "X" can be any amino acid residue. The response might be "homeostatic," that is, LPS might increase TRH and TRH-like peptide release, thereby moderating the cell damaging effects of this bacterial cell wall constituent. On the other hand, LPS might impair the synthesis and release of these neuropeptides, thus facilitating the induction of early response genes, cytokines, and other downstream biochemical changes that contribute to the "sickness syndrome." Sprague-Dawley rats (300 g) received a single intraperitoneal injection of 100 microg/kg LPS. Animals were then decapitated 0, 2, 4, 8, and 24 h later. Serum cytokines and corticosterone peaked 2 h after intraperitoneal LPS along with a transient decrease in serum T3. TRH and TRH-like peptides were measured by a combination of high-performance liquid chromatography and radioimmunoassay. TRH declined in the nucleus accumbens and amygdala in a manner consistent with LPS-accelerated release and degradation. Various TRH-like peptide levels increased at 2 h in the anterior cingulate, hippocampus, striatum, entorhinal cortex, posterior cingulate, and cerebellum, indicating decreased release and clearance of these peptides. These brain regions are part of a neuroimmunomodulatory system that coordinates the behavioral, endocrine, and immune responses to the stresses of sickness, injury, and danger. A sustained rise in TRH levels in pancreatic beta-cells accompanied LPS-impaired insulin secretion. TRH and Leu-TRH in prostate and TRH in epididymis remained elevated 2-24 h after intraperitoneal LPS. We conclude that these endogenous neuroprotective and antidepressant-like peptides both mediate and moderate some of the behavioral and toxic effects of LPS.

Comprehensive Analytical Method for the Determination of Hydrophilic Metabolites by High-Performance Liquid Chromatography and Mass Spectrometry

A method for the comprehensive analysis of hydrophilic metabolites, based on a combination of high-performance liquid chromatography and mass spectrometry, is described. We evaluated three types of stationary phases to achieve the separation of highly hydrophilic metabolites. Good chromatographic retention and separation of these metabolites were achieved on a pentafluorophenylpropyl-bonded silica column with gradient elution, using 0.1% aqueous formic acid and acetonitrile as the mobile phase. The optimized conditions allowed the comprehensive determination of the standard 49 kinds of amino acids, 6 kinds of amines, 45 kinds of organic acids, 18 kinds of nucleic bases, 5 kinds of nucleosides, and 14 kinds of nucleotides, and then the linearity, dynamic range, detection limit, and precision of the retention time and the peak area were validated. We applied this method for the targeted analysis of the components in soy sauce. The results from the quantitative determination of amino acids were compared to those obtained with an amino acid analyzer, and the accuracy was in the range between 85 and 119%. The accuracy of other detected components was confirmed to be 105-133% by the recovery rate after the addition of standard compounds. We also applied the method for the nontargeted metabolic profiling of the components in several kinds of soy sauces with the principal component analysis. They were classified by the manufacturing methods, and the components that corresponded to the differences were identified. This method could be useful for the targeted analysis of hydrophilic metabolites as well as their nontargeted metabolic profiling.

Secondary metabolism of olive secoiridoids. New microcomponents detected in drupes by electrospray ionization and high‐resolution tandem mass spectrometry

Olive drupe tissues have been selected as a possible source of information about the secondary metabolism of oleaceae secoiridoids. The structures of a number of new microcomponents, detected and isolated by a combined high-performance liquid chromatography (HPLC)-fraction collector/ultraviolet electrospray ionization mass spectrometry (UV/ESI-MS) apparatus, can be inferred from the data obtained by high-resolution tandem mass spectrometry in a QqTOF instrument. Some of them (1, 2, 3 and 4) are closely correlated to oleuropein. Others, such as neo-nuzhenide (5) and 2''-hydroxyoleuropein (6), typical of other oleaceae families, have never been found in olive tissues.