Authentic Compounds Research Articles

AMDORAP: Non-targeted metabolic profiling based on high-resolution LC-MS

BackgroundLiquid chromatography-mass spectrometry (LC-MS) utilizing the high-resolution power of an orbitrap is an important analytical technique for both metabolomics and proteomics. Most important feature of the orbitrap is excellent mass accuracy. Thus, it is necessary to convert raw data to accurate and reliable m/z values for metabolic fingerprinting by high-resolution LC-MS.ResultsIn the present study, we developed a novel, easy-to-use and straightforward m/z detection method, AMDORAP. For assessing the performance, we used real biological samples, Bacillus subtilis strains 168 and MGB874, in the positive mode by LC-orbitrap. For 14 identified compounds by measuring the authentic compounds, we compared obtained m/z values with other LC-MS processing tools. The errors by AMDORAP were distributed within ±3 ppm and showed the best performance in m/z value accuracy.ConclusionsOur method can detect m/z values of biological samples much more accurately than other LC-MS analysis tools. AMDORAP allows us to address the relationships between biological effects and cellular metabolites based on accurate m/z values. Obtaining the accurate m/z values from raw data should be indispensable as a starting point for comparative LC-orbitrap analysis. AMDORAP is freely available under an open-source license at http://amdorap.sourceforge.net/.

Bioconversion of car-3-ene by a dioxygenase of Pleurotus sapidus

Mycelium of the basidiomycete Pleurotus sapidus known to contain a novel dioxygenase was used for the bioconversion of car-3-ene [I]. After 4h of incubation 25.3mgL(-1) car-3-en-5-one [V], 5.4mgL(-1) car-3-en-2-one [VII], and 7.3mgL(-1) car-2-en-4-one [XV] accumulated as major oxidation products. The identity of the respective carenones and their corresponding alcohols was confirmed by comparison with MS and NMR spectral data obtained for synthesized authentic compounds. The peak areas of oxidation products were at least five times higher as compared with autoxidation. A radical mechanism similar to lipoxygenase catalysis was proposed and substantiated with detailed product analyses. The reduction of assumed car-3-ene hydroperoxides to the corresponding alcohols evidenced the radical initiated formation of hydroperoxides and confirmed the regio- and stereo-selectivity of the dioxygenase. The introduction of molecular oxygen into the bicyclic car-3-ene [I] molecule occurred at allylic positions of a cyclic isopentenyl moiety with a pronounced preference for the position adjacent to the non-substituted carbon atom of the C-C-double bond. This co-factor independent selective oxygenation presents an alternative to P450 mono-oxygenase based approaches for the production of terpene derived flavor compounds, pharmaceuticals and other fine chemicals.

Preparation, characterization, and biological evaluation of 6I,6IV-di-O-[α-l-fucopyranosyl-(1→6)-2-acetamido-2-deoxy-β-d-glucopyranosyl]-cyclomaltoheptaose and 6-O-[α-l-fucopyranosyl-(1→6)-2-acetamido-2-deoxy-β-d-glucopyranosyl]-cyclomaltoheptaose

6I,6IV-Di-O-[α-l-fucopyranosyl-(1→6)-2-acetamido-2-deoxy-β-d-glucopyranosyl]-cyclomaltoheptaose (βCD) {6I,6IV-di-O-[α-l-Fuc-(1→6)-β-d-GlcNAc]-βCD (5)} and 6-O-[α-l-fucopyranosyl-(1→6)-2-acetamido-2-deoxy-β-d-glucopyranosyl]-βCD {6-O-[α-l-Fuc-(1→6)-β-d-GlcNAc]-βCD (6)} were chemically synthesized using the corresponding authentic compounds, bis(2,3-di-O-acetyl)-pentakis(2,3,6-tri-O-acetyl)-βCD as the glycosyl acceptor and 2,3,4-tri-O-benzyl-α-l-fucopyranosyl-(1→6)-3,4-di-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-d-glucopyranosyl trichloroacetimidate as the fuco-glucosaminyl donor. NMR confirmed that α-l-Fuc-(1→6)-d-GlcNAc was bonded by β-linking to the βCD ring. To evaluate biological efficiency, the biological activities of the new branched βCDs were examined. The cell detachment activity of 5 was lower than that of 6 in real-time cell sensing (RT-CES) assay, indicating that 5 has lower toxicity. In SPR analysis, 5 had a higher special binding with AAL, a fucose-recognizing lectin. These results suggest that 5 could be an efficient drug carrier directed at cells expressing fucose-binding proteins.

Characterization and determination of the major constituents in Belamcandae Rhizoma by HPLC–DAD–ESI-MS n

Belamcandae Rhizoma, derived from the rhizome of Belamcanda chinensis (L.) DC., has been used as traditional Chinese medicine for the treatment of coughing and pharyngitis. However, there have been few studies dealing with the systematic analysis of the bioactive constituents in Belamcandae Rhizoma. In this work, high performance liquid chromatography–diode array detection–electrospray ionization multiple-stage mass spectrometry (HPLC-DAD-ESI-MS n ) combined with liquid chromatography–time of flight-mass spectrometry (HPLC–TOF/MS) was established for profiling and characterization of multi-constituent in Belamcandae Rhizoma. The ESI-MS n fragmentation behaviors of the authentic references were proposed for aiding the structural identification of components in the extract. Thirty-five flavonoids, including 30 isoflavones and five xanthones, were identified or tentatively identified by comparing their retention times, UV and MS spectra with those of authentic compounds or literature data. Twelve of the identified compounds (neomangiferin, mangiferin, tectoridin, iristectorin B, iristectorin A, iridin, tectorigenin, iristectorigenin A, irigenin, irisflorentin, irilone and dichtomitin) were determined by HPLC–DAD using a C 18 column. The results indicated that the developed analysis method could be employed as a rapid, effective technique for structural characterization of chemical constituents in herbal medicine. This work is expected to provide comprehensive information for the quality evaluation of Belamcandae Rhizoma, which would be a valuable reference for the further study and development of this herb and related medicinal products.

Metabolomics Reveals Attenuation of the SLC6A20 Kidney Transporter in Nonhuman Primate and Mouse Models of Type 2 Diabetes Mellitus

To enhance understanding of the metabolic indicators of type 2 diabetes mellitus (T2DM) disease pathogenesis and progression, the urinary metabolomes of well characterized rhesus macaques (normal or spontaneously and naturally diabetic) were examined. High-resolution ultra-performance liquid chromatography coupled with the accurate mass determination of time-of-flight mass spectrometry was used to analyze spot urine samples from normal (n = 10) and T2DM (n = 11) male monkeys. The machine-learning algorithm random forests classified urine samples as either from normal or T2DM monkeys. The metabolites important for developing the classifier were further examined for their biological significance. Random forests models had a misclassification error of less than 5%. Metabolites were identified based on accurate masses (<10 ppm) and confirmed by tandem mass spectrometry of authentic compounds. Urinary compounds significantly increased (p < 0.05) in the T2DM when compared with the normal group included glycine betaine (9-fold), citric acid (2.8-fold), kynurenic acid (1.8-fold), glucose (68-fold), and pipecolic acid (6.5-fold). When compared with the conventional definition of T2DM, the metabolites were also useful in defining the T2DM condition, and the urinary elevations in glycine betaine and pipecolic acid (as well as proline) indicated defective re-absorption in the kidney proximal tubules by SLC6A20, a Na(+)-dependent transporter. The mRNA levels of SLC6A20 were significantly reduced in the kidneys of monkeys with T2DM. These observations were validated in the db/db mouse model of T2DM. This study provides convincing evidence of the power of metabolomics for identifying functional changes at many levels in the omics pipeline.

Simultaneous Identification of Multiple Celangulins from the Root Bark of Celastrus angulatus Using High‐performance Liquid Chromatography–Diode Array Detector–Electrospray Ionisation–Tandem Mass Spectrometry

Celangulins are a small family of β-dihydroagarofuran sesquiterpenoids endowed with diverse polyoxygenated polyol esters and various biological properties. Since our research focuses on celangulins, the development of rapid and sensitive online analytical methods to analyse and characterise them is of great significance. To develop an HPLC-DAD-ESI-MS/MS method capable of simple and rapid analysis of celangulins in crude extract of root bark of C. angulatus extracts. High-performance liquid chromatography coupled with a diode array detector and electrospray ionisation tandem mass spectrometry was established for the efficient and rapid identification of the celangulins. Chromatographic separations of celangulins were performed on a Hypersil Gold C(18) reverse-phase column by gradient elution with acetonitrile-water as mobile phase at a flow-rate 0.2 mL/min. ESI/MS/MS analysis of sodium adduct ion ([M + Na](+) ) of each celangulin shows that all the celangulins produced very similar fragmentation profiles, and that the characteristic fragments at m/z 245, m/z 229 and m/z 231 were defined as the diagnostic ions for celangulins. Simultaneously, 46 components in the extracts of this plant were separated, and 36 of them were characterised as celangulins by online ESI/MS/MS and by comparing their retention times, UV and MS spectra with those of authentic compounds. HPLC-DAD-ESI-MS/MS was demonstrated to be a powerful tool for the characterisation of minor celangulins in complex samples.

Erratum to: Mass balance method for purity assay of phthalic acid esters: development of primary reference materials as traceability sources in the Japan Calibration Service System

Purity assay of high-purity materials (HPMs) of phthalic acid esters (PAEs) was carried out by means of a mass balance method. In this method, chromatographic methods such as gas chromatography-flame ionization detector (GC-FID) and/or high-performance liquid chromatography (HPLC) in combination with other methods such as Karl-Fischer (KF) titration and vacuum evaporation (VE) were applied. The sum of the impurities estimated by these methods allowed the estimation of the purity of the main component by difference. Seven PAEs with varying side chain structures and levels of impurities were analysed on a systematic way in which impurities were classified into several groups in terms of their abundance, availability of qualitative information and availability of authentic compounds, etc. The absolute quantity of each impurity was determined by GC-FID and/or HPLC based on the calibration made by the authentic compounds of impurities whenever available. The purities in mass fraction of these PAEs were certified at the National Metrology Institute of Japan (NMIJ), and the PAEs were registered as primary reference materials playing an essential role in linking the metrological traceability of the Japan Calibration Service System (JCSS) to the International System of Units (SI).

Identification of the major constituents in Xuebijing injection by HPLC‐ESI‐MS

Xuebijing injection (XBJ) is a traditional Chinese herbal prescription widely used in the treatment of sepsis. This is the first report concerning the identification of XBJ constituents. In addition, to evaluate XBJ's quality, partial least square discrimination analysis (PLS-DA) was performed on chemical fingerprint data. Establish an LC-MS method to identify the components in XBJ for the purpose of quality control. Compounds were separated by HPLC using a C(18) column and gradient elution of acetonitrile-methanol (60:40, v/v) and water-acetic acid (100 : 0.5, v/v) in 80 min. HPLC equipped with diode array detector (DAD) coupled with time-of-flight (TOF) tandem mass spectrometry and HPLC electrospray ionisation (ESI) multi-stage tandem ion-trap mass spectrometry (IT-MS(n) ) method was developed to analyse XBJ's major components. Both positive and negative ionisation modes were employed. Twenty-one compounds including amino acids, phenolic acids, flavonoid glycoside, terpene glycoside and phthalide were identified or tentatively characterised. Their retention times, UV and MS spectra were compared with those of authentic compounds or literature data. The score plot of PLS-DA clearly revealed variations among samples produced in different commercial batches. The analytical method developed is highly effective for the discrimination and quality control of XBJ.

Microsomal formation of N-benzyl-4-hydroxymethylaniline from N-benzyl-4-methylaniline.

The in-vitro metabolism of N-benzyl-4-methylaniline was re-examined using male hamster and rabbit hepatic microsomes; both species generated N-benzyl-4-hydroxymethylaniline, confirmed by TLC and HPLC, comparison with authentic compound. Further confirmation of the formation of this metabolite was achieved by use of a rapid scan UV detector.

Identification and measurement of desethylamiodarone in blood plasma specimens from amiodarone-treated patients.

Desethylamiodarone has been identified as the principal lipophilic metabolite of amiodarone present in plasma specimens from amiodarone-treated patients. This structure has been confirmed by probe-injection mass spectrometry of column effluent fractions and comparison with the authentic compound using both chromatographic and mass spectrometric techniques. Although there is no information available as to the pharmacological activity of desethylamiodarone in man, the plasma concentrations of this metabolite attained during chronic amiodarone therapy are similar to those of the parent compound (0.1-4 mg litre-1).

Detection, isolation and characterization of normorphine and norcodeine as morphine metabolites in man.

Abstract Normorphine and norcodeine were identified by two-dimensional thin-layer chromatography (t.l.c.) as human urinary morphine metabolites from 24 h urine samples of three patients who had received 190–210 mg morphine sulphate intravenously and from one patient who had chronically received 220 mg morphine sulphate orally per day. Both metabolites were isolated by preparative t.l.c. Comparison of the isolated norcodeine and normorphine with their respective authentic compounds, prepared by chemical synthesis, showed identical melting points, mass fractionation patterns, and chemical ionization spectra, confirming t.l.c. identification.

Zirconium-Catalyzed Polymerization of a Styrene: Catalyst Reactivation Mechanisms Using Alkenes and Dihydrogen

Zirconium salicylaldiminates in the presence of AliBu3/[Ph3C][B(C6F5)4] are inactive for the homopolymerization of p-tert-butylstyrene (TBS), but the addition of hexene leads to a highly active system. Analysis of the polymers by NMR and MALDI indicates surprisingly that most of the product chains contain no hexene, while some contain a single hexene unit. Addition of dihydrogen also leads to the production of poly(TBS) without substantial reduction in molecular weight and with main-chain end-group unsaturation. These and other observations lead us to two related mechanisms for the hexene- and dihydrogen-promoted reactions. Synthetic and catalytic studies indicate that the active species is not a typical bis(salicylaldiminato) compound but an amido/alkoxido system. An authentic compound of this class is highly active for the TBS polymerization even in the absence of added aluminum alkyl.

Phytochemical investigation and hair growth studies on the rhizomes of Nardostachys jatamansi DC

Nardostachys jatamansi DC rhizomes were subjected to extraction, fractionation, and isolation of terpenoid compounds. Three terpenoid compounds were isolated which are nardal, jatamansic acid, and nardin. These compounds were identified based on physical and spectral data (UV, IR,1H and13C NMR, 2D NMR, Mass) and comparison with authentic compounds. The crude extract, fractions, and two of the isolated compounds were tested for their hair growth activity. The hair growth studies showed good activities for the extract, fraction, and the isolated compounds.

Analysis of the Essential Oils of Some Centaurea Species (Asteraceae) Growing Wild in Algeria and Greece and Investigation of their Antimicrobial Activities

The volatile composition of Centaurea pullata L., C. grisebachii (Nyman) Heldr. subsp. grisebachii and C. affinis Friv. have been studied. The essential oils were obtained by hydrodistillation in a modified Clevenger-type apparatus, and their analyses were performed by GC and GC-MS. Identification of the components was made by comparison of mass spectra and retention indices with literature records and by co-chromatography with authentic compounds. Their main constituents were caryophyllene oxide for C. pullata, 6,10,14-trimethyl pentadecan-2-one and spathulenol for C. grisebachi subsp. grisebachii, thymol and dodecanol for C. affinis. The essential oils were evaluated for their antimicrobial potential and showed a moderate activity against all microorganisms tested.

Induction of Biologically Active Flavonoids in Cell Cultures of Morus nigra and Testing their Hypoglycemic Efficacy

The antidiabetic activity of both leaves and MJ-treated cell cultures of Morus nigra was evaluated after their oral administration to streptozotocin-induced diabetic rats. The antidiabetic activity of extracts from leaves given to streptozotocin (STZ)-diabetic rats for 10 days increased with increasing doses of leaves extract up to 500 mg/kg/day. The administration of 500 mg/kg/day of leaves extract reduced the concentration of glucose from 370 ± 7.31 mg/dl (control) to 154 ± 6.27 mg/dl, and a significant increase in the insulin level from 11.3 ± 0.31 μU/ml (control) to 14.6 ± 0.43 μU/ml was recorded. Cell suspension cultures were established from the young leaves of Morus nigra cultivated on modified MS medium supplemented with 2.0 mg/l 1-naphthaleneacetic acid (NAA), 0.2 mg/l 6-(furfurylamino)purine (kinetin). The changes in cell weight and flavonoid content were monitored between day zero and 12. The linear increase in fresh weight was found to be parallel to flavonoids production. Cell cultures treated with 100 μM methyl jasmonate for 24 hours showed a noticeable increase in level of flavonoids and significant and more effective hypoglycemic activity than that for extract from leaves. The major flavonoids were isolated by TLC and HPLC and identified as rutin, quercetin, Morusin and cyclomorusin by co-chromatography and mass spectrometry in comparison to samples of authentic reference compounds.

Differential Radiation Sensitivity of Shikonin Derivatives from Lithospermum erythrorhizon S.

Differences in radiation sensitivity and color degradation of shikonin derivatives between the extracts of Lithospermum erythrorhizon root and authentic compounds were investigated. The degradation of shikonin derivatives in both root extracts and authentic compounds were drastically increased to above 8 kGy. HPLC analysis revealed radiation sensitivities of root extracts and authentic compounds differed significantly. Both shikonin and deoxyshikonin were the most sensitive with a degradation rate of 87–91%, while other derivatives in the extracts were the most resistant with a rate of 44–67%. Mixture of shikonin and deoxyshikonin showed a degradation rate of 20–24% at 4 kGy and around 60% at 8 kGy, whereas other shikonin derivatives showed a 48–62% degradation rate at 4 kGy and an 89–94% degradation at 8 kGy. The degradation pattern of shikonin derivatives in the mixture exhibited a similar pattern to those of the extract, but showed opposite pattern in individual derivative. The order of radiation sensitivity of shikonin derivatives are as follows: shikonin, deoxyshikonin>acetylshikonin, dimethylacrylshikonin, isobutyrylshikonin, isovalerylshikonin, 2-methyl-n-butyrylshikonin>hydroxyisovalerylshikonin in authentic compounds and isobutyrylshikonin, acetylshikonin, hydroxyisovalerylshikonin, isovalerylshikonin, 2-methyl-n-butyrylshikonin>deoxyshikonin, dimethylacrylshikonin>shikonin in root extracts.

Regioselective deglycosylation of onion quercetin glucosides by Saccharomyces cerevisiae

Bioconversion of quercetin glucosides using four generally recognized as safe (GRAS) organisms (Aspergillus oryzae, Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae) was evaluated by measuring changes in the levels of quercetin compounds of onion. Of the four organisms, S. cerevisiae increased the content of quercetin-3-O-β-D-glucoside (III; isoquercitrin) and quercetin (IV), whereas decreasing quercetin-3,4'-O-β-D-glucoside (I) and quercetin-4'-O-β-D-glucoside (II). Also, S. cerevisiae converted authentic compound I to III, and II to IV, respectively. These results suggest that S. cerevisiae can be used to increase the levels of isoquercitrin (III), the most bioavailable quercetin compound in onion.

Biomarker discovery in biological specimens (plasma, hair, liver and kidney) of diabetic mice based upon metabolite profiling using ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry

BackgroundThe number of diabetic patients has recently been increasing worldwide. Diabetes is a multifactorial disorder based on environmental factors and genetic background. In many cases, diabetes is asymptomatic for a long period and the patient is not aware of the disease. Therefore, the potential biomarker(s), leading to the early detection and/or prevention of diabetes mellitus, are strongly required. However, the diagnosis of the prediabetic state in humans is a very difficult issue, because the lifestyle is variable in each person. Although the development of a diagnosis method in humans is the goal of our research, the extraction and structural identification of biomarker candidates in several biological specimens (i.e., plasma, hair, liver and kidney) of ddY strain mice, which undergo naturally occurring diabetes along with aging, were carried out based upon a metabolite profiling study. MethodsThe low-molecular-mass compounds including metabolites in the biological specimens of diabetic mice (ddY-H) and normal mice (ddY-L) were globally separated by ultra-performance liquid chromatography (UPLC) using different reversed-phase columns (i.e., T3-C18 and HS-F5) and detected by electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The biomarker candidates related to diabetes mellitus were extracted from a multivariate statistical analysis, such as an orthogonal partial least-squares-discriminant analysis (OPLS-DA), followed by a database search, such as ChemSpider, KEGG and HMDB. ResultsMany metabolites and unknown compounds in each biological specimen were detected as the biomarker candidates related to diabetic mellitus. Among them, the elucidation of the chemical structures of several possible metabolites, including more than two biological specimens, was carried out along with the comparison of the tandem MS/MS analyses using authentic compounds. One metabolite was clearly identified as N-acetyl-l-leucine based upon the MS/MS spectra and the retention time on the chromatograms. ConclusionsN-acetyl-l-leucine is an endogenous compound included in all biological specimens (plasma, hair, liver and kidney). Therefore, this metabolite appears to be a potential biomarker candidate related to diabetes. Although the structures of other biomarker candidates have still not yet determined, the present approach based upon a metabolite profiling study using UPLC-ESI-TOF-MS could be helpful for understanding the abnormal state of various diseases.

Characterization of alkaloids in Sophora flavescens Ait. by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

Sophora flavescens Ait., a well-known Chinese herbal medicine, is widely used in clinical practice for the treatment of viral hepatitis, cancer, gastrointestinal hemorrhage, and skin diseases. This paper is the first report on a method based on the combined use of high-performance liquid chromatography, photodiode array detection, and electrospray ionization tandem mass spectrometry for the comprehensive and systematic separation and characterization of bioactive alkaloids in Sophora flavescens Ait. A total of 22 constituents were identified on the basis of the extracted ion chromatograms for different [M+H] + ions of the alkaloids present in S. flavescens Ait. Among these, 5 constituents were unambiguously identified by comparing the experimental data on their retention times and MS n spectra with those of the authentic compounds, and 17 other constituents were tentatively identified on the basis of their MS n fragmentation behaviors and/or molecular weight information from literatures. Furthermore, some characteristic fragmentation pathways of the alkaloids in S. flavescens Ait. were detected and examined. This information may be useful for characterizing the bioactive alkaloids present in S. flavescens Ait. and for possible applications in formulations.

Retraction: Stable Aziridinium Salts as Versatile Intermediates: Isolation and Regio‐ and Stereoselective Ring‐Opening and Rearrangement

The following article from Angewandte Chemie International Edition, “Stable Aziridinium Salts as Versatile Intermediates: Isolation and Regio- and Stereoselective Ring-Opening and Rearrangement”, published online on January 13, 2009 in Wiley Online Library (www.onlinelibrary.wiley.com, DOI: 10.1002/anie.200805244) and in print (Angew. Chem. Int. Ed. 2009, 48, 1328–1330), has been retracted by agreement between the corresponding author, the journal Editor, Dr. Peter Gölitz, and Wiley-VCH. The retraction has been agreed upon because by performing additional NMR analyses and preparing authentic reference compounds, the authors discovered that the structures of the aziridinium ions 3 were incorrectly assigned.