Authentic Compounds Research Articles

IDENTIFICATION AND DETERMINATION OF THE CHEMICAL CONSTITUENTS IN A HERBAL PREPARATION, COMPOUND KUSHEN INJECTION, BY HPLC AND LC-DAD-MS/MS

The qualitative and quantitative analyses by HPLC and LC-DAD-MS/MS were conducted for the quality consistency evaluation of Compound Kushen Injection (CKI). For fingerprint analysis, 8 peaks were selected as the characteristic peaks in the HPLC chromatograms of 27 different batches of CKI samples and their similarities were more than 0.90, except the four samples. For the typical HPLC profile, peak identification was carried out by HPLC and LC-MS/MS analyses and as a result, 21 chromatographic peaks were identified or characterized based on the comparison of retention time and UV spectra with authentic compounds as well as by summarized MS fragmentation rules. Six of these identified compounds, including oxymatrine, oxysophocarpine, N-methylcytisine, matrine, sophocarpine, and trifolirhizin, were simultaneously determined and the results showed that the total amounts of six marker compounds were variable, to some extent, which was deduced to be related to the quality of the crude materials and the manufacturing process employed. Supplemental materials are available for this article. Go to the publisher's online edition of the Journal of Liquid Chromatography & Related Technologies to view the supplemental file.

Identification of volatiles from oxidised phosphatidylcholine molecular species using headspace solid-phase microextraction (HS-SPME) and gas chromatography–mass spectrometry (GC-MS)

Headspace solid-phase microextraction (HS-SPME) followed by gas chromatography-mass spectrometry analysis (GC-MS) was used to investigate the volatile compounds from oxidised phosphatidylcholine molecular species. 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) and 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC) were chosen as models. The influence of several parameters on the efficiency of volatile oxidised compounds (VOCs) microextraction, such as type of fibre, extraction duration and temperature were studied. The best results were obtained with a polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibre used at 50 °C during 25 min. The effect of oxidation temperature on the yield of VOCs from SOPC and SLPC was investigated. Oxidative kinetics of SOPC and SLPC were investigated by measuring both the production of VOCs and the degradation of starting materials. More than 30 VOCs were detected by means of the reference mass spectra of the National Institute of Standards and Technology mass spectral library, and most of them were further confirmed by comparing their mass spectra and retention time with those obtained from authentic reference compounds under the same analytical conditions. Moreover, the origins of VOCs from oxidised PLs were studied by comparing those obtained from their corresponding triacylglycerides under the same experimental conditions. The main VOCs identified from oxidised SOPC were (E)-2-decenal, nonanal and octanal and from oxidised SLPC were (E)-2-heptenal, (E)-2-octenal and (E, E)-2,4-decadienal. The proposed method was applied to a real food sample, soy lecithin.

Distribution and Quantification of Flavan-3-ols and Procyanidins with Low Degree of Polymerization in Nuts, Cereals, and Legumes

The monomeric flavan-3-ols catechin and epicatechin as well as procyanidins are of great interest due to their potential beneficial health effects observed in epidemiological studies. However, the occurrence and concentration of these compounds is not well-known due to the fact that reference compounds are not commercially available. In this study we determined the pattern and concentration of catechin, epicatechin, and different dimeric and trimeric procyanidins in 38 food samples (nuts, cereals, legumes) using a reversed phase high-performance liquid chromatography electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS) approach based on isolated authentic reference compounds. Of the analyzed food samples 21 were found to contain dimeric and trimeric procyanidins and their monomeric building units catechin and epicatechin. Mainly the monitored nut samples contained the analyzed procyanidins as well as catechin and epicatechin whereas only 3 cereals were identified as sources of the analyzed compounds. The concentration ranged from 148 μg/100 g in macadamia nut to 55 mg/100 g in pinto bean. Catechin and procyanidin B3 were found to be the most abundant analytes. The only A-type procyanidin that could be identified was procyanidin A2, which was found in peanut. The achieved data could be used for authenticity control and furthermore in combination with dietary studies to calculate the daily intake of monomeric flavan-3-ols and procyanidins. To our knowledge this is the first detailed study quantifying monomeric flavan-3-ols and dimeric and trimeric procyanidins in various nuts, cereals, and legumes.

Identification and Quantification of Tar Compounds in Plant Cell Cultures of Taxus chinensis

In this study, the tar compounds derived from the plant cell cultures of Taxus chinensis were first identified and then quantified via gas chromatography/mass spectrometry (GC/MS) and gas chromatography (GC). 2-Picoline, 2,5-xylenol, acenaphthene, 1-methylnaphthalene and o-xylene were found to be the major tar compounds by biomass. These compounds were identified and confirmed by comparing their retention times with those of authentic compounds. Each compound also spiked with the pure standard. The contents of 2-picoline, 2,5-xylenol, acenaphthene, 1-methylnaphthalene, and o-xylene in biomass were 0.2512, 0.1586, 0.1240, 0.0942 and 0.0525 wt%, respectively. Liquid-liquid extraction and adsorbent treatment were able to remove 42% and 94% of the tars from biomass, respectivly. After hexane precipitation, all of the tars were perfectly removed.

Isolation and structural elucidation of novel natural compounds from twigs of Pinus banksiana

Pinus banksiana (Pinaceae), also called Jack pine, is an evergreen and fast-growing conifer, with a sparse, variable crown and spreading branches at maturity. The species is widely distributed in subpolar and polar areas of northern hemisphere, including Taibai, Daxing'an and Xiaoxing'an Mountains of China, northwest territories of America and many forestry regions of Canada [1]. The conifer has long been used in traditional medicines to enhance cardiovascular functions, improve immunity, cure and prevent cancer and aging diseases [2]. Literatures reported that extracts of wood knot of P. banksiana exhibited significant anti-tumor and antioxidative activities to human cells [3]. In the current work, phytochemical investigation of P. banksiana twigs led to the isolation and purification of five known phenolics and a new lignan derivatives named phillygenin-4-rutinoside (Fig. 1). The structures of the new and known natural compounds were extensively elucidated and determined by their NMR and mass spectral evidence and a careful comparasion with authentic compounds. To the best of our knowledge, the six secondary metabolites were found in P. banksiana for the first time.

Characterization of major flavonoids, triterpenoid, dipeptide and their metabolites in rat urine after oral administration of Radix Astragali decoction

To characterize of the constituents in rat urine after oral administration of Radix Astragali decoction, a HPLC-DAD-MS/MS technique had been developed. Urine collected from 0 to 24 h, after administration, was purified using a C18 solid-phase extraction cartridge, and then detected by an on-line MS/MS detector. By comparing the retention times and MS/MS data with those obtained from authentic compounds and the published data, a total of 11 compounds including six flavonoids, one dipeptide, one triterpenoid and three of their metabolites in urine were identified. Compounds daidzein, genistein, quercetin, L-asparamide-D-phenylalanine, 4,2′,4′-trihydroxychalcone, Astragaloside-IV, daidzein sulfate, and kaempferol sulfate were for the first time detected by HPLC-MS/MS technique in urine. The present research results success-fully narrowed the range of effective constituents to be found in Astragalus membranaceus and would be useful for the following action mechanism research of this traditional Chinese medicine in treating various diseases.

Assesment of antidermatophyte activity of oil from Curcuma longa L. in vitro

Curcuma longa L. is a rhizomatous herbaceous perennial plant, and its essential oil showed antifungal, antibacterial, antioxydant, antivenom, and antitumor effects. The antidermatophytes of oil from turmeric was demonstrated. This study was carried out in order to evaluate inhibition activity of oil from Vietnamese turmeric against two strains dermatophyte, Trychophytol mentargrohytes and Candida albicans. The oil from Vietnamese turmeric was obtained by hydrodistillation method and its chemical composition was determined by GC/MS. By comparing with authentic reference compounds in mass spectra library Nist 98.1 and Wiley 275.L, 13 compounds of oil from Vietnamese turmeric were identified. The received results showed that, the oil is rich in turmerones (about 44%), which has been recognized as major constituent attributing to antimicrobial activity. By disc diffusion method, the antifungal activity of the turmeric essential oil against T. mentargrophyte and C. albicans was demonstrated. The MICs of the turmeric oil for T. mentargrophytes và C. albicans were 0.5%. The minimal killing time for C. albicans was 60 minutes and for T. mentargrophyte was 90 minutes after treatment with the turmeric oil.

Analysis of Multiple Constituents in Cong‐Ming‐Tang, a Chinese Herbal Formula for the Treatment of Amnesia, by High‐performance Liquid Chromatography with Quadrupole Time‐of‐flight Mass Spectrometry

Cong-Ming-Tang (CMT), named smart-soup in English, is a well-known traditional Chinese medicine formula for the treatment of amnesia in China. However, the isolation, purification and identification procedures of the major bioactive constituents in CMT are difficult and time consuming. To establish a rapid and sensitive high-performance liquid chromatography/electrospray ionisation with quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF/MS/MS) method that could be applied to rapidly separate and identify the major bioactive constituents in CMT. Methanolic extract of CMT was used for HPLC-QTOF/MS/MS analysis. Separation was performed on an Agilent Poroshell 120 EC- C18 column (2.7 ×100 mm .i.d., 2.7 µm) with 0.1% formic acid aqueous solution and acetonitrile as the mobile phase under gradient conditions. Both positive and negative ion modes were employed. This analytical tool allowed the identification of 55 compounds from CMT formulae by comparing their retention times and MS spectra with those of authentic compounds or literature data in both positive and negative ion modes, including 4 xanthone C-glycosides, 4 sucrose esters, 11 oligosaccharide multi-esters,15 triterpene saponins, 15 triterpene acids, 2 lignans and 4 phenylpropanoids. Onjisaponin MF was tentatively elucidated as a new triterpene saponin based on the summarised fragmentation rules. HPLC-QTOF/MS/MS provides a new powerful approach to identify the major chemical constituents in CMT rapidly and accurately. This study proposed a series of potential bioactive components without preparative isolation from the crude extract of CMT and indicated that the HPLC-QTOF/MS/MS method also can be a promising tool for the analysis of other traditional Chinese medicines.

Glycosylated derivatives of substituted hydroxylamine. II. The phase transfer synthesis and the study of the glycosyl transfer reaction of glucosaminides of substituted hydroxylamine

The interaction of 1-(2-acetamido-3,4,6,-tri-O-acetyl-2-deoxy-β-D-glucopyranosyloxy)benzotriazole with primary and secondary aliphatic and cycloaliphatic alcohols or diisopropylidenegalactose in refluxing methylene chloride under the catalysis of Lewis acids resulted in alkyl-O-glucosaminides with the 1,2-trans-configuration of the glycoside bond. Other glucosaminides of substituted hydroxylamine were shown not to react under these conditions. The structures of the synthesized glucosaminides were confirmed by 1H NMR spectroscopy and comparison with the authentic compounds.

Development and validation of a UPLC-DAD-MS method for characterization and quantification of alkaloids in Menispermi Rhizoma and its preparations

Menispermi Rhizoma (MR), a well known traditional Chinese medicine, is widely used to prevent and treat sore throat, enteritis, dysentery and rheumatoid arthralgia clinically. However, many rhizomes of Chinese herbal medicines are mistaken as MR due to their similar appearance, which could affect MR quality and cause serious consequences for patients. To guarantee the quality of MR products, an ultra-high-performance liquid chromatography–diode array detector–tandem mass spectrometry (UPLC-DAD-MS) method was established for the characterization of major active ingredients in MR and its preparations. By comparing their retention times and characteristic fragmentations with those of authentic compounds, nine alkaloids in MR were unequivocally identified as acutumidine, acutumine, magnoflorine, menisperine, dauricine, menisporphine, N-demethyl-N-formyldehydronuciferine, 6-O-demethylmenisporphine and dauriporphine. Quantitative analysis of the nine alkaloids in MR and its preparations was accomplished by UPLC-DAD. A UPLC C18 column was employed for the chromatographic separation which was effected by a gradient elution with acetonitrile and 0.1% aqueous formic acid solution containing 5mM ammonium acetate at a flow rate of 0.3mL/min. This quantitative method was validated with good linearity (R2≥0.9991), desirable intra- and inter-day precisions (RSD≤3.32%), and acceptable recoveries (97.90–106.8%). The method was also successfully applied to quantify nine alkaloids in eight batches of MR, six batches of MR capsules and two batches of MR pills. A counterfeit MR sample from Henan province was identified by the validated method, followed by further verification by appearance and microscopic identification. The developed UPLC-DAD-MS method overcame the shortcomings of other quality control methods, such as scant chemical marker, long analytical time, consumption of large amounts of organic solvents and limitation to MR or its single dosage form.

Structural identification of the metabolites of ganoderic acid B from Ganoderma lucidum in rats based on liquid chromatography coupled with electrospray ionization hybrid ion trap and time‐of‐flight mass spectrometry

Ganoderic acid B (GAB), a representative triterpenoid in Ganoderma lucidum, possesses various pharmaceutical effects and has been used as a chemical marker in quality control of G. lucidum and related products. The metabolites of GAB in vivo after its oral administration to rats were investigated by liquid chromatography coupled with electrospray ionization hybrid ion trap and time-of-flight mass spectrometry. A total of 14 metabolites of GAB in rat plasma, bile and various organs were detected and identified by direct comparison with the authentic compounds and their characteristic mass fragmentation patterns. The results showed that oxidization and hydroxylation were the common metabolic pathways for GAB in rats. Moreover, some reduction metabolites of GAB were detected in rat kidney and stomach and glucuronidation only appeared in rat bile. This is the first report on the metabolites of GAB in vivo.

Essential Oil of Indian Propolis: Chemical Composition and Repellency against the Honeybee Apis florea

Hitherto unknown biological properties and the chemical composition of the essential oil isolated from propolis of Indian origin were established. GC/MS Analysis of the essential oil revealed the presence of 32 constituents, of which ten were major compounds, nine had intermediate contents, and 13 were minor compounds. With the exception of six minor constituents, that could not be identified, their identification was based on the comparison of their mass spectra and Kovats retention indices with those listed in the NIST and Wiley mass spectral libraries. Their structural assignment was confirmed by GC/MS co-injection of the essential oil with authentic compounds. Quantification of the components was done by GC-FID analyses. Moreover, the essential oil was shown to possess repellent activity against the honeybee Apis florea. The activity was found to be dose dependent. The average repellency (ΔR) increased with increasing essential-oil concentration up to 24 μg/ml and remained constant for the formulation with the higher concentration. These findings established the chemical constitution of the essential oil and might be useful to beekeepers for the improvement of the bee management.

Biocontrol of Late Blight (Phytophthora capsici) Disease and Growth Promotion of Pepper by Burkholderia cepacia MPC-7.

A chitinolytic bacterial strain having strong antifungal activity was isolated and identified as Burkholderia cepacia MPC-7 based on 16S rRNA gene analysis. MPC-7 solubilized insoluble phosphorous in hydroxyapatite agar media. It produced gluconic acid and 2-ketogluconic acid related to the decrease in pH of broth culture. The antagonist produced benzoic acid (BA) and phenylacetic acid (PA). The authentic compounds, BA and PA, showed a broad spectrum of antimicrobial activity against yeast, several bacterial and fungal pathogens in vitro. To demonstrate the biocontrol efficiency of MPC-7 on late blight disease caused by Phytophthora capsici, pepper plants in pot trials were treated with modified medium only (M), M plus zoospore inoculation (MP), MPC-7 cultured broth (B) and B plus zoospore inoculation (BP). With the sudden increase in root mortality, plants in MP wilted as early as five days after pathogen inoculation. However, plant in BP did not show any symptom of wilting until five days. Root mortality in BP was markedly reduced for as much as 50%. Plants in B had higher dry weight, P concentration in root, and larger leaf area compared to those in M and MP. These results suggested that B. cepacia MPC-7 should be considered as a candidate for the biological fertilizer as well as antimicrobial agent for pepper plants.

Development of an UPLC-QTOF-MS method for qualitative and quantitative analysis ofSaussurea eopygmaea

Summary An ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry UPLC-QTOF-MS method has been developed for qualitative and quantitative analysis major compounds in Saussurea eopygmaea Hand-Mazz, which has long been used as a traditional Tibetan medicine. This method was validated to be sensitive, precise, and accurate with the limits of detection of compounds 2,3,4,6, and 7 with 0.67–1.90 μg mL−1, the overall intra-day and inter-day variations less than 8.45%, and the overall recovery over 93.8%, respectively. The correlation coefficients (R2) of the calibration curves were higher than 0.998. In addition, by comparison MS and MS/MS spectra with those of authentic compounds and literatures, a total of 14 main peaks were identified within 6 min. These results demonstrate that this approach has the potential for quality control of S. eopygmaea and other Tibetan herbal medicines.

Identification of Volatile Oil Components of Nepeta binaludensis Jamzad by GC-MS and 13C-NMR Methods and Evaluation of its Antimicrobial Activity

The essential oil of the aerial parts of Nepeta binaludensis Jamzad, an endemic species to Iran, was obtained by hydro-distillation and analyzed by GC, GC-MS and then 13C-NMR spectra of the main compounds. Sixty-five components, representing 97.42 % of the oil were identified. The major components of the oil were 1,8-cineol (68.31 %), α-terpineol (5.24 %), β-pinene (4.74 %), δ-terpineol (2.57 %), α-pinene (1.54 %). The minimum inhibitory concentrations (MICs) and minimum cidal concentrations (MCCs) of the essential oil and it's major component, 1,8-cineol, as authentic compound were determined using broth dilution method against four bacteria and one fungus. The essential oil was moderately active against Bacillus cereus (3.125 mg/ml), Escherichia coli (3.125 mg/ml), Staphylococcus aureus (6.25 mg/ml) and Candida albicans (12.5 mg/ml) with the same MIC and MCC values in each case. Another gram negative bacteria, Pseudomonas aeroginosa, appeard not to be susceptible to inhibitory effects of the essential oil. The obtained MIC and MCC values for 1,8-cineol were closely near to the essential oil values.

Miniaturized HPTLC of Vitamin B12 Compounds in Foods

High-performance thin-layer chromatography (HPTLC) is a separation technique commonly used to identify and quantify compounds in chemical mixtures. Preliminary experiments indicated that Lichrospher silica gel 60 F254s HPTLC sheets were the most suitable for analyzing vitamin B12 compounds. This study revealed the advantages of miniaturized Lichrospher HPTLC for analyzing authentic vitamin B12 compounds as short migration distances (5 cm) and short development times ( 25 pmol at 254 nm). The practicability of using miniaturized HPTLC was demonstrated by the separation and identification of vitamin B12 compounds purified from foods using an immunoaffinity column.

Prioritization of putative metabolite identifications in LC‐MS/MS experiments using a computational pipeline

One of the major bottle-necks in current LC-MS-based metabolomic investigations is metabolite identification. An often-used approach is to first look up metabolites from databases through peak mass, followed by verification of the obtained putative identifications using MS/MS data. However, the mass-based search may provide inappropriate putative identifications when the observed peak is from isotopes, fragments, or adducts. In addition, a large fraction of peaks is often left with multiple putative identifications. To differentiate these putative identifications, manual verification of metabolites through comparison between biological samples and authentic compounds is necessary. However, such experiments are laborious, especially when multiple putative identifications are encountered. It is desirable to use computational approaches to obtain more reliable putative identifications and prioritize them before performing experimental verification of the metabolites. In this article, a computational pipeline is proposed to assist metabolite identification with improved metabolome coverage and prioritization capability. Multiple publicly available software tools and databases, along with in-house developed algorithms, are utilized to fully exploit the information acquired from LC-MS/MS experiments. The pipeline is successfully applied to identify metabolites on the basis of LC-MS as well as MS/MS data. Using accurate masses, retention time values, MS/MS spectra, and metabolic pathways/networks, more appropriate putative identifications are retrieved and prioritized to guide subsequent metabolite verification experiments.

Inhibitory Effects of Lamiaceae Plants on the Formation of Advanced Glycation Endproducts (AGEs) in Model Proteins

Protein glycation and oxidative stress caused by chronic hyperglycemia play vital role in diabetic complications. This study focused on the evaluation of the antiglycation effect of four Lamiaceae plants (Ocimum sanctum, O. basilicum, O. americanum and Metha cordifolia opiz.). Among the ethanolic extracts, O. sanctum extract exhibited high content of phenolic compounds and strong antioxidant activity. Chemical composition analyzed by HPLC revealing two major phenolic conpounds in O. sanctum extracts as rosmarinic acid (4.43 mg/g) and luteolin (0.96 mg/g). In the antiglycation assays, bovine serum albumin (BSA) and histone which were used as model proteins for investigation in the presence of methylglyoxal (MGO) with or without the extracts comparing with the authentic phenolic compounds. The results showed that O. sanctum extract possessed a potent antiglycation activity in both BSA and histone models with 23.4% and 43.0 % inhibition at the concentration of 500 and 250μg/mL, respectively. The results indicated that O. sanctum which contained high phenolic compounds has potential to prevent protein glycation caused by oxidative stress.

INTEGRATED LC-MS/MS SYSTEM FOR PLANT METABOLOMICS

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is highly sensitive, selective, and enables extensive detection of metabolites within a sample. The result allows us to characterize comprehensive metabolite accumulation patterns without dependence on authentic standard compounds and isolation of the individual metabolites. A reference database search is essential for the structural assignment process of un-targeted MS and MS/MS data. Moreover, the characterization of unknown metabolites is challenging, since these cannot be assigned a candidate structure by using a reference database. In this case study, integrated LC-MS/MS based plant metabolomics allows us to detect several hundred metabolites in a sample; and integrated omics analyses, e.g., large-scale reverse genetics, linkage mapping, and association mapping, provides a powerful tool for candidate structure selection or rejection. We also examine emerging technology and applications for LC-MS/MS-based un-targeted plant metabolomics. These activities promote the characterization of massive extended detectable metabolites.

Abundant naphthenic acids in oil sands process‐affected water: studies by synthesis, derivatisation and two‐dimensional gas chromatography/high‐resolution mass spectrometry

The large volumes of 'supercomplex' mixtures of reputedly toxic organic compounds in acidic extracts of oil sands process-affected waters (OSPW) represent a challenging goal for complete characterisation. To date, comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GCxGC/TOFMS) has allowed the acquisition and interpretation of numerous electron ionisation mass spectra including many of those confirmed to be tricyclic and pentacyclic carboxylic acids by reference to the spectra and retention positions of authentic or synthetic compounds. This has allowed the toxicities of some of the identified acids to be determined and their environmental significance to be better assessed. Synthesis, derivatisation (methyl, trideuteriomethyl and trimethylsilyl esters) and GCxGC/TOFMS with nominal mass and higher mass accuracy (ca. 5 ppm) were used to study three abundant unknown acids present in OSPW samples from two different industrial suppliers. GCxGC/TOFMS with nominal mass accuracy, of methyl, trideuteriomethyl and trimethylsilyl esters of three abundant acids in two OSPW samples, produced mass spectra consistent with their assignment as either C(16) tetracyclic acids or as isobaric pentacyclic C(15) hydroxy acids ('O(3)') or sulfur-containing ('SO(2)') species. The synthesis of several isomeric pentacyclic C(15) hydroxy acids and examination of the GCxGC retention times and mass spectra (nominal mass) of their derivatives suggested that the unknown OSPW acids were not hydroxy acids, and GCxGC/TOFMS with higher mass accuracy ruled out the possibility. The possibility that they were isobaric 'SO(2)' species could also be dismissed as this was inconsistent with accurate masses, the derivatisation reactions observed, the fragmentation patterns and the isotope distributions, which excluded the presence of sulphur. The data support the contention that the three abundant unknowns were indeed C(16) tetracyclic acids. An equally rigorous approach will be necessary to characterise further acids in such mixtures. This is important so that chemistry can be used to guide the search for toxic modes of action.