Characterization and identification of diarylheptanoids in ginger (Zingiber officinale Rosc.) using high-performance liquid chromatography/electrospray ionization mass spectrometry

In our continuing investigation of diarylheptanoids in Zingiberaceae plants using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS), 26 diarylheptanoids were identified from fresh ginger rhizome. Of the 26 compounds, 15 diarylheptanoids appear to be new compounds. In addition, the majority of these compounds (18) were acetylated, which is different from our investigation of diarylheptanoids from turmeric, another member of the Zingiberaceae, which did not possess any acetylated diarylheptanoids. In all, five distinct groups (homologous series) of diarylheptanoids were found in extracts from ginger rhizome. These groups were differentiated by structural differences on the heptane skeletons, whereas homologs within each group differed by substitution patterns on the aromatic rings. Diagnostic fragmentation behavior in (+)- and (-)ESI-MS/MS analyses for each group of homologs, as well as information regarding polarity obtained from retention time data, allowed us to classify compounds by group and identify them based on key structural features.     

Mass spectrometric analysis of chemical warfare agents and their degradation products in soil and synthetic samples

A packed capillary liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the identification of chemical warfare agents, their degradation products and related compounds in synthetic tabun samples and in soil samples collected from a former mustard storage site. A number of organophosphorus and organosulfur compounds that had not been previously characterized were identified, based on acquired high-resolution ESI-MS data. At lower sampling cone voltages, the ESI mass spectra were dominated by protonated, sodiated and protonated acetonitrile adducts and/or their dimers that could be used to confirm the molecular mass of each compound. Structural information was obtained by inducing product ion formation in the ESI interface at higher sampling cone voltages. Representative ESI-MS mass spectra for previously uncharacterized compounds were incorporated into a database as part of an on-going effort in chemical warfare agent detection and identification. The same samples were also analyzed by capillary column gas chromatography (GC)-MS in order to compare an established method with LC-ESI-MS for chemical warfare agent identification. Analysis times and full-scanning sensitivities were similar for both methods, with differences being associated with sample matrix, ease of ionization and compound volatility. GC-MS would be preferred for organic extracts and must be used for the determination of mustard and relatively non-polar organosulfur degradation products, including 1,4- thioxane and 1,4-dithiane, as these compounds do not ionize during ESI-MS. Diols, formed following hydrolysis of mustard and longer-chain sulfur vesicants, may be analyzed using both methods with LC-ESI-MS providing improved chromatographic peak shape. Aqueous samples and extracts would, typically, be analyzed by LC-ESI-MS, since these analyses may be conducted directly without the need for additional sample handling and/or derivatization associated with GC-MS determinations. Organophosphorus compounds, including chemical warfare agents, related compounds and lower volatility hydrolysis products may all be determined during a single LC-ESI- MS analysis. Derivatization of chemical warfare agent hydrolysis products and other compounds with hydroxyl substitution would be required prior to GC-MS analysis, giving LC-ESI-MS a definite advantage over GC-MS for the analysis of samples containing chemical warfare agents and/or their hydrolysis products.     

Application of an efficient strategy based on MAE,HPLC‐DAD‐MS/MS and HSCCC for the rapid extraction,identification, separation and purification of flavonoids from Fructus Aurantii Immaturus

This study presents an efficient strategy based on microwave‐assisted extraction (MAE), HPLC‐DAD‐MS/MS and high‐speed counter‐current chromatography (HSCCC) for the rapid extraction, identification, separation and purification of active components from the traditional Chinese medicine Fructus Aurantii Immaturus. An LC‐DAD‐MS/MS method was applied for the screening and structural identification of main components in crude extract, and five components were preliminarily identified as neoeriocitrin, narirutin, naringin, hesperidin and neohesperidin according to their UV and mass spectra. An efficient MAE method for the extraction of the three most abundant components (narirutin, naringin and neohesperidin) was optimized by the combination of univariate and multivariate approaches. The crude extract was then separated and purified by HSCCC and a total of 61.6 mg of narirutin, 207.3 mg of naringin and 159.5 mg of neohesperidin at high purities of 98.1, 97.2 and 99.5%, respectively, were obtained from 1.42 g of crude extract. The recoveries of these compounds were 86, 93 and 89%, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of synthetic phosphodiesterase-5 inhibitors found in a dietary supplement and pre-mixed bulk powders for dietary supplements using high-performance liquid chromatography with diode array detection and liquid chromatography-electrospray ionization tandem mass spectrometry

A high-performance liquid chromatography-diode array detection (HPLC-DAD) method and a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method were developed to screen for the presence of synthetic phosphodiesterase-5 (PDE-5) inhibitors and their analogues, namely sildenafil, vardenafil, tadalafil, homosildenafil, acetildenafil and hydroxyhomosildenafil. The methods were applied to pre-market samples submitted to the Health Sciences Authority of Singapore (HSA) for testing. One sample was in the form of capsules while six other samples were pre-mixed bulk powder samples for dietary supplements to be repackaged or formulated into the final dosage forms (usually capsules). Identification of PDE-5 inhibitors and their analogues was achieved by comparing individual peak retention times, UV spectra and mass spectra with those of reference standards. The seven samples were found to contain at least one of the following compounds: sildenafil, vardenafil, hydroxyhomosildenafil, homosildenafil and acetildenafil. The five compounds were simultaneously determined by LC-ESI-MS/MS in multiple reactions monitoring (MRM) scan mode. The method has been validated for accuracy, precision, linearity and sensitivity.     

Direct characterization of aqueous extract of Hibiscus sabdariffa using HPLC with diode array detection coupled to ESI and ion trap MS

The phenolic fraction and other polar compounds of the Hibiscus sabdariffa were separated and identified by HPLC with diode array detection coupled to electrospray TOF and IT tandem MS (DAD‐HPLC‐ESI‐TOF‐MS and IT‐MS). The H. sabdariffa aqueous extract was filtered and directly injected into the LC system. The analysis of the compounds was carried out by RP HPLC coupled to DAD and TOF‐MS in order to obtain molecular formula and exact mass. Posterior analyses with IT‐MS were performed and the fragmentation pattern and confirmation of the structures were achieved. The H. sabdariffa samples were successfully analyzed in positive and negative ionization modes with two optimized linear gradients. In positive mode, the two most representative anthocyanins and other compounds were identified whereas the phenolic fraction, hydroxycitric acid and its lactone were identified using the negative ionization mode.     

Qualitative and quantitative analysis of Swertia herbs by high performance liquid chromatography-diode array detector-mass spectrometry (HPLC-DAD-MS)

We evaluated the composition of Swertia herbs using high performance liquid chromatography-diode array detector-mass spectrometry (HPLC-DAD-MS). Eleven peaks of 6 species were unequivocally identified by comparing their retention times, UV spectra, on-line electrospray ionization mass (ESI-MS) spectra, and collision-induced dissociation mass spectrometry/mass spectrometry (CID-MS/MS) data with those of authentic compounds. We adopted wavelengths of 254 nm, 340 nm and 230 nm to simultaneously determine these 11 compounds. By comparing the overall DAD and total ion current (TIC) profiles of various samples, the 6 species were differentiated in terms of the occurrence and/or relative concentrations of the eleven compounds. Our novel validated HPLC-DAD-MS method not only facilitates quality control and identification of Swertia herbs, but is also applicable to systematic investigations of the distribution of secoiridoids, flavonoids, and xanthones in the genus Swertia.     

Characterization and identification of saikosaponins in crude extracts from three Bupleurum species using LC-ESI-MS

A LC-diode array detection (DAD)-ESI-MS/MS method was established for the online characterization and identification of saikosaponins (SSs) from extracts of roots of Bupleurum scorzonerifolium Willd, B. marginatum var. stenophyllum and B. komarovianum. In ESI-MS/MS spectra of SSs, [M-H](- )ions were subjected to the cleavage of glycosidic bonds and produced Y type ions, which can be used to elucidate the structures of saccharide chains and aglycones. Fragmentation of aglycones provided mass information about their major substitutions. For three structural types of SSs, type III can be easily identified by their fragmentation behaviors; while type I and II often occur as isomers and they can be discriminated by their typical UV absorption data. The only sugar ring-cross cleavage corresponding to 76 Da took place at a furanose sugar moiety. As a result, more than 75 SSs, including eight novel compounds, were identified or tentatively characterized based on their UV and mass spectra and retention times. The approach established here allows a comprehensive analysis of the SSs in the genus of Bupleurum and will be helpful for quality control of the crude materials and their related preparations.     

Use of high-performance liquid chromatography with diode array detection coupled to electrospray-Qq-time-of-flight mass spectrometry for the direct characterization of the phenolic fraction in organic commercial juices

We have developed a direct method for the qualitative analysis of polyphenols in commercial organic fruit juices. The juices were diluted with water (50/50), filtered and directly injected. The analysis of phenolic compounds was carried out by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to photodiode array detection (DAD) and electrospray ionisation-Qq-time-of-flight mass spectrometry (ESI-Qq-TOF-MS). A unique gradient program has been optimized for the separation of several phenolic classes and the analysis time was only 5 min. The fruit juice samples were successfully analysed in positive and negative ionisation modes. In positive mode the anthocyanins were identified whereas the vast majority of polyphenols were identified using the negative ionisation mode. The sensitivity, together with mass accuracy and true isotopic pattern of the Qq-TOF-MS, allowed the identification of the phenolic compounds. Moreover, the advantage of the proposed method is the combined search of MS and MS/MS spectra, which improves the identification of compounds considerably, reducing ambiguities and false positive hits. Therefore the total fragmentation of the compound ion leading to the aglycone ion or other fragments was corroborated by MS–MS. The method was successfully employed to characterize diverse phenolic families in commercially available organic juices from four different fruits and consequently could be used in the future for the quantification purposes to compare different content of polyphenols in juices.     

Metabolomic Analysis of the Lichen <Emphasis Type="Italic">Everniopsis trulla</Emphasis> Using Ultra High Performance Liquid Chromatography-Quadrupole-Orbitrap Mass Spectrometry (UHPLC-Q-OT-MS)

A new depside was identified in the methanolic extract from the lichen Everniopsis trulla based on the metabolomics UHPLC–DAD–MS analysis and HESI–MS–MS fragmentation patterns along with thirty-two known compounds for the first time. The compounds were structurally characterized by UV and high resolution quadrupole orbitrap mass spectra and by comparison with literature. According to the characteristic fragmentation patterns, the presence of two simple aromatic compounds, six lipid derivatives, eight depsidones, thirteen depsides, a chromone, two diphenylethers, and a dibenzofuran were identified. To our knowledge, this is the first study of the lichen E. trulla by liquid chromatography hyphenated with tandem mass spectrometry.     

Quality assessment of Cortex Phellodendri by high‐performance liquid chromatography coupled with electrospray ionization mass spectrometry

A simple method based on liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (LC‐DAD‐ESI‐MS) was developed for the quality assessment of Cortex Phellodendri (CP), which was mainly derived from two species of Phellodendron chinense Schneid and Phellodendron amurense Rupr. Total 41 compounds, including 14 phenols, 24 alkaloids and three liminoidal triterpenes were identified or tentatively characterized from the 75% methanol extract of CP samples by online ESI‐MSⁿ fragmentation and UV spectra analysis. Among them, two phenols and six alkaloids were simultaneously quantified using HPLC‐DAD method. The validated HPLC‐DAD method showed a good linearity, precision, repeatability and accuracy for the quantification of eight marker compounds. Furthermore, the plausible fragmentation pathway of the representative compounds were proposed in the present study. The differences of the chemical constituents content and the comprehensive HPLC profiles between the two CP species using LC‐DAD‐ESI‐MS method are reported for the first time, indicating that the CP drugs from different resources should be used separately in the clinic. Copyright © 2009 John Wiley & Sons, Ltd.     

天麻中对羟基苯类化合物的高效液相色谱-质谱研究

天麻中对羟基苯类化合物的(EsI(-))一级质谱图中除分子离子峰[M-1]外，还可见明显的[M-1 20] m[2m-1]峰，由此规律可方便确证未知同类样品的分子离子峰。由二级质谱(ESI/MS/MS)的特征碎片离子93、105和107等，结合紫外光谱及核磁结果确定了3个不纯样品中主要成分的结构，同时初步确定了两个同类杂质的结构。     

Systematic screening and characterization of the major bioactive components of Poria cocos and their metabolites in rats by LC-ESI-MS(n)

Poria cocos is a well-known medicinal plant widely used in China and other East Asian countries owing to its various therapeutic effects. However, the bioactive constituents responsible for the pharmacological effects of Poria cocos and their metabolites in vivo are still unclear to date. The aim of the present study was to develop a practical method based on the combined use of the liquid chromatography coupled with electrospray ionization multistage tandem mass spectrometry (LC-ESI-MS(n) ) for the comprehensive and systematic separation and characterization of the bioactive constituents of Poria cocos extract and their metabolites in rats. Based on the proposed strategy, a total of 34 compounds were characterized from the extract of Poria cocos. Among them, eight were unambiguously identified by comparing their retention times and mass spectra with those of reference standards, and 26 were tentatively identified on the basis of their MS(n) fragmentation behaviors and molecular weight information from literatures. In vivo, seven compounds were successfully detected in rat urine whereas one was found in rat plasma. This study proposed a series of potential bioactive components and provided helpful chemical information for further research on the action mechanism of traditional Chinese medicine.     

Analysis of phenolic compounds from different morphological parts of Helichrysum devium by liquid chromatography with on‐line UV and electrospray ionization mass spectrometric detection

A simple and rapid method has been used for the screening and identification of the main phenolic compounds from Helichrysum devium using high‐performance liquid chromatography with on‐line UV and electrospray ionization mass spectrometric detection (LC‐DAD/ESI‐MSⁿ). The total aerial parts and different morphological parts of the plant, namely leaves, flowers and stems, were analyzed separately. A total of 34 compounds present in the methanolic extract from Helichrysum devium were identified or tentatively characterized based on their UV and mass spectra and retention times. Three of these compounds were positively identified by comparison with reference standards. The phenolic compounds included derivatives of quinic acid, O‐glycosylated flavonoids, a caffeic acid derivative and a protocatechuic acid derivative. The characteristic loss of 206 Da from malonylcaffeoyl quinic acid was used to confirm the malonyl linkage to the caffeoyl group. This contribution presents one of the first reports on the analysis of phenolic compounds from Helichrysum devium using LC‐DAD/ESI‐MSⁿ and highlights the prominence of quinic acid derivatives as the main group of phenolic compounds present in these extracts. We also provide evidence that the methanolic extract from the flowers was significantly more complex when compared to that of other morphological parts. Copyright © 2009 John Wiley & Sons, Ltd.     

Metabolite profiling of licorice (Glycyrrhiza glabra) from different locations using comprehensive two-dimensional liquid chromatography coupled to diode array and tandem mass spectrometry detection

Profiling of the main metabolites from several licorice (Glycyrrhiza glabra) samples collected at different locations is carried out in this work by using comprehensive two-dimensional liquid chromatography (LC × LC) coupled to diode array (DAD) and mass spectrometry (MS) detectors. The optimized method was based on the application of a HILIC-based separation in the first dimension combined with fast RP-based second dimension separation. This set-up was shown to possess powerful separation capabilities allowing separating as much as 89 different metabolites in a single sample. Identification and grouping of metabolites according to their chemical class were achieved using the DAD, MS and MS/MS data. Triterpene saponins were the most abundant metabolites followed by glycosylated flavanones and chalcones, whereas glycyrrhizic acid, as expected, was confirmed as the main component in all the studied samples. LC × LC-DAD-MS/MS was able to resolve these complex licorice samples providing with specific metabolite profiles to the different licorice samples depending on their geographical origin. Namely, from 19 to 50 specific compounds were exclusively determined in the 2D-chromatograms from the different licorice samples depending on their geographical origin, which can be used as a typical pattern that could potentially be related to their geographical location and authentication.     

Identification and characterization of impurities in an insecticide,bifenthrin technical

The HPLC‐DAD and GC/MS methods were successfully used for the identification and characterization of the impurities in an agrochemical insecticide, bifenthrin technical. Three impurities ranging from 0.175%–0.541% were detected by the HPLC‐DAD method. The LC/MS technique with ESI or APCI source failed to detect the impurities detected by HPLC‐DAD, due to lack of ionization in ESI or APCI. The three impurities were enriched by prep‐HPLC, and then their structures were elucidated based on the GC/EIMS and CIMS data. The EI mass spectra of bifenthrin and its impurities displayed molecular ion and provided structure indicative fragment ions; the CIMS data further confirmed their molecular weight. The identity of the impurity 1 was further confirmed by the synthesis of the authentic sample followed by NMR and GC/MS data.     

Quantification of sugars and sugar phosphates in Arabidopsis thaliana tissues using porous graphitic carbon liquid chromatography-electrospray ionization mass spectrometry

This work reports the development and optimisation of a negative ion mode on-line LC-ESI-MS/MS method for the sensitive targeted analysis of the key glycolytic intermediates, sugars and sugar phosphates from plants, using a porous graphitic carbon (PGC) stationary phase and an MS compatible mobile phase. Using this newly developed method, separation and detection of a solution of standard compounds is achieved in less than 20min. Target metabolite compounds were identified in plant extracts from their characteristic retention times, and product ion spectra. This on-line PGC-ESI-MS/MS method shows good linearity over the concentration range 0-100microM, selectivity, short analysis time, and limits of detection of 0.1microM for disaccharides trehalose (Tre), sucrose (Suc), and maltose, and 1.5microM for hexose phosphates fructose-6-phosphate (Fru6P), glucose-1-phosphate (Glc1P), and glucose-6-phosphate (Glc6P), and phosphoenolpyruvate (PEP). This paper describes details of our method and its application to the simultaneous quantitative analysis of soluble sugars and sugar phosphates from Arabidopsis thaliana tissues. We have demonstrated the utility of our method for the analysis of biological samples by applying it to the simultaneous quantitation of changes in soluble sugars and sugar phosphates in A. thaliana Columbia-0 (Col-0) and its starchless phosphoglucomutase (pgm) mutant over a 12-h light/12-h dark growth cycle.     

Determination of 19 rhubarb constituents by high-performance liquid chromatography-ultraviolet-mass spectrometry

Rhubarb (Rhei rhizoma), a commonly used Chinese herb, contains anthraquinones, anthrones, galloylglucoses, stilbenes, and flavan-3-ols compounds, etc. as major constituents. Using 19 of these compounds as markers, an HPLC-UV-MS method was developed to estimate the quality of rhubarb samples within a period of 70 min. Extracts were analyzed with a Cosmosil 5C18-MS column and eluted with a gradient comprising an aqueous solution of acetic acid and methanol at a flow rate of 0.9 mL/min. Peaks were detected by absorbance measurements at 254 nm (6 and 8-19) and 280 nm (1-5 and 7), and the peaks of the marker substances were identified from their UV spectra and MS fragmentation patterns. The proposed method yielded a peak-area ratio RSD value with an intraday SD falling within 0.71-1.78% and an interday SD within 0.78-1.98% at a detection limit of 0.2-3.2 microg/mL. The ESI negative ion mode was used to collect data (molecular weight, CID fragments from MS and MS/MS spectra) for 19 compounds from four types of structure categories: anthraquinones, dianthrone glycosides, stilbenes, and galloylglucosides. The information gathered can be used to identify the structures of various peaks appearing in the LC chromatograms of rhubarb samples.