Chromatography-electrospray Ion Trap Mass Spectrometry Research Articles

Structural analysis of glycosaminoglycans from Colla corii asini by liquid chromatography-electrospray ion trap mass spectrometry.

Colla corii asini (CCA) made from donkey-hide has been widely used as a traditional animal-based Chinese medicine. Chondroitin sulfate (CS), dermatan sulfate (DS) and hyaluronic acid (HA) are structurally complex classes of glycosaminoglycans (GAGs) that have been implicated in a wide range of biological activities. However, their possible structural characteristics in CCA are not clear. In this study, GAG fractions containing CS/DS and HA were isolated from CCA and their disaccharide compositions were analyzed by high sensitivity liquid chromatography-ion trap/time-of-flight mass spectrometry (LC-MS-ITTOF). The result showed that CS/DS/HA disaccharides were detected in the three lower salt fractions from anion-exchange chromatography. The sulfation patterns and densities of CS/DS chains in these fractions differed greatly, while HA chains varied in their chain lengths. The quantitative analysis first revealed that the amount of GAGs in CCA varied significantly in total and in each fraction. This novel structural information could help clarify the possible involvement of these polysaccharides in the biological activities of CCA.

Novel Neuroprotective Lead Compound Ligustrazine Derivative Mass Spectrometry Fragmentation Rule and Metabolites in Rats by LC/LTQ-Orbitrap MS.

The neuroprotective evaluation of ligustrazine derivatives has become a research focus all over the world. A novel ligustrazine derivative, (3,5,6-Trimethylpyrazin-2-yl)methyl(E)-3-(4-((3,5,6-trimethylpyrazin-2-l)methoxy)phenyl)acrylate (T-CA), has shown protective effects against CoCl2-induced neurotoxicity in a differentiated PC12 cell model and middle cerebral artery occlusion (MCAO) model in our previous studies. However, nearly none of the parent drugs existed after rapid metabolism due to uncertain reasons. Thus, the fragmentation regularities of mass spectra, and metabolites, of T-CA in rats were examined using liquid chromatography-electrospray ionizationion trap mass spectrometry (LC/LTQ-Orbitrap MS) in this research. The main fragment ion, mass spectrum characteristics, and the structural information were elucidated. When compared with a blank sample, we identified five kinds of T-CA metabolites, including three phase I metabolites and two phase II metabolites. The results showed that the metabolic pathways of T-CA in rats via oral administration were hydrolysis (ether bond rupture, ester bond rupture), oxidation, reduction, glucose aldehyde acidification, etc. In addition, three main metabolites were synthesized and their structures were identified by superconducting high-resolution NMR and high-resolution mass spectroscopy (HR-MS). The neuroprotective activity of these metabolites was validated in a PC12 cell model. One of the metabolites (M2) showed significant activity (EC50 = 9.67 μM), which was comparable to the prototype drug T-CA (EC50 = 7.97 μM). The current study provides important information for ligustrazine derivatives, pertaining to the biological conversion process in vivo.

Characterization of fortimicin aminoglycoside profiles produced from Micromonospora olivasterospora DSM 43868 by high-performance liquid chromatography-electrospray ionization-ion trap-mass spectrometry.

In this study, an efficient high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-ion trap-tandem mass spectrometry (MS/MS) was developed for the identification of the biosynthetic congeners involved in the aminocyclitol aminoglycosidic fortimicin pathway from Micromonospora olivasterospora fermentation. The usage of both acid extraction (pH ∼2.5) followed by an cationic-exchanging SPE cleanup and pentafluoropropionic acid mediated ion-pairing chromatography with ESI-ion trap-MS/MS detection was determined to be sufficiently practical to profile the fortimicin (FOR) congeners produced in a culture broth. The limit of the quantification for the fortimicin A (FOR-A) standard spiked in the culture broth was ∼1.6 ng mL(-1). The average recovery rate was 93.6%, and the intra- and inter-day precisions were <5% with accuracy in the range from 87.1 to 94.2%. Moreover, the epimeric mixtures including FOR-KH, FOR-KR, and FOR-B were separately resolved through a macrocyclic glycopeptide (teicoplanin)-bonded chiral column. As a result, ten natural FOR pseudodisaccharide analogs were identified and semi-quantified in descending order as follows: FOR-A, FOR-B, DCM, FOR-KH plus FOR-KR, FOR-KK1, FOR-AP, FOR-KL1, FOR-AO, and FOR-FU-10. This is the first report on both the simultaneous characterization of diverse structurally closely related FORs derived from bacterial fermentation using HPLC-ESI-ion trap-MS/MS analysis and the chromatographic separation of the three FOR epimers.

Microwave-assisted Extraction Coupled with High Performance Liquid Chromatography-electrospray Ion Trap Mass Spectrometry for the Determination of Three Flavone di-C-glycosides in Viola yedoensis

The flavone C-glycosides in Viola yedoensis have been reported as the major active antiinflammatory, anti-cancer, and antioxidant components. However, due to the difficulty in obtaining standard compounds, little work has been done on the quantitative analysis of the multiple major flavone C-glycosides in V. yedoensis. In this study, a microwave-assisted extraction (MAE) and high performance liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry (HPLC-ESI-MS/MS) method in the selected reaction-monitoring (SRM) mode was developed and validated for the simultaneous separation and quantification of three major active flavone di-Cglycosides (apigenin 6,8-di-C-β-D-glucoside, apigenin 6-C-β-D-glucosyl-8-C-α-L-arabinoside, and apigenin 6,8-di-C-α-L-arabinoside) in V. yedoensis. The MAE conditions were optimized using an orthogonal design L9 (34). The extraction yields by MAE were the same or even higher than traditional extraction methods. All calibration curves showed good linearity (r2 > 0.9986) over their corresponding test ranges. The LOD ranged from 17 to 34 ng/mL and LOQ ranged from 34 to 68 ng/mL. The intra- and inter-day precisions (%RSD) were within 2.64% for the three analytes. The recovery of the assay was in the range of 97.67-98.93% with RSD values less than 2.54%. The developed HPLC-MS/MS method was used to quantitatively analyze the three di-C-glycosides in 14 batches of V. yedoensis samples. This newly developed HPLC-MS/MS method coupled with MAE is sensitive and accurate for simultaneous determination of multiple flavone C-glycosides in V. yedoensis. Keywords: Flavone C-glycosides, HPLC-MS/MS, microwave-assisted extraction, quantification, selected reaction-monitoring, Viola yedoensis.

Simultaneous determination of four designer drugs and their major metabolites by liquid chromatography-mass spectrometry.

A sensitive liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-ITMS) method was utilized for the simultaneous analysis of four designer drugs and their in vitro metabolites in rat liver microsome S9 fraction. Four designer drugs, including methcathinone (MC), 3,4-methylenedioxymethcathinone (MDMC), 3,4-methylenedioxy-pyrovalerone (MDPV) and 4'-methyl-α-pyrrolidinopropiophenone (MPPP), were individually incubated with rat liver microsome S9 fraction, and the incubation mixtures were pooled together and analyzed by LC-ESI-ITMS simultaneously. Besides four designer drugs, five of their main metabolites were identified via the analysis of protonated molecules and tandem mass spectrometry data. Meanwhile, the quantification analysis of four designer drugs in rat liver microsome S9 fraction was performed, the calibration curves showed good linearity in the range of 0.01-5.0μg/mL and the detection limits were below 0.03μg/mL with RSDs less than 5.9% and recovery ratios above 77.4%. The experimental results not only showed that these designer drugs could be easily metabolized in rat liver microsome, and also displayed the superiorities of the method including time and cost saving, high efficiency, sensitivity and selectivity. The studies in this study indicated that the approach could be applied in the determination of illicit drugs and their metabolites in medical, pharmaceutical and forensic investigations.

Online capillary solid-phase microextraction coupled liquid chromatography-mass spectrometry for analysis of chiral secondary alcohol products in yeast catalyzed stereoselective reduction cell culture

An online solid-phase microextraction coupled liquid chromatography-electrospray ionization-ion trap mass spectrometry was developed for the analysis of trace R- and S-4-phenyl-2-butanol (R- and S-pbol) in salt rich cell culture of Saccharomyces cerevisiae catalyzed stereoselective reduction of 4-pheny-2-butanone (pbone). A Supel-Q PLOT capillary column was used for the extraction and deionized distilled water was used as the extraction mobile phase. The extraction flow rate and extraction time were at 0.1mLmin−1 and 0.95min, respectively. The three target analytes, pbone, R-pbol, and S-4-pbol, were desorbed and eluted by the mobile phase of water/methanol/isopropanol (55/25/20, v/v/v) with a flow rate of 0.5mLmin−1 and analyzed by a chiral column. The mass spectrometric detection of the three target analytes was in positive ion mode with the signal [M+Na]+. The matrix-matched external standard calibration curves with linear concentration range between 0 and 50μgmL−1 were used for quantitative analysis. The linear regression correlation coefficients (r2) of the standard calibration curves were between 0.9950 and 0.9961. The yeast mediated reduction was performed with a recation culture of yeast incubation culture/glycerol (70/30, v/v) for 4 days. This biotransformation possessed 82.3% yield and 92.9% S-enantomeric excess. The limit of detection (LOD)/limit of quantification (LOQ) for pbone, R-pbol, and S-pbol was 0.02/0.067, 0.01/0.033, and 0.01/0.033μgmL−1, respectively. The intra-day and inter-day precisions from repeated measurements were 10.8–21.1% and 11.6–18.7%, respectively. The analysis accuracy from spike recovery was 84–91%.

Determination of Small Phenolic Compounds in Tequila by Liquid Chromatography with Ion Trap Mass Spectrometry Detection

Tequila is elaborated from Agave tequilana Weber blue variety and it is commercialized at different stages of aging. Chemical composition of this product has often been addressed; however, data on phenolic compounds are scarce. In this work, a high-performance liquid chromatography–electrospray ionization-ion trap mass spectrometry (HPLC–ESI-ITMS) procedure has been established for the determination of 34 small phenolic compounds. The combination of suitable separation conditions with extraction of chromatograms at individual m/z values has enabled for total analysis run of 17 min (11 min separation plus 6 min column cleaning/equilibration) with the detection limits in the range 1.28–75.0 μg l−1 (0.07–6.1 pmol on-column). Commercial tequilas analyzed included 6 white, 12 rested, and 4 aged. The following acids were found and quantified: gallic, procatechuic, 4-hydroxybenzoic, vanillic, syringic, homovanillic, 3-hydroxybenzoic, ferulic, salicylic, and benzoic. The white tequilas contained fewer compounds and lower total phenolics concentrations (range 36–408 μg l−1) as compared to the rested and aged liquors (515–4,296 and 2,048–3,249 μg l−1, respectively). In the latter products, syringic, vanillic, procatechuic, and gallic acids were the most abundant, which indicates that maturation in wooden barrels is the main source of small phenolics in tequila. On the other part, homovanillic acid was found in all tequila types (medians for white, rested, and aged products 82, 153, and 162 μg l−1, respectively), suggesting that some phenolics may originate from the raw material or might be formed during liquor elaboration.

Determination and quantitative analysis of designer drugs in human urine by liquid chromatography-mass spectrometry

A robust liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-ITMS) method was developed and utilized for the determination and quantitative analysis of four designer drugs in human urine.

A sensitive HPLC assay for quantitative determination of polyamidoamine dendrimers after pre-column derivatization using FITC

A pre-column derivatization method was developed to analyze polyamidoamine (PAMAM) dendrimers. The fluorescein isothiocyanate (FITC) derivation method improved the retention of PAMAM dendrimers on the reversed-phase column of high performance liquid chromatography (RP-HPLC). The derivation benefited the UV-Vis detection of PAMAM, although the fluorescence of FITC was quenched by PAMAM dendrimers. The primary amine-terminated PAMAM dendrimers, generation 3 (PAMAM-NH2 (G3)), were quantitatively determined by UV-Vis spectroscopy over the concentration range of 10–80 μg mL−1, with the calibration curve expressed as A = 131.6C (μg mL−1) − 295.6 (R2 = 0.9901) and the detection limit of 2.7 μg mL−1. The binding ratio of FITC to PAMAM-NH2 (G3) was determined as 8 : 1 by liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-IT-MS).

Simultaneous analysis of analgesics and their major metabolites by liquid chromatography-electrospray ion trap mass spectrometry

A high-throughput analysis method applying liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-ITMS) for the simultaneous analysis of analgesics and their in vitro metabolites is described. Four analgesics, including tramadol, ketamine, pethidine and methadone, were individually incubated with rat liver microsome S9 fraction initially. The incubation samples were pooled together for analysis by LC-ESI-ITMS in positive ion and full-scan detection modes. The analgesics and their metabolites were identified from the detection of the protonated molecules. The metabolite detection was confirmed by tandem mass spectrometry (MSn) analysis. In addition, the metabolic stabilities of the analgesics were determined by comparing the peak intensities between the control and incubated samples. The obtained results indicated that the method could provide a significant increase in analytical throughput and can be applied in the detection of samples in forensic analysis and in clinically addicted cases.

Simultaneous determination of 24 or more acidic and alkaline phytohormones in femtomole quantities of plant tissues by high-performance liquid chromatography–electrospray ionization–ion trap mass spectrometry

Phytohormones act at relatively low concentrations as major regulatory factors of plant growth and development, and cross talk of phytohormones is currently of great interest throughout the plant science community. To meet this demand, a method that is capable of simultaneously analyzing diverse plant hormones is essential. This paper introduces a high-performance liquid chromatographic separation technique coupled with sensitive and selective ion trap mass spectrometry to simultaneously determine 24 or more acidic and alkaline phytohormones, including auxin, cis- and trans-abscisic acid, 11 cytokinins, and 10 gibberellins, in a single injection of sample. A binary solid-phase extraction using Oasis MCX cartridges for cations and Oasis MAX cartridges for anions was used to prepurify more than 24 acidic and alkaline phytohormones from a single plant extract. The method showed good linearity for all 24 phytohormones with R(2) values ranging from 0.9903 to 0.9997. Limits of detection for most of the phytohormones were in the femtomole range with some extending into the sub-femtomole range. This method was applied to hundreds of plant samples comprising different tissues from various plants, including herbaceous, woody climbing, and woody plants to demonstrate feasibility and to validate the methodology.

In Vitro Studies on the Oxidative Metabolism of 20(S)-Ginsenoside Rh2 in Human, Monkey, Dog, Rat, and Mouse Liver Microsomes, and Human Liver S9

20(S)-Ginsenoside Rh2 (Rh2)-containing products are widely used in Asia, Europe, and North America. However, extremely limited metabolism information greatly impedes the complete understanding of its clinical safety and effectiveness. The present study aims to systematically investigate the oxidative metabolism of Rh2 using a complementary set of in vitro models. Twenty-five oxidative metabolites were found using liquid chromatography-electrospray ionization ion-trap mass spectrometry. Six metabolites and a metabolic intermediate were synthesized. The metabolites were structurally identified as 26-hydroxy Rh2 (M1-1), (20S,24S)-epoxydammarane-12,25-diol-3-β-d-glucopyranoside (M1-3), (20S,24R)-epoxydammarane-12,25-diol-3-β-d-glucopyranoside (M1-5), 26,27-dihydroxy Rh2 (M3-6), (20S,24S)-epoxydammarane-12,25,26-triol-3-β-d-glucopyranoside (M3-10), (20S,24R)-epoxydammarane-12,25,26-triol-3-β-d-glucopyranoside (M3-11), and 26-aldehyde Rh2 on the basis of detailed mass spectrometry and nuclear magnetic resonance data analysis. Double-bond epoxidation followed by rearrangement and vinyl-methyl group hydroxylation represent the initial metabolic pathways generating monooxygenated metabolites M1-1 to M1-5. Further sequential metabolites (M2-M5) from the dehydrogenation and/or oxygenation of M1 were also detected. CYP3A4 was the predominant enzyme involved in the oxidative metabolism of Rh2, whereas alcohol dehydrogenase and aldehyde dehydrogenase mainly catalyzed the metabolic conversion of alcohol to the corresponding carboxylic acid. No significant differences were observed in the phase I metabolite profiles of Rh2 among the five species tested. Reactive epoxide metabolite formation in both humans and animals was evident. However, GSH conjugate M6 was detected only in cynomolgus monkey liver microsomal incubations. In conclusion, Rh2 is a good substrate for CYP3A4 and could undergo extensive oxidative metabolism under the catalysis of CYP3A4.

Mass spectrometry‐based chemotaxonomic classification of Penicillium species (P. echinulatum, P. expansum, P. solitum, and P. oxalicum) and its correlation with antioxidant activity

In this study, 4 Penicillium species (17 strains) were classified on the basis of metabolite profile (chemotaxonomy) by using liquid chromatography-electrospray ionization ion trap-mass spectrometry (LC-ESI-MS), gas chromatography-ion trap-mass spectrometry (GC-IT-MS) and multivariate statistical analysis. The LC-ESI-MS-based dendrogram was similar to the internal transcribed spacer (ITS)-based dendrogram, in that Penicillium oxalicum was separated from the other 3 species. Moreover, vermiculidiol, meleagrin, oxaline, glandicolin A and B, and secalonic acid D were identified as metabolites that enable discrimination of Penicillium species by partial least squares discriminant analysis (PLS-DA). Evaluation of the species-specific metabolites produced by P. expansum, P. echinulatum, and P. solitum revealed that the 3 species differed from each other. On the other hand, GC-IT-MS-based dendrogram revealed that P. expansum was clearly classified separately from the other 3 species, and this result correlated with the antioxidant activity of the 4 species: P. expansum had a higher radical scavenging activity than the other 3 species. The metabolites produced in higher amounts in P. expansum were gluconic acid (12, 29, 33); andrastin A (16), B (15), and C (17); chaetoglobosin C (14), a class of sugar (31, 32); and salicylic acid (28). The results of this study demonstrated that metabolite-based chemotaxonomy could be used not only as a classification method but also as a tool for evaluation of species-specific activities.

Simultaneous Analysis of Strychnine and Brucine and their Major Metabolites by Liquid Chromatography-Electrospray Ion Trap Mass Spectrometry

A liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-ITMS) method was developed for the simultaneous analysis of strychnine, brucine and their major metabolites. Strychnine and brucine were individually incubated with rat liver S9 fraction. The incubation samples were pooled together and analyzed with LC-ESI-ITMS in positive ion and full-scan detection mode. The calibration curves of strychnine and brucine in rat liver showed good linearity in ranges of 0.020 to 8.0 µg/mL for strychnine and 0.020 to 8.5 µg/mL for brucine. The limits of detections were both 0.008 µg/mL and the recoveries were 88.3 and 83.2% for strychnine and brucine, respectively. Two metabolites were identified as strychnine N-oxide and brucine N-oxide by comparing the molecular mass, retention time, full-scan mass spectra, tandem MS and MS(3) spectra with those of strychnine and brucine. The developed method provided high sensitivity and selectivity for the determination of poisonous alkaloids and their major metabolites and can be applied in the determination of samples in forensic and clinically toxicological cases.

Determination of Tinuvin 292 in Acrylic Resins by Reversed-phase High-performance Liquid Chromatography–Electrospray Ionization–Ion Trap Mass Spectrometry

Abstract Due to recent reports on toxicity of hindered-amine light stabilizers (HALS) in polymer materials, demand for the regulation of the dosage of HALS is increasing. Therefore development of a quantitation method for low concentration of HALS is required either for toxicity study of HALS in water or saliva extracts from polymeric materials or for accurate determination of reduced amounts of HALS in polymeric materials. Here we present a novel quantitation method for Tinuvin 292, a worldwide used HALS, in acrylic resins using high-performance liquid chromatography–electrospray ionization–ion trap mass spectrometry at a broad concentration ranging from 10 ppb to 50 ppm. Collision-induced dissociation was performed using helium gas, and the multiple reaction monitoring parameters were optimized for detection of parent/daughter ions of Tinuvin 292. Sample preparation employed a very simple extraction of Tinuvin 292 from acrylic resins with considerable recovery. The method was successfully applied to quantitate Tinuvin 292 in various acrylic polymers.

Investigation of antibacterial phytochemicals in the bark and leaves of Ficus coronata by high‐performance liquid chromatography‐electrospray ionization–ion trap mass spectrometry (HPLC‐ESI‐MSn) and ESI‐MSn

The ethyl acetate extracts of the bark and leaves of Ficus coronata were separated by column chromatography and the resulting fractions tested for their bioactivity toward methicillin-resistant-Staphylococcus aureus (MRSA) and M. luteus. The bioactive column chromatography fractions were further separated by preparative thin layer chromatography (TLC) and the resulting bands investigated by high-performance liquid chromatography-electrospray ionization-ion trap mass spectrometry (HPLC-ESI-MS(n) ) and ESI-MS(n) . The resulting retention times, molecular masses, their fragmentation patterns, and the chemnet database (www.chemnetbase.com) were then used in the dereplication process by structural elucidation of some of the compounds when compared with known structures of natural origin. Some molecular masses and the corresponding fragmentations were found that did not correlate with any known compounds thus revealing potentially novel natural products that could be investigated on a larger scale and could ultimately find application as new drugs against MRSA and other multidrug-resistant microorganisms. Structures are also proposed for known compounds that have not been previously reported for F. coronata.

Characterization of Gallotannins from Astronium Species by Flow Injection Analysis-Electrospray Ionization-Ion Traptandem Mass Spectrometry and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

The species Astronium urundeuva (Allemao) Engl. and Astronium graveolens Jacq., which are used in Brazilian folk medicine to treat allergies, inflammation, diarrhea and ulcers, were investigated for their composition. The aim of this study was to define a rapid and reliable analytical approach, based on the flow-injection analysis-electrospray ionization-ion trap-tandem mass spectrometry (FIA-ESI-IT-MS-MS) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF-MS), to investigate the full range of hydrolyzable tannins present in the extracts of these Astronium species. The MALDI-ToF-MS analysis allowed us to ascertain the presence of hydrolysable tannins in both Astronium species as a series of gallotannins with degrees of polymerization of 7 to 13 galloyl units. Moreover, the analysis by FIA-ESI-IT-MS-MS, as well as confirming this result and chemically defining gallotannins as galloylglucose compounds, highlighted the presence of further classes of hydrolysable tannins, such as hexahydrodiphenoyl esters of glucose and some gallic acid derivatives, providing information about their structure by a careful study of their fragmentation patterns. Finally, the evaluation of the number of positional isomers of gallotannins occurring in both Astronium species was obtained by high-performance liquid chromatography-electrospray ionization-ion trap mass spectrometry (HPLC/ESI-IT-MS). This is the first mass spectrometric evidence relating to the existence of gallotannins in Astronium genus.

HPLC-ESI/MS/MS를 이용한 생양파와 흑양파의 퀘세틴 배당체 분석

퀘세틴은 양파에 있는 주요한 플라보노이드이고, 항산화제 역할을 한다. 양파에 있는 퀘세틴은 주로 배당체 형태로 존재한다. 흑양파는 습도 90%와 온도 <TEX>$60^{\circ}C$</TEX> 조건 하에서 30일 동안의 숙성처리과정으로 만들어진다. 흑양파 제조과정 전후의 양파 속에 있는 퀘세틴과 퀘세틴 배당체들은 HPLC-ESI/MS/MS를 이용하여 분석되었다. 생양파 속에는 퀘세틴 단당체와 퀘세틴 이당체가 동정되었고, 반면에 흑양파 속에는 퀘세틴만이 존재하였다. 그러한 결과들은 생양파에 있는 퀘세틴 배당체(단당체와 이당체)들은 숙성과정 동안 해당과정이 일어난다는 것을 의미한다. 이러한 분석프로파일은 숙성과정 동안 발생되는 양파의 주요한 두 개의 퀘세틴 배당체들의 변화를 추정하는데 용이한 방법을 제공할 것이다. Quercetin is a major flavonoid present in onions, which acts as an antioxidant. Quercetin exists both as a free compound and conjugated with carbohydrates, primarily as glucosides in onion. Aged black onion was made through a 30 day aging process in which the onions were kept in an environment of <TEX>$60^{\circ}C$</TEX> and high humidity (90% RH). Quercetin and quercetin glucosides were assayed in onion bulbs before and after the aging process, using high performance liquid chromatography-electrospray ion trap mass spectrometry (HPLC-ESI/MS/MS). Quercetin mono- and diglucosides were identified in fresh onion bulbs, whereas quercetin aglycone was the only form present in aged black onion bulbs. These findings indicate that the quercetin mono- and di-glucosides present in fresh onions undergo complete deglycosylation during the aging process. Such profiling will provide a rapid method that can be used to assess changes in the two major quercetin glycosides during the aging process of onion bulbs.

Elucidation of the Structures of Metabolites of Picroside II in Rat Bile by LC–ESI–IT–MS

Picroside II is one of the main active constituents of Picrorhiza kurroa, which has hepatoprotective, anticholestatic, antioxidant, and immune-modulating activity. To gain an understanding of the biotransformation of picroside II in vivo, liquid chromatography–electrospray ionization ion-trap mass spectrometry (LC–ESI–IT–MS) was used to investigate the metabolism of picroside II in rats after intravenous administration of a single dose. This method could simultaneously determine picroside II and its metabolites in rat bile. The bile samples were purified by use of a C18 solid-phase extraction (SPE) cartridge and were separated on a Hypersil ODS2 C18 analytical column. Two phase II metabolites of picroside II in rat bile were characterized, and elucidation of their structures was performed by comparing changes in molecular masses (ΔM), retention times, and MS2 spectral patterns of metabolites with those of the parent drug. Two metabolites identified for the first time in this research were glucuronide and sulfate conjugates.

Simultaneous quantification of lignans in Arabidopsis thaliana by highly sensitive capillary liquid chromatography-electrospray ionization-ion trap mass spectrometry

Lignans are phenylpropanoid dimers in which the monomers are linked by the central carbon (C8) atoms. Because many lignans have physiological effects, including antioxidant activity, they are now in demand as components in health foods. However, the lignan biosynthetic pathways in plants are only now being understood. Recently, lariciresinol was detected in Arabidopsis thaliana. This observation indicated the existence of common lignan biosynthetic pathways in A. thaliana, despite a low amount of lignans other than lariciresinol glycosides. In this study, we established a highly sensitive analytical method that enables quantification of both glycoside and aglycone forms of lignans in A. thaliana simultaneously using capillary liquid chromatography-electrospray ionization-ion trap mass spectrometry. Some lignans not previously detected in A. thaliana were quantified in extracts of both roots and shoots. Our method can be used for the comprehensive analysis of lignans in small samples from mutants and transformants. This method will be utilized to elucidate the metabolic pathways and physiological roles of lignans as well as the regulation of their biosynthesis in plants.