Study of the in vitro metabolism of TJ0711 using ultra high performance liquid chromatography with quadrupole time‐of‐flight and ultra fast liquid chromatography with quadrupole linear ion trap mass spectrometry

TJ0711 (1‐[4‐(2‐methoxyethyl)phenoxy]‐3‐[2‐(2‐methoxyphenoxy)ethylamino]‐2‐propanol) is a novel β‐adrenoreceptor blocker with vasodilating activity. The aim of this study was to investigate the in vitro metabolic properties of TJ0711 from both qualitative and quantitative aspects using mouse, rat, dog, and human liver microsomes as well as rat hepatocytes. Two modern liquid chromatography with tandem mass spectrometry systems, ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry and ultra fast liquid chromatography with quadrupole linear ion trap mass spectrometry, were utilized for the analysis. To better characterize the metabolic pathways of TJ0711, two major metabolites were incubated under the same conditions as that for TJ0711. TJ0711 was extensively metabolized in vitro, and a total of 34 metabolites, including 19 phase I and 15 phase II metabolites, were identified. Similar metabolite profiles were observed among species, and demethylation, hydroxylation, carboxylic acid formation, and glucuronidation were proposed as the major metabolic routes. Significant interspecies differences were observed in the metabolic stability studies of TJ0711. Furthermore, gender differences were significant in mice, rats, and dogs, but were negligible in humans. The valuable information provided in this work will be useful in planning and interpreting further pharmacokinetic, in vivo metabolism and toxicological studies of this novel β‐blocker.     

Characterization of tropane and cinnamamide alkaloids from Scopolia tangutica by high‐performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry

Scopolia tangutica is a traditional Chinese medicine used for antispasmodic, anesthesia, analgesia, and sedation. Its medicinal activity is associated to alkaloid constituents, including tropane and cinnamamide types. Low content of alkaloids in plant makes them difficult to be isolated and identified. The present work developed an effective method to quickly characterize alkaloids from Scopolia tangutica by high‐performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry. Thirteen reference compounds were studied for their fragmentation pathways, including five tropane alkaloids and eight cinnamamide ones. Alkaloid constituent was analyzed by an optimized high‐performance liquid chromatography method and mass spectrometry analysis to achieve systematic characterization of alkaloids from Scopolia tangutica. As a result, 53 compounds were identified, including 21 tropane alkaloids (eight new ones), 18 caffeoyl ones (ten new ones) and 14 dicaffeoyl ones (seven new ones). It was important to provide rich information in phytochemical study and structure‐guided isolation of important compounds from this plant.     

Optimization of a quadrupole ion storage trap as a source for time‐of‐flight mass spectrometry

Designs of a quadrupole ion trap (QIT) as a source for time‐of‐flight (TOF) mass spectrometry are evaluated for mass resolution, ion trapping, and laser activation of trapped ions. Comparisons are made with the standard hyperbolic electrode ion trap geometry for TOF mass analysis in both linear and reflectron modes. A parallel‐plate design for the QIT is found to give significantly improved TOF mass spectrometer performance. Effects of ion temperature, trapped ion cloud size, mass, and extraction field on mass resolution are investigated in detail by simulation of the TOF peak profiles. Mass resolution (m/Δm) values of several thousand are predicted even at room temperature with moderate extraction fields for the optimized design. The optimized design also allows larger radial ion collection size compared with the hyperbolic ion trap, without compromising the mass resolution. The proposed design of the QIT also improves the ion–laser interaction volume and photon collection efficiency for fluorescence measurements on trapped ions. Copyright © 2011 John Wiley & Sons, Ltd.     

Metabolic profiling of quercetin in rats using ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry

Quercetin, a kind of major flavonoid found in many traditional chinese medicines, is an effective substance for treatments such as lowering blood lipids. However, the studies on quercetin have been mainly focused on its pharmacological effect; the treatment of diseases on a material basis, particularly the metabolites derived from quercetin in vivo , has not been evaluated. In this study, we determined the levels, distributions and types of quercetin's metabolites in plasma, urine, feces and bile of rats after a single oral administration of quercetin at a dose of 80 mg/kg, using ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (UPLC‐Q‐TOF/MS). A total of 36 metabolites of quercetin were identified, including 11 metabolites in plasma, 34 metabolites in urine, 12 metabolites in feces and 21 metabolites in bile. The results showed that phase I metabolites were reduction metabolites and phase II metabolites mainly included glucuronidation, sulfation and methylation metabolites. These results provide important information on the metabolism of quercetin, which will be helpful for its further development and utilization.     

Characterization of in vitro metabolites of trimethoprim and diaveridine in pig liver microsomes by liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry

Trimethoprim (TMP) and diaveridine (DVD) are used in combination with sulfonamides and sulfaquinoxlaine as an effective antibacterial agent and antiprotozoal agent, respectively, in humans and animals. To gain a better understanding of the metabolism of TMP and DVD in the food-producing animals, the metabolites incubated with liver microsomes of pigs were analyzed for the first time with high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry. Seven TMP-related and six DVD-related metabolites were characterized based on the accurate MS2 spectra and known structure of the parent drug, respectively. The metabolites of TMP were identified as two O-demethylation metabolites, a di-O-demethylation metabolite, two N-oxides metabolites, a hydroxylated metabolite on the methylene carbon and a hydroxylated metabolite on the methyl group. DVD was also biotransformed to two O-demethylation metabolites, a di-O-demethylation metabolite, an N-oxide metabolite, a hydroxylation metabolite on the methylene carbon and a hydroxylation metabolite followed by O-demethylation. The results indicate that the two compounds have similar biotransformation pathways in pigs. O-Demethylation was the major metabolic route of TMP and DVD in the pig liver microsomes. The proposed metabolic pathways of TMP and DVD in liver microsomes will provide a basis for further studies of the in vivo metabolism of the two drugs in food-producing animals.     

An introduction to hybrid ion trap/time‐of‐flight mass spectrometry coupled with liquid chromatography applied to drug metabolism studies

Metabolism studies play an important role at various stages of drug discovery and development. Liquid chromatography combined with mass spectrometry (LC/MS) has become a most powerful and widely used analytical tool for identifying drug metabolites. The suitability of different types of mass spectrometers for metabolite profiling differs widely, and therefore, the data quality and reliability of the results also depend on which instrumentation is used. As one of the latest LC/MS instrumentation designs, hybrid ion trap/time‐of‐flight MS coupled with LC (LC‐IT‐TOF‐MS) has successfully integrated ease of operation, compatibility with LC flow rates and data‐dependent MSⁿ with high mass accuracy and mass resolving power. The MSⁿ and accurate mass capabilities are routinely utilized to rapidly confirm the identification of expected metabolites or to elucidate the structures of uncommon or unexpected metabolites. These features make the LC‐IT‐TOF‐MS a very powerful analytical tool for metabolite identification. This paper begins with a brief introduction to some basic principles and main properties of a hybrid IT‐TOF instrument. Then, a general workflow for metabolite profiling using LC‐IT‐TOF‐MS, starting from sample collection and preparation to final identification of the metabolite structures, is discussed in detail. The data extraction and mining techniques to find and confirm metabolites are discussed and illustrated with some examples. This paper is directed to readers with no prior experience with LC‐IT‐TOF‐MS and will provide a broad understanding of the development and utility of this instrument for drug metabolism studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Rapid separation and identification of Strychnos alkaloids metabolites in rats by ultra high performance liquid chromatography with linear ion trap Orbitrap mass spectrometry

An in vivo study of Strychnos alkaloids metabolites in rats by ultra high performance liquid chromatography with linear ion trap Orbitrap MS is reported for the first time. Two major Strychnos alkaloids compounds including strychnine and brucine were investigated. To obtain optimal extraction efficiency, samples were pretreated by using an SPE plate. The structures of metabolites and their fragment ions were characterized based on the accurate mass and MSⁿ data. Forty‐seven metabolites were identified in rat urine, of which 25 were reported for the first time. Four new metabolism pathways were proposed on the basis of the identified metabolites. This study provides a practical approach for rapidly identifying complicated metabolites, a methodology that could be widely applied not only in forensic and clinically toxicological relevant cases, but also for the structural characterization of metabolites of other compounds.     

In vivo metabolite identification of acotiamide in rats using ultra‐performance liquid chromatography–quadrupole/time‐of‐flight mass spectrometry

Acotiamide hydrochloride (ACT) is a drug used for the treatment of functional dyspepsia. Understanding which metabolites are likely to be formed in vivo is essential for interpreting pharmacology, pharmacokinetic and toxicology data. The metabolism of ACT has been investigated using a specific and sensitive liquid chromatography positive ion electrospray ionization high‐resolution tandem mass spectrometry method. In vivo samples including rat plasma, urine and feces were collected separately after dosing healthy Sprague–Dawley rats at a dose of 20 mg kg ⁻¹ ACT at different time points up to 24 h. The metabolites were enriched by optimized sample preparation involving protein precipitation using acetonitrile followed by solid‐phase extraction. The mass defect filter technique was used for better detection of both predicted and unexpected drug metabolites with the majority of interference ions removed. The structural elucidation of the metabolites was performed by comparing their [M + H]⁺ ions and their product ions with those of the parent drug. As a result, a total of seven hitherto unknown metabolites were characterized from the biosamples. The only phase I metabolite detected was N‐ despropyl acotiamide, whereas six phase II glucuronide conjugate metabolites were identified.     

Development of a tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source

A new tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source ‘ESI‐TOF/quadTOF’ was designed and constructed to achieve the desired aim of structural elucidation via high‐energy collision‐induced dissociation (CID), and the simultaneous detection of all fragment ions. The instrument consists of an orthogonal acceleration‐type ESI ion source, a linear TOF mass spectrometer, a collision cell, a quadratic‐field ion mirror and a microchannel plate detector. High‐energy CID spectra of doubly protonated angiotensin II and bradykinin were obtained. Several fragment ions such as a‐, d‐, v‐ and w‐type ions, characteristic of high‐energy CID, were clearly observed in these spectra. These high‐energy CID fragment ions enabled confirmation of the complete sequence, including leucine–isoleucine determinations. It was demonstrated that high‐energy CID of multiply protonated peptides could be achieved in the ESI‐TOF/quadTOF. Copyright © 2010 John Wiley & Sons, Ltd.     

Comprehensive identification and structural characterization of target components from Gelsemium elegans by high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry based on accurate mass databases combined with MS/MS spectra

This study reports an applicable analytical strategy of comprehensive identification and structure characterization of target components from Gelsemium elegans by using high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (LC‐QqTOF MS) based on the use of accurate mass databases combined with MS/MS spectra. The databases created included accurate masses and elemental compositions of 204 components from Gelsemium and their structural data. The accurate MS and MS/MS spectra were acquired through data‐dependent auto MS/MS mode followed by an extraction of the potential compounds from the LC‐QqTOF MS raw data of the sample. The same was matched using the databases to search for targeted components in the sample. The structures for detected components were tentatively characterized by manually interpreting the accurate MS/MS spectra for the first time. A total of 57 components have been successfully detected and structurally characterized from the crude extracts of G. elegans , but has failed to differentiate some isomers. This analytical strategy is generic and efficient, avoids isolation and purification procedures, enables a comprehensive structure characterization of target components of Gelsemium and would be widely applicable for complicated mixtures that are derived from Gelsemium preparations. Copyright © 2017 John Wiley & Sons, Ltd.     

Chemical fingerprint of Ganmaoling granule by double‐wavelength ultra high performance liquid chromatography and ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

A method incorporating double‐wavelength ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry was developed for the investigation of the chemical fingerprint of Ganmaoling granule. The chromatographic separations were performed on an ACQUITY UPLC HSS C₁₈ column (2.1 × 50 mm, 1.8 μm) at 30°C using gradient elution with water/formic acid (1%) and acetonitrile at a flow rate of 0.4 mL/min. A total of 11 chemical constituents of Ganmaoling granule were identified from their molecular weight, UV spectra, tandem mass spectrometry data, and retention behavior by comparing the results with those of the reference standards or literature. And 25 peaks were selected as the common peaks for fingerprint analysis to evaluate the similarities among 25 batches of Ganmaoling granule. The results of principal component analysis and orthogonal projection to latent structures discriminant analysis showed that the important chemical markers that could distinguish the different batches were revealed as 4,5‐di‐O‐caffeoylquinic acid, 3,5‐di‐O‐caffeoylquinic acid, and 4‐O‐caffeoylquinic acid. This is the first report of the ultra high performance liquid chromatography chemical fingerprint and component identification of Ganmaoling granule, which could lay a foundation for further studies of Ganmaoling granule.     

Characterization of ginsenoside compound K metabolites in rat urine and feces by ultra‐performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Ginsenoside compound K (CK) is an active metabolite of ginsenoside and has been shown to have ameliorative property in various diseases. However, the detailed in vivo metabolism of this compound has rarely been reported. In the present study, a method using liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry together with multiple data processing techniques, including extracted ion chromatogram, multiple mass defect filter and MS/MS scanning, was developed to detect and characterize the metabolites of CK in rat urine and feces. After oral administration of CK at a dose of 50 mg/kg, urine and feces were collected for a period of time and subjected to a series of pretreatment. A total of 12 metabolites were tentatively or conclusively identified, comprising 11 phase I metabolites and a phase II metabolite. Metabolic pathways of CK has been proposed, including oxidation, deglycosylation, deglycosylation with sequential oxidation and dehydrogenation and deglycosylation with sequential glucuronidation. Relative quantitative analyses suggested that deglycosylation was the main metabolic pathway. The result could offer insights for better understanding of the mechanism of its pharmacological activities.     

Structural elucidation of new urinary tamoxifen metabolites by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In this study, tamoxifen metabolic profiles were investigated carefully. Tamoxifen was administered to two healthy male volunteers and one female patient suffering from breast cancer. Urinary extracts were analyzed by liquid chromatography quadruple time‐of‐flight mass spectrometry using full scan and targeted MS/MS techniques with accurate mass measurement. Chromatographic peaks for potential metabolites were selected by using the theoretical [M + H]⁺ as precursor ion in full‐scan experiment and m/z 72, 58 or 44 as characteristic product ions for N,N‐dimethyl, N‐desmethyl and N,N‐didesmethyl metabolites in targeted MS/MS experiment, respectively. Tamoxifen and 37 metabolites were detected in extraction study samples. Chemical structures of seven unreported metabolites were elucidated particularly on the basis of fragmentation patterns observed for these metabolites. Several metabolic pathways containing mono‐ and di‐hydroxylation, methoxylation, N‐desmethylation, N,N‐didesmethylation, oxidation and combinations were suggested. All the metabolites were detected in the urine samples up to 1 week. Copyright © 2014 John Wiley & Sons, Ltd.     

Detecting changes in arabidopsis cell wall composition using time‐of‐flight secondary ion mass spectrometry

Time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) was previously used to characterize lignocellulosic materials, including woody biomass. ToF‐SIMS can acquire both rapid spectral and spatial information about a sample's surface composition. In the present study, ToF‐SIMS was used to characterize the cell walls of stem tissue from the plant model organism, Arabidopsis thaliana. Using principal component analyses, ToF‐SIMS spectra from A. thaliana wild‐type (Col‐0), cellulose mutant (irx3), and lignin mutant (fah1) stem tissues were distinguished using ToF‐SIMS peaks annotated for wood‐derived lignocellulose, where spectra from the irx3 and fah1 were characterized by comparatively low polysaccharide and syringyl lignin content, respectively. Spatial analyses using ToF‐SIMS imaging furthermore differentiated interfascicular fiber and xylem vessels based on differences in the lignin content of corresponding cell walls. These new data support the applicability of ToF‐SIMS peak annotations based on woody biomass for herbaceous plants, including model plant systems like arabidopsis. Copyright © 2015 John Wiley & Sons, Ltd.     

Systematic screening and characterization of astragalosides in an oral solution of Radix Astragali by liquid chromatography with quadrupole time‐of‐flight mass spectrometry and Peakview software

Liquid chromatography with quadrupole time‐of‐flight mass spectrometry coupled with automated data analysis by Peakview software was employed to systematically screen and characterize the astragalosides in Radix Astragali, a Chinese medical preparation. The separation was performed on a poroshell 120 SB‐C18 column equipped in a conventional liquid chromatography system. After being separated using a general gradient elution, the analytes were detected by the triple quadrupole time‐of‐flight mass spectrometer in both positive‐ and negative‐ion modes. The mass defect filtering function built in the Peakview software was utilized to rapidly screen the potential ions of interest, while some functions of Peakview such as Formula Finder, XIC manager, and IDA Explorer were employed to facilitate the assignment or characterization of the screened astragalosides. A total of 42 astragalosides were screened and tentatively characterized or assigned, and 20 of them were firstly detected in Radix Astragali. According to the screened astragalosides, acetylation, glycosidation, hydrogenation, oxidation, and hydration were considered to be the major secondary metabolic pathways involved in the formation of the astragalosides. The combination of liquid chromatography with quadrupole time‐of‐flight mass spectrometry and automated Peakview analysis is a feasible and efficient tool to screen and identify the constituents in complex matrices of herbal medicines.     

Evaluation of the chemical consistency of Yin‐Chen‐Hao‐Tang prepared by combined and separated decoction methods using high‐performance liquid chromatography and quadrupole time‐of‐flight mass spectrometry coupled with multivariate statistical analysis

In this study, Yin‐Chen‐Hao‐Tang prepared by two decoction methods, namely, combined decoction (modern decoction method) and separated decoction (traditional decoction method), was analyzed by high‐performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry. The acquired datasets containing sample codes, t_R‐m/z pairs and ion intensities were processed with multivariate statistical analyses, such as principal component analysis and an orthogonal partial least squared discriminant analysis model, to globally compare the chemical differences between the different decoction samples. Then, the chemical differences between the combined and separated decoctions were screened out by S‐plots generated from the orthogonal partial least squared discriminant analysis model and compared with chemical information from an established in‐house library. The six components that contributed the most to the chemical differences were identified as chlorogenic acid, caffeic acid, geniposide, genipin, scopoletin, and 3,5‐dicaffeoylquinic acid. The concentrations of genipin and caffeic acid from the separated decoction were higher than those from the combined decoction, indicating that the separated decoction may present a stronger hepatoprotective effect. However, the results still require further investigation through pharmacological and clinical studies. Our findings not only establish a strategy to evaluate chemical consistency of Yin‐Chen‐Hao‐Tang but also provide the scientific basis for using traditional separated decoction method.     

Determination of multiple pesticide residues in teas by gas chromatography with accurate time‐of‐flight mass spectrometry

In this work, gas chromatography tandem with electron ionization and full‐scan high‐resolution mass spectrometry with a time‐of‐flight mass analyzer was evaluated for analyzing pesticide residues in teas. The relevant aspects for mass spectrometry analysis, including the resolution and mass accuracy, acquisition rate, temperature of ion source, were investigated. Under acquisition condition in 2‐GHz extended dynamic range mode, accurate mass spectral library including 184 gas chromatography detectable pesticides was established and retrieval parameters were optimized. The mass spectra were consistent over a wide concentration range (three orders) with good match values to those of NIST (EI‐quadrupole). The methodology was verified by the validation of 184 pesticides in four tea matrices. A wide linear range (1–1000 μg/kg) was obtained for most compounds in four matrices. Limit of detection, limit of quantification, and limit of identification values acquired in this study could satisfy the requirements for maximum residue levels prescribed by the European Community. Recovery studies were performed at three concentrations (10, 50, and 100 μg/kg). Most of the analytes were recovered at an acceptable range of 70–120% with relative standard deviations ≤ 20% in four matrices. The potential extension of qualitative screening scope makes gas chromatography tandem with electron ionization and mass spectrometry with a time‐of‐flight mass analyzer a more powerful tool compared with gas chromatography with tandem mass spectrometry.     

Solid acids assisted matrix solid‐phase dispersion microextraction of alkaloids by capillary electrophoresis coupled with quadrupole time‐of‐flight mass spectrometry

The quantification of three alkaloids is important because quantitative study is a means of assessing the reliability of the experimental method, and three alkaloids of peimine, peiminine, and peimisine are main active ingredients in Chinese Pharmacopoeia 2015. An effective method based on the matrix solid‐phase dispersion microextraction was developed for the extraction of alkaloid compounds in Fritillariae Thunbergii Bulbus. Target analytes were analyzed by capillary electrophoresis coupled with quadrupole time‐of‐flight mass spectrometry. The optimized experimental condition was that 50 mg Fritillariae Thunbergii Bulbus was blended homogeneously with 10 mg citric acid for 5 min. Two hundred microliters of water acidized by 1 mol/L hydrochloric acid (pH = 4.5) was selected to elute tested alkaloids. The results demonstrated that the investigated method had low limits of detection (1.32–1.59 ng/mL), good recoveries (86.63–98.12%), and reproducibility (relative standard deviations of peak areas < 0.87%). The proposed matrix solid‐phase dispersion microextraction coupled with capillary electrophoresis combined with quadrupole time‐of‐flight mass spectrometry was successfully applied for the extraction of alkaloids in plants.