Characterization of the potential new phthalides in Ligusticum chuanxiong Hort. using ultra‐performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry

The characterization of unknown compounds is still a great challenge currently. A strategy for deduction of potential new phthalides through the characterization of isomers based on ultra‐performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry was proposed here to characterize the unknown compounds of Ligusticum chuanxiong Hort. (Chuanxiong). This proposed strategy consisted of four steps: (1) the high resolution MS data was collected, and the peaks were screened preliminarily by UNIFI^TM platform based on the in‐house database; (2) the fragmentation patterns and the characteristic fragments were summarized based on the representative standards; (3) the target compounds were identified based on the fragmentation rules, standards comparison and false positive exclusion; (4) the unknown components were structurally characterized according to the accurate mass and fragmentation patterns analysis. This strategy was successfully applied to the identification and deduction of phthalides in Chuanxiong. A total of 81 phthalides were detected. Fifty‐five known phthalides were identified, and 26 potential new phthalides were characterized. This research enriched the material basis of Chuanxiong, and provided a liquid chromatography tandem mass spectrometry‐oriented method for the discovery of the potential new compounds.     

Profiling and identification of the absorbed constituents and metabolites of schisandra lignans by ultra‐performance liquid chromatography coupled to mass spectrometry

Schisandra chinensis Baill grows wild in Russia, China, Korea and Japan, and its fruit has been found to be effective in amnesia and insomnia. It is enriched in schisandra lignans (SL) that are major components responsible for therapeutic action. However, there are no reports on the biotransformation analysis of SL. An ultra‐performance liquid chromatography/electrospray‐ionization high‐definition mass spectrometry (UPLC‐Q‐TOF‐HDMS) method was developed to investigate the metabolism of SL in vivo. MS was performed on a Waters Micromass high‐definition system with an electrospray ionization source in positive ion mode and automated MetaboLynx software analysis with excellent MS accuracy and enhanced MS data acquisition. An improved mass defect filter (MDF) method employing both drug and core structure filter templates was applied to the processing of UPLC‐Q‐TOF‐HDMS data for the detection and structural characterization of metabolites. In this study, 30 metabolites were detected and identified in vivo, and demethylation and hydroxylation were confirmed as the primacy metabolic pathway for SL in rat plasma. In conclusion, the presently developed methodology was suitable for biotransformation research of SL and will find wide use in metabolic studies for other herbal medicines. Copyright © 2013 John Wiley & Sons, Ltd.     

液相色谱-串联四极杆飞行时间质谱和超高效液相色谱-串联三重四极杆质谱用于检测牛肉中的刚果红

建立了牛肉中刚果红的检测方法。定性方法采用液相色谱-串联四极杆飞行时间质谱对未知物进行质谱谱图库匹配,定量分析采用超高效液相色谱-串联三重四极杆质谱。牛肉样品中的刚果红经液液萃取净化后,采用Agilent ZORBAX Eclipse Plus C₁₈ Rapid Resolution HD色谱柱(50 mm×2.1 mm, 1.8 μm)进行分离,流动相为95%(体积分数)甲醇,流速为0.2 mL/min。AB 4000⁺三重四极杆质谱仪在电喷雾负离子化(ESI)及MRM模式下定量。结果显示,刚果红在0.03~1 mg/L浓度范围内,线性关系良好(相关系数为0.9998),精密度良好(RSD小于5%),回收率为88%~91%,检出限约为0.01 mg/L。本方法快速简便,重现性好,可以为牛肉及其他肉制品中刚果红的定量提供良好的解决方案。     

Detection of buffalo mozzarella adulteration by an ultra‐high performance liquid chromatography tandem mass spectrometry methodology

Over the past years, LC–MS‐based approaches have gained a growing interest in food analysis by using different platforms and methodologies. In particular, enhanced selectivity and sensitivity of multiple reaction monitoring (MRM) scan function offer powerful capabilities in detecting and quantifying specific analytes within complex mixtures such as food matrices. The MRM approach, traditionally applied in biomedical research, is particularly suitable for the detection of food adulteration and for the verification of authenticity to assure food safety and quality, both recognized as top priorities by the European Union Commission. Increasingly stringent legislation ensure products safety along every step ‘from farm to fork’, especially for traditional foods designed with the Protected Designation of Origin certification. Therefore, there is a growing demand of new methodologies for defining food authenticity in order to preserve their unique traits against frauds. In this work, an ultra performance liquid chromatopgraphy‐electrospray ionization‐tandem mass spectrometry (MS/MS) methodology based on MRM has been developed for the sensitive and selective detection of buffalo mozzarella adulteration. The targeted quantitative analysis was performed by monitoring specific transitions of the phosphorylated β‐casein f33‐48 peptide, identified as a novel species‐specific proteotypic marker. The high sensitivity of MRM‐based MS and the wide dynamic range of triple quadrupole spectrometers have proved to be a valuable tool for the analysis of food matrices such as dairy products, thus offering new opportunities for monitoring food quality and adulterations. Copyright © 2012 John Wiley & Sons, Ltd.     

Rapid analysis and characterization of multiple constituents of corn silk aqueous extract using ultra‐high‐performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry

Corn silk is a well‐known traditional Chinese medicine that has been widely used for its antidiabetic, antioxidant, antihyperlipidemic, and other effects in China for thousands of years. Numerous studies have revealed that corn silk contains multiple bioactive constituents that are beneficial for human health. However, the constituents of corn silk in vivo remain ambiguous. In this study, high‐throughput ultra‐high‐performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry technology using multivariate statistical analysis was established to systematically investigate the constituents migrating into blood from corn silk aqueous extract. As a result, 76 compounds were identified, including caffeic acid and ten of its derivatives, (E)‐p‐coumaric acid and two of its derivatives, ferulic acid and four of its derivatives, and five flavones. Among the identified constituents, 21 constituents, including nine prototype components and 12 metabolites derived from eight components, were characterized in sequence. Based on the significance of the results, the applied approach was powerful for the accurate determination and rapid screening of bioactive components from corn silk aqueous extract. The obtained results are valuable for the in‐depth understanding and further pharmacological study of corn silk aqueous extract.     

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.     

Study of tamoxifen urinary metabolites in rat by ultra‐high‐performance liquid chromatography time‐of‐flight mass spectrometry

Tamoxifen (TMX) is a nonsteroidal estrogen antagonist drug used for the treatment of breast cancer. It is also included in the list of banned substances of the World Anti Doping Agency (WADA) prohibited in and out of competition. In this work, the excretion of urinary metabolites of TMX after a single therapeutic dose administration in rats has been studied using ultra‐high‐performance liquid chromatography electrospray time‐of‐flight mass spectrometry (UHPLC‐TOFMS). A systematic strategy based on the search of typical biotransformations that a xenobiotic can undergo in living organisms, based on their corresponding molecular formula modification and accurate mass shifts, was applied for the identification of TMX metabolites. Prior to UHPLC‐TOFMS analyses, a solid‐phase extraction step with polymeric cartridges was applied to urine samples. Up to 38 TMX metabolites were detected. Additional collision induced dissociation (CID) MS/MS fragmentation was performed using UHPLC‐QTOFMS. Compared with recent previous studies in human urine and plasma, new metabolites have been reported for the first time in urine. Metabolites identified in rat urine include the oxygen addition, owing to different possibilities for the hydroxylation of the rings in different positions (m/z 388.2271), the incorporation of two oxygen atoms (m/z 404.2220) (including dihydroxylated derivatives or alternatives such as epoxidation plus hydroxylation or N‐oxidation and hydroxylation), epoxide formation or hydroxylation and dehydrogenation [m/z 386.2114 (+O –H₂)], hydroxylation of the ring accompanied by N‐desmethylation (m/z 374.2115), combined hydroxylation and methoxylation (m/z 418.2377), desaturated TMX derivate (m/z 370.2165) and its N‐desmethylated derivate (m/z 356.2009), the two latter modifications not previously being reported in urine. These findings confirm the usefulness of the proposed approach based on UHPLC‐TOFMS. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of bilobetin metabolites,in vivo and in vitro,based on an efficient ultra‐high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry strategy

Bilobetin, a natural compound extracted from Ginkgo biloba, has various pharmacological activities such as antioxidation, anticancer, antibacterial, antifungal, anti‐inflammatory, antiviral, and promoting osteoblast differentiation. However, few studies have been conducted and there are no reports on its metabolites owing to its low content in nature. In addition, it has been reported to have potential liver and kidney toxicity. Therefore, this study aimed to identify the metabolites of bilobetin in vitro and in vivo. Bilobetin was incubated with liver microsomes to determine metabolites in vitro, and faeces and urine were collected after oral administration to rats to determine metabolites in vivo. After the samples were processed, they were measured using ultra‐high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. As a result, a total of 21 and 9 metabolites were detected in vivo and in vitro, respectively. Demethylation, demethylation and loss of water, demethylation and hydrogenation, demethylation and glycine conjugation, oxidation, methylation, oxidation and methylation, and hydrogenation were the main metabolic pathways. This study is the first to identify the metabolites of bilobetin and provides a theoretical foundation for the safe use of bilobetin in clinical application and the development of new drugs.     

Rapid characterization of the chemical constituents and rat metabolites of the Wen‐Jing decoction by ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

The Wen‐Jing decoction, a traditional Chinese medicine formula, has been used as a blood‐activating and stasis‐eliminating drug to treat gynaecological syndromes, such as dysmenorrhea, amenorrhea, and menstrual disorders. However, its pharmacodynamic material basis and mechanism of action have not been thoroughly elucidated to date. The goal of this study was to characterize and identify multiple constituents and metabolites in Wen‐Jing decoction. An ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry method was established and validated in the present study for the first time. A total of 101 compounds, including 11 monoterpene glycosides, 19 flavonoids, 49 triterpene saponins, 5 phthalides, 3 phytoecdysones, and 14 others, were unambiguously or tentatively characterized by comparing their retention times and MS data with reference standards or with data reported in the literature. After oral administration of Wen‐Jing decoction, 27 compounds, including nine prototype compounds and 18 metabolites were detected in rat plasma. Thus, the ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry method was found to be efficient for in‐depth structural elucidation of chemical compounds in complex matrices of herbal medicines, which will provide useful chemical information for quality control and mechanism‐of‐action research.     

Characterization of chemical constituents and identification of absorbed components and metabolites in rat plasma of Fu‐Ke‐Zai‐Zao pills by ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

Fu‐Ke‐Zai‐Zao pills, the famous traditional Chinese medicine formula, composed of 42 medicinal herbs, have been widely used to treat various gynecological diseases. However, the chemical constituents and metabolic profiling of Fu‐Ke‐Zai‐Zao pills remain largely unknown, which hampers improvement of the quality control and pharmacological elucidation of this formula. In the present study, a sensitive and selective ultra high performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry method was developed to separate and identify the comprehensive chemical constituents of Fu‐Ke‐Zai‐Zao pills. According to the results, a total of 83 compounds were identified, including phenylpropionic acids, flavonoids, terpenoids, triterpene saponins, and phthalides, and 81 compounds were first reported in Fu‐Ke‐Zai‐Zao pills. Moreover, the absorbed components and metabolites in rat plasma after intragastric administration of Fu‐Ke‐Zai‐Zao pills were also detected by the same analytical method. A total of 36 compounds were identified, including 21 prototypes and 15 metabolites. The latter were generated through the metabolic pathways of methylation and glucuronidation, and glucuronidated metabolites were the main constituents in the plasma. This is the first systematic study on the chemical constituents and metabolic profiling of Fu‐Ke‐Zai‐Zao pills, and the results provide valuable chemical information for further elucidating pharmacological effects and mechanism of action of Fu‐Ke‐Zai‐Zao pills.     

Identification of four cornua by ultra‐performance liquid chromatography with time‐of‐flight mass spectrometry coupled with principal component analysis

An ultra‐high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry method coupled with principal component analysis was developed and applied to the identification of Cornu Antelopis, Cornu Bubali, Cornu Naemorhedi, and Cornu Bovis. The data obtained from the trypsin‐digested samples were subjected to principal component analysis to classify these four cornua. Additionally, marker peptides of the cornua were determined by orthogonal partial least‐squares discriminant analysis, and fragmentation tandem mass spectra of these marker peptides were evaluated. The results from this study indicate that the proposed method is reliable, and it has been successfully applied to the identification of variants of cornua commonly used in traditional Chinese medicine.     

Rapid annotation and structural characterization of saponins in the active fraction of Albizia julibrissin by high‐performance liquid chromatography coupled with quadrupole time of flight mass spectrometry based on accurate mass database

The purified active fraction of Albizia julibrissin saponin was proved to be a promising adjuvant candidate for vaccine. In this study, a simple, convenient, and practical strategy was established for characterizing the saponins in this purified active fraction. The personal accurate mass database including chemical structure, molecular formula, and theoretical mass was first constructed by collecting 110 reported known saponins from genus Albizia species. The raw data was obtained by high‐performance liquid chromatography coupled with quadrupole time of flight mass spectrometry. The potential compounds were extracted from raw data, and matched with the accurate mass databases. A series of saponin compounds were predicted and their chemical structures were characterized by interpreting the tandem mass spectrometry data. A total of 29 saponins including 10 new compounds and 5 first found saponins from A. julibrissin were successfully characterized in this purified active fraction using this new strategy.     

Evaluation and validation of an ion mobility quadrupole time‐of‐flight mass spectrometry pesticide screening approach

An ion mobility quadrupole time‐of‐flight mass spectrometry‐based pesticide suspect screening methodology was developed and validated covering 20 plant‐derived food matrices deriving from six commodity groups of different complexity according to the actual European Commission document SANTE/11813/2017 applying a QuEChERS sample preparation protocol. The method combines ultra‐performance liquid chromatography, traveling wave ion mobility, and quadrupole time‐of‐flight mass spectrometry. Besides the determination of the physicochemical property collision cross‐section and the establishment of a corresponding scientific suspect screening database comprising 280 pesticides for several pesticides, different protomers, sodium adducts, as well as dimers were identified in ion mobility spectrometry traces. Additionally, collision cross‐section values were included in the validation requirements regarding chromatography and mass spectrometry for the detection of pesticides. A collision cross‐section value window was analyzed within a tolerable error of ±2%. For this cross‐matrix validation, screening detection limits were determined at concentration levels of 0.100 mg/kg (84% of the original pesticide scope), 0.010 mg/kg (56%), and 0.001 mg/kg (21%). By application of ion mobility spectrometry, the compound identification was improved due to independence of commodity of concern and concentration levels of analyte molecules, as false assignments are reduced by application of a collision cross‐section range.     

An ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry identification and characterization of the active constituents from Abrus mollis Hance

Abrus mollis Hance is a traditional Chinese medicine that is widely used to treat acute and chronic hepatitis, steatosis, and fibrosis. Its therapeutic qualities of it have long been acknowledged, although the active ingredients responsible for its efficacy and the mechanisms of its action are unknown. In this study, the chemical constituents absorbed into the blood from Abrus mollis Hance were assessed by using liquid chromatography-quadrupole-time-of-flight mass spectrometry and the data was analyzed with the UNIFI screening platform. The results obtained were compared to existing chromatographic-mass spectrometry information, including retention times and molecular weights as well as known reference compounds. 41 chemical constituents were found in Abrus mollis Hance, and these included 16 flavonoids, 13 triterpenoids, five organic acids, and two alkaloids. Experimentally it was found that Abrus mollis Hance had a therapeutic benefit when treating α-naphthalene isothiocyanate-induced acute liver injury in rats. In addition, 11 blood prototypical constituents, including six flavonoids, three triterpenoids, and two alkaloids, were found in serum samples following intragastric administration of Abrus mollis Hance extracts to rats. This novel study can be used for the quality control and pharmacodynamic assessment of Abrus mollis Hance in order to assess its efficacy in the therapeutic treatment of patients.     

Metabolic profiling comparison of isovitexin in normal and kidney stone model rats by ultra‐high‐performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry

Isovitexin, a bioactive flavonoid constituent isolated from Desmodii Styracifolii, is considered an adjuvant for antiurolithiasis diseases. In this study, an ultra‐high‐performance liquid chromatography coupled with hybrid triple quadruple time‐of‐flight mass spectrometry method was developed to characterize and compare the metabolic profiling of isovitexin experimented on normal and kidney stone model rats. The comparative research indicated that 28 metabolites (18 phase I and 10 phase II) in normal rats and 33 metabolites (20 phase I and 13 phase II) in kidney stone model rats were initially identified. The results of relative quantitative determination reflected that the contents of metabolites produced by deglycosylation, reduction, and isomerization in kidney stone model rats were greater than those in healthy rats. Instead, the levels of oxidative and dehydrogenated metabolites in normal groups were higher than those in kidney stone model groups. The results of this study are valuable and important for understanding the metabolic process of isovitexin in clinical application, and especially the metabolism study in kidney stone model rats could provide a beneficial reference for the further search of effective substances associated with the treatment of kidney stones.     

Characterization of the multiple components of Acanthopanax Senticosus stem by ultra high performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry

Acanthopanax Senticosus Harms. has been used widely in traditional Chinese medicine for the treatment of chronic bronchitis, neurasthenia, hypertension and ischemic heart disease. However, the in vivo constituents of the stem of Acanthopanax Senticosus remain unknown. In this paper, ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry and the MarkerLynx^TM software combined with multiple data processing approach were used to study the constituents in vitro and in vivo. The aqueous extract from the Acanthopanax Senticosus stem and the compositions in rat serum after intragastric administration were completely analyzed. Consequently, 115 compounds in the aqueous extract from Acanthopanax Senticosus stem and 41 compounds absorbed into blood were characterized. Of the 115 compounds in vitro, 54 were reported for first time, including sinapyl alcohol, sinapyl alcohol diglucoside, and 1‐O‐sinapoyl‐β‐d ‐glucose. In the 41 compounds in vivo, 7 were prototype components and 34 were metabolites which were from 21 components of aqueous extract from Acanthopanax Senticosus stem, and the metabolic pathways of the metabolites were elucidated for first time. The results narrowed the range of screening the active components and provided a basis for the study of action mechanism and pharmacology.     

超高效液相色谱-四极杆飞行时间质谱法用于食物中毒的快速筛查与确证

建立了一种利用超高效液相色谱-四极杆飞行时间质谱筛查食物中毒的方法。试样用乙腈提取,QuEChERS净化,以0.1%(v/v)甲酸水溶液和0.1%(v/v)甲酸乙腈溶液为流动相进行梯度洗脱,经Acquity UPLC BEH C18柱(100 mm×2.1 mm,1.7μm)分离。采用四极杆飞行时间质谱信息依赖性扫描(information dependent acquisition,IDA)模式进行分析。该模式可以实现一次进样分析同时获得分析物的一级和二级碎片的精确质量质谱图,结合SCIEX OS软件可对581种目标物质进行快速筛查,其中包括546种农药、24种真菌毒素、11种鼠药,以一级离子精确质量数、一级离子同位素丰度比以及二级碎片进行标准质谱库匹配。应用建立的快速筛选确认检测方法对一起突发性食物中毒安全事件样本进行检测,共检测11份样本,其中9份均检测出呋喃丹。进一步以呋喃丹标准品确认保留时间,结果表明,样品与标准品保留时间一致。呋喃丹的精确质量数偏差均小于3.7×10-6,在0.5~500 ng/mL范围内线性关系良好,相关系数为0.998,仪器检出限(S/N=3)为0.3μg/kg,定量限(S/N=10)为1μg/kg,在10、50、200μg/kg 3个添加水平的回收率为75.6%~95.9%,相对标准偏差为3.6%~6.9%(n=6)。该方法快速、简便、准确、灵敏,适用于突发性公共安全事件的快速筛查与检测要求。     

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.