Benzylisoquinoline alkaloid analysis using high‐resolution Orbitrap LC‐MSn

Benzylisoquinoline alkaloids (BIAs) have profound implications on human health owing to their potent pharmacological properties. Notable naturally occurring BIAs are the narcotic analgesics morphine, the cough suppressant codeine, the potential anticancer drug noscapine, the muscle relaxant papaverine, and the antimicrobial sanguinarine, all of which are produced in opium poppy (Papaver somniferum). Thebaine, an intermediate in the biosynthesis of codeine and morphine, is used in the manufacture of semisynthetic opiates, including oxycodone and naloxone. As the only commercial source of pharmaceutical opiates, opium poppy has been the focus of considerable research to understand BIA metabolism in the plant. The elucidation of several BIA biosynthetic pathways has enabled the development of synthetic biology platforms aimed at the alternative commercial production of valuable phytochemicals in microorganisms. The detection and identification of BIA pathway products and intermediates in complex extracts is essential for the continuing advancement of research in plant specialized metabolism and microbial synthetic biology. Herein, we report the use of liquid chromatography coupled with linear trap quadrupole and high‐resolution Orbitrap multistage mass spectrometry to characterize 44 authentic BIAs using collision‐induced dissociation (CID), higher‐energy collisional dissociation (HCD), and pulsed Q collision‐induced dissociation (PQD) MS² fragmentation, with MS² CID followed by MS³ and MS⁴ fragmentation. Our deep library of diagnostic spectral data constitutes a valuable resource for BIAs identification. In addition, we identified 22 BIAs in opium poppy latex and roots extracts.     

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.     

Rapid screening and identification of lycodine‐type alkaloids in Lycopodiaceae and Huperziaceae plants by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

Lycodine‐type alkaloids have gained significant interest owing to their unique skeletal characteristics and acetylcholinesterase activity. This study established a rapid and reliable method using ultra‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐ESI‐Q/TOF‐MS/MS) for comprehensive characterization of lycodine‐type alkaloids for the first time. The lycodine‐type alkaloids were detected successfully from Lycopodiastrum casuarinoides, Huperzia serrata and Phlegmarirus carinatus in seven plants of the Lycopodiaceae and Huperziaceae families, based on the established characteristic MS fragmentation of five known alkaloids. Furthermore, a total of 13 lycodine‐type alkaloids were identified, of which three pairs of isomers were structurally characterized and differentiated. This study further improves mass analysis of lycodine‐type alkaloids and demonstrates the superiority of UPLC with a high‐resolution mass spectrometer for the rapid and sensitive structural elucidation of other trace active compounds. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization of the chemical constituents in Da‐Huang‐Gan‐Cao‐Tang by liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry and liquid chromatography coupled with ion trap mass spectrometry

In this work, the chemical constituents in Da‐Huang‐Gan‐Cao‐Tang, a traditional Chinese formula, were studied by liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry and liquid chromatography coupled with ion trap mass spectrometry for the first time. Among the 146 compounds detected in Da‐Huang‐Gan‐Cao‐Tang, 104 compounds were identified unambiguously or tentatively based on their accurate molecular weight and multistage MS data, including one potential novel compound and two reported in Glycyrrhiza genus for the first time. The possible fragmentation pathways were proposed and fragmentation rules of the major types of compounds were concluded. This study provided an example to facilitate the tedious identification of chemical composition in traditional Chinese medicine, and maybe a promising reference approach to research the analogous formulae.     

Chemical constituents of Meconopsis horridula and their simultaneous quantification by high‐performance liquid chromatography coupled with tandem mass spectrometry

Meconopsis horridula Hook.f. Thoms has been used as a traditional Tibetan medicine to clear away heat, relieve pain, and mobilize static blood. In this study, a reliable method based on high‐performance liquid chromatography with diode array detection and electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry was established for the identification of components in this herb. A total of 40 compounds (including 17 flavonoids, 15 alkaloids, and eight phenylpropanoids) were identified or tentatively identified. Among them, 17 components were identified in the herb for the first time. Compound 39 appears to be a novel compound, which is confirmed as 3‐(kaempferol‐8‐yl)‐2,3‐epoxyflavanone by NMR spectroscopy and mass spectrometry. Moreover, seven major constituents were simultaneously quantified by the developed high‐performance liquid chromatography with tandem triple‐quadrupole mass spectrometry method. The quantitative method was validated and quality parameters were established. The study provides a comprehensive approach for understanding this herbal medicine.     

Rapid discovery of absorbed constituents and metabolites in rat plasma after the oral administration of Zi Shen Wan using high‐throughput UHPLC–MS with a multivariate analysis approach

Zi Shen Wan is a typical formula consisting of three herbs, Phellodendri Amurensis Cortex, Rhizoma Anemarrhenae, and Cortex Cinnamomi, and has been widely used for treating prostatitis and infection diseases. However, it lacks in‐depth research of the constituents of Zi Shen Wan in vivo and in vitro. In this work, ultra high performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry and MassLynx software was established to characterize the chemical compositions of Zi Shen Wan in vivo and in vitro. In total, 92 peaks were characterized in vitro and 33 peaks were characterized in vivo based on mass spectrometry and tandem mass spectrometry data. Among the 33 compounds characterized in rat plasma, 22 prototype components absorbed in rat serum and 11 metabolites were identified in vivo. This work was fully reports the chemical constituents of traditional Chinese formula of Zi Shen Wan, it demonstrated that ultra high performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry coupled to MassLynx software and multivariate data processing approach could be successfully applied for rapid screening and comprehensive analysis of chemical constituents in vitro and prototype components or metabolites in vivo of traditional Chinese medicine.     

Rapid identification of efavirenz metabolites in rats and humans by ultra high performance liquid chromatography combined with quadrupole time‐of‐flight tandem mass spectrometry

An in vivo study of efavirenz metabolites in rats and human patients with ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry combined with MetabolitePilot^MT software is reported for the first time. Considering the polarity differences between the metabolites, solid‐phase extraction and protein precipitation were both applied as a part of the sample preparation method. The structures of the metabolites and their fragment ions were identified or tentatively characterized based on the accurate mass and MS² data. As a result, a total of 15 metabolites, including 11 from rat samples and 13 from human samples, were identified or tentatively characterized. Two metabolites and several new metabolism pathways are reported for the first time. This study provides a practical approach for identifying complicated metabolites through the rapid and reliable ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry technique, which could be widely used for the investigation of drug metabolites.     

UHPLC coupled with mass spectrometry and chemometric analysis of Kang‐Ai injection based on the chemical characterization,simultaneous quantification,and relative quantification of 47 herbal alkaloids and saponins

Kang‐Ai injection, which is composed of Astragali Radix, Ginseng Radix et Rhizoma, and kushenin, is extensively used in China as an adjuvant therapy for many types of cancer and chronic hepatitis B. In the present study, 47 herbal compounds (11 alkaloids, 8 astragalosides, and 28 ginsenosides), were detected in Kang‐Ai injection by ultra‐high‐performance liquid chromatography coupled to quadrupole time‐of‐flight tandem mass spectrometry, of which 31 were identified using authentic standards. Additionally, a practical ultra‐high‐performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry method was employed for simultaneous quantitative detection (31 available compounds), and relative quantitative detection (16 unavailable compounds) within 10 min. The limit of detection and limit of quantification was 0.11‐2.22 and 0.53‐11.08 ng/mL, respectively. Altogether, content levels of each compound ranged from 0.03 to 9835.57 μg/mL. Furthermore, chemometric analysis indicated oxymatrine, astragaloside IV, ginsenosides Rg1 and Re, and matrine had the greatest effect on concentration fluctuation. Therefore, we suggested these five compounds should be monitored during the manufacturing process. This method can be applied to provide crucial chemical profiles and quality assessments for Kang‐Ai injection, guaranteeing the safety, effectiveness, and controllability of the drug in clinics.     

Rapid identification of erythrocyte phospholipids in Sprague–Dawley rats by ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

A rapid, sensitive, and reliable approach for analyzing five kinds of erythrocyte phospholipids in Sprague–Dawley rats was provided by ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry with MassLynx^TM MassFragment. Improving conventional high performance liquid chromatography techniques, ultra high performance liquid chromatography integrated with quadrupole time‐of‐flight tandem mass spectrometry offers high sensitivity and increased analytical speed by using columns packed with sub‐2 μm particles (1.7 μm), which allows a faster separation to be achieved. Through this method, 83 phospholipids were tentatively characterized based on their mass spectra and tandem mass spectra, as well as by matching the in‐house formula database within a mass error of 5 ppm, including 40 phosphatidylcholines, 24 phosphatidyl ethanolamines, three phosphatidylinositols, six phosphatidylserines, and ten sphingomyelins. Our present results proved that the established method could be used to qualitatively analyze complex erythrocyte phospholipids in Sprague–Dawley rats and provide a useful data base for pharmacology and phospholipidomics to seek potential biomarkers of disease prediction.     

Qualitative and quantitative analysis of major constituents from Dazhu Hongjingtian capsule by UPLC/Q‐TOF‐MS/MS combined with UPLC/QQQ‐MS/MS

In this work, a sensitive and efficient method was established and validated for qualitative and quantitative analysis of major bioactive constituents in Dazhu Hongjingtian capsule by liquid chromatography tandem mass spectrometry. A total of 32 compounds were tentatively identified using ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. Furthermore, 12 constituents, namely gallic acid, 3,4‐dihydroxybenzoic acid, salidroside, p‐ coumaric acid‐4‐O ‐β ‐d ‐glucopyranoside, bergeninum, 4‐hydroxybenzoic acid, 4‐hydroxyphenylacetic acid, syringate, 6′′‐O ‐galloylsalidroside, rhodiosin, rhodionin and kaempferol‐7‐O ‐α ‐l ‐rhamnoside, were simultaneously quantified by the developed ultra‐performance liquid chromatography coupled with a triple quadrupole mass spectrometry method in 9 min. All of them were analyzed on an Agilent ZorBax SB‐C₁₈ column (3.0 × 100 mm, 1.8 μm) with linear gradient elution of methanol–0.1% formic acid water. The proposed method was applied to analyze three batches of samples with acceptable linearity (R , 0.9979–0.9997), precision (RSD, 1.3–4.7%), repeatability (RSD, 1.7–4.9%), stability (RSD, 2.2–4.9%) and recovery (RSD, 0.6–4.4%) of the 12 compounds. As a result, the analytical method possessing high throughput and sensitivity is suitable for the quality control of Dazhu Hongjingtian capsule.     

Analysis and Identification of the Chemical Constituents of Ding‐Zhi‐Xiao‐Wan Prescription by HPLC‐IT‐MSn and HPLC‐Q‐TOF‐MS

Ding‐Zhi‐Xiao‐Wan (DZXW) is a famous traditional Chinese medicine (TCM) formula, which is composed of four herbs, Ginseng Radix, Poria, Polygala Radix and Acori Tatarinowii Rhizoma. It has been popularly used for the treatment of emotional disease, like Alzheimer's disease, Parkinson's disease, depression, anxiety, forgetfulness and neurasthenia. In this research, a high‐performance liquid chromatography coupled with ion‐trap tandem mass spectrometry (HPLC‐IT‐MSⁿ) method along with a high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry (HPLC‐Q‐TOF‐MS) method in negative ion mode was established to investigate the major constitutions in DZXW. The extracts were prepared by ultra‐sonication in ethyl acetate, n‐butanol, 95% ethanol and deionized water sequentially as well as in deionized water directly. A Kromasil C18 column was used to separate the extracts of DZXW. Acetonitrile and 0.1% aqueous formic acid (V/V) were used as the mobile phase. A total of 64 components were characterized, including 16 triterpenoids, 14 Polygala saponins, 10 oligosaccharide esters, 6 sucrose esters, 2 xanthone C‐glycosides and 16 ginsenosides.     

Simultaneous qualitative and quantitative analysis of flavonoids and alkaloids from the leaves of Nelumbo nucifera Gaertn. using high‐performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

A reliable method, combining qualitative analysis by high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry and quantitative assessment by high‐performance liquid chromatography with photodiode array detection, has been developed to simultaneously analyze flavonoids and alkaloids in lotus leaf extracts. In the qualitative analysis, a total of 30 compounds, including 12 flavonoids, 16 alkaloids, and two proanthocyanidins, were identified. The fragmentation behaviors of four types of flavone glycoside and three types of alkaloid are summarized. The mass spectra of four representative components, quercetin 3‐O‐glucuronide, norcoclaurine, nuciferine, and neferine, are shown to illustrate their fragmentation pathways. Five pairs of isomers were detected and three of them were distinguished by comparing the elution order with reference substances and the mass spectrometry data with reported data. In the quantitative analysis, 30 lotus leaf samples from different regions were analyzed to investigate the proportion of eight representative compounds. Quercetin 3‐O‐glucuronide was found to be the predominant constituent of lotus leaf extracts. For further discrimination among the samples, hierarchical cluster analysis, and principal component analysis, based on the areas of the eight quantitative peaks, were carried out.     

Comparison of the chemical profiles of fresh‐raw and dry‐processed Juglans mandshurica

The processing of Juglans mandshurica Maxim. is important to reduce its toxicity and enhance its efficacy. Simple, efficient, and sensitive ultra‐high performance liquid chromatography coupled with a time‐of‐flight mass spectrometry based chemical profiling approach was proposed to rapidly evaluate the chemical difference between fresh and dry samples. Under the optimized ultra‐high performance liquid chromatography and quadrupole time‐of‐flight tandem mass spectrometry conditions, 81 significantly different compounds were rapidly discovered using principal component analysis, and then tentatively identified by comparison with reference substances or inferred through mass spectral fragment ion analysis and literature data. These compounds included 35 naphthoquinones, 11 diarylheptanoids, nine flavonoids, eight triterpenes, 12 phenolic acids, and six aliphatics. The results demonstrated that chemical reactions occurring during processing could be used to elucidate the processing mechanism of Juglans mandshurica Maxim. This study provides a novel approach to identifying complicated components of various complex mixtures in fresh‐raw and dry‐processed traditional Chinese medicines, which could be used as a valid analytical method to further understand the processing mechanisms of these medicines, as well as providing intrinsic quality control of the medicines and their processed products.     

Mass spectrometric detection,identification, and fragmentation of arseno‐phytochelatins

Phytochelatins (PC) are cystein‐rich oligopeptides in plants for coordination with toxic metals and metalloids via their thiol groups. The composition, structure, and mass spectrometric fragmentation of arseno‐PC (As‐PC) with PC of different degree of oligomerization (PC2–PC5) in solution were studied using liquid chromatography coupled in parallel to inductively coupled plasma mass spectrometry and electrospray ionization quadrupole time‐of‐flight mass spectrometry. As‐PC were detected from As(PC2) to As(PC5) with an increasing number of isomers that differ in the position of thiol groups bound to As. Thermodynamic modeling supported the identification process in case of these isomers. Mass spectrometric fragmentation of the As‐PC does not follow the established pattern of peptides but is governed by the formation of series of As‐containing annular cations, which coordinate to As via S, N, or O. Structure proposals for 30 As‐PC fragment ions in the range m/z 147.92 to m/z 1290.18 are elaborated. Many of these fragment ions are characteristic to several As‐PC and may be suited for a screening for As‐PC in plant extracts. The mass spectrometric data offer the perspective for a future more sensitive determination of As‐PC by means of liquid chromatography tandem mass spectrometry with multiple reaction monitoring. Copyright © 2014 John Wiley & Sons, Ltd.     

High‐throughput ultra high performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry method for the rapid analysis and characterization of multiple constituents of Radix Polygalae

Radix Polygalae, the dried roots of Polygala tenuifolia and P. sibirica , is one of the most well‐known traditional Chinese medicinal plants. It is an important medicinal plant that has been used as a sedative and to improve memory for a number of years in most of Asia. However, the in vivo constituents of the multiple constituents from Radix Polygalae remain unknown. In the current study, ultra high performance liquid chromatography coupled to 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. A rapid and efficient method for the characterization of multiple constituents in the herbal medicine Radix Polygalae by ultra high performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry is described. In total, 35 compounds in the Radix Polygalae and 13 compounds absorbed into blood were characterized. Of the 35 compounds in vitro, ten were reported for first time. In the 13 compounds in vivo, six were prototype components and seven were metabolites were also elucidated for first time. This work narrowed the range of screening the potentially bioactive components and provided a basis for the quality control and mechanism of action.     

Online high‐pH reversed‐phase liquid chromatography × low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension for the analysis of alkaloids in Macleaya cordata (willd.) R. Br

An online high‐pH reversed‐phase liquid chromatography× low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension was constructed to separate and identify alkaloids from Macleaya cordata (willd.) R. Br. The modulation was performed by using a dual second dimensional columns interface combined with a make‐up dilution pump, which is responsible for dilution and neutralization of the first dimensional effluent, and the dual second dimensional columns integrated the trapping and the separation function to reduce the second dimension system dead volume. Taking advantage of the dissociable characteristics of alkaloids, mobile phases with different pH values were applied in the first dimension (pH 9.0) and the second dimension (pH 2.6) to improve the orthogonality of two‐dimension separation. Besides, the pulse elution gradient in first dimension and second dimensional gradient were carefully optimized and much better separation was achieved compared to the separation with the traditional two‐dimensional liquid chromatography approach. Finally, mass measurement was performed for alkaloids in M. cordata (willd.) R. Br. by coupling proposed two‐dimensional liquid chromatography system with triple quadrupole mass spectrometry, and 39 alkaloids were successfully identified by comparing the obtained result with the former reported results.     

Application of characteristic fragment filtering with ultra high performance liquid chromatography coupled with high‐resolution mass spectrometry for comprehensive identification of components in Schisandrae chinensis Fructus

An integrated strategy of characteristic fragment filtering combined with target database screening based on ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry was proposed for comprehensive profiling of components in Schisandrae chinensis Fructus. The strategy consisted of following five steps: (1) Representative standards were analyzed by ultra high performance liquid chromatography coupled with linear ion trap‐Orbitrap mass spectrometer for characteristic fragments and fragmentation rules of each structure type. (2) The raw data of 70% methanol extract was collected by ultra high performance liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry. (3) The chemical components database that consisted of names, chemical formulas and structures of potential components in Schisandrae chinensis Fructus was established by summarizing previous literature to screen the collected liquid chromatography with mass spectrometry data and obtain matched compounds. (4) Characteristic fragments, literature, and reference standards were used to verify the matches. (5) Characteristic fragment filtering combined with online database querying was used to deduce potential new compounds. As a result, a total of 94 compounds were identified or characterized and 16 of them were potential new compounds. The study provided a reference for comprehensive characterization of ingredients in herbal medicine and formed the foundation for pharmacodynamic study of Schisandrae chinensis Fructus.     

Establishment of a search library about benzylisoquinoline alkaloids based on selective separation on the binaphthyl column and standard analysis on C18 column

A search library about benzylisoquinoline alkaloids was established based on preparation of alkaloid fractions from Rhizoma coptidis, Cortex phellodendri, and Rhizoma corydalis. In this work, two alkaloid fractions from each herbal medicine were first prepared based on selective separation on the “click” binaphthyl column. And then these alkaloid fractions were analyzed on C18 column by liquid chromatography coupled with tandem mass spectrometry. Many structure‐related compounds were included in these alkaloids fractions, which led to easy separation and good MS response in further work. Therefore, a search library of 52 benzylisoquinoline alkaloids was established, which included eight aporphine, 19 tetrahydroprotoberberine, two protopine, two benzyltetrahydroisoquinoline, and 21 protoberberine alkaloids. The information of the search library contained compound names, structures, retention times, accurate masses, fragmentation pathways of benzylisoquionline alkaloids, and their sources from three herbal medicines. Using such a library, the alkaloids, especially those trace and unknown components in some herbal medicine could be accurately and quickly identified. In addition, the distribution of benzylisoquinoline alkaloids in the herbal medicines could be also summarized by searching the source samples in the library.     

Simultaneous determination of niacin and pyridoxine at trace levels by using diode array high‐performance liquid chromatography and liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry

A highly sensitive and simple diode‐array high‐performance liquid chromatography and liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry method was developed for the simultaneous determination of niacin and pyridoxine in pharmaceutical drugs, tap water, and wastewater samples. To determine the in vivo behavior of niacin and pyridoxine, analytes were subjected to simulated gastric conditions. The calibration plots of the diode‐array high‐performance liquid chromatography and liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry method showed good linearity over a wide concentration range with close to 1.0 correlation coefficients for both analytes. The limit of detection/limit of quantitation values for liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry analysis were 1.98/6.59 and 1.3/4.4 μg/L for niacin and pyridoxine, respectively, while limit of detection/limit of quantitation values for niacin and pyridoxine in high‐performance liquid chromatography analysis were 3.7/12.3 and 5.7/18.9 μg/L, respectively. Recovery studies were also performed to show the applicability of the developed methods, and percentage recovery values were found to be 90–105% in tap water and 94–97% in wastewater for both analytes. The method was also successfully applied for the qualitative and quantitative determination of niacin and pyridoxine in drug samples.