Characterization and identification of multiple constituents in Yinhuang granules by high-performance liquid chromatography with diode-array and time-of-flight mass spectrometry detection

A fast high-performance liquid chromatography (HPLC) method with diode-array detection (DAD) and time-of-flight mass spectrometry (TOF/MS) has been developed for the analysis of multi-constituent in Yinhuang granules, a well-known combined herbal remedy prepared from the extract mixtures of Flos Lonicerae and Radix Scutellariae. The fast HPLC analysis was performed on an Agilent ZorBax SB-C(18) column (4.6×50 mm, 1.8 μm) and 0.2% aqueous formic acid and acetonitrile was the optimum mobile phase for gradient elution in 17 min, which is five times faster than the performance of conventional columns packed with 5.0 μm particles. With various fragmentor voltages in TOF/MS, accurate mass measurements (<5 ppm error) for molecular ions and characteristic fragment ions represented reliable identification criteria for different constituents. A total of 28 compounds, including nine phenolic acids, three iridoid glycosides and nine saponins from Flos Lonicerae and seven flavonoids from Radix Scutellariae, were identified or tentatively characterized in the extract of Yinhuang granules. The established fast HPLC-DAD-TOF/MS method turns out to be useful and efficient for quality control of this commonly used Chinese herbal preparation.     

Ultra‐performance liquid chromatography coupled with electrospray ionization/quadrupole time‐of‐flight mass spectrometry for the rapid analysis of constituents in the traditional Chinese medical formula Danggui San

In this work, ultra‐performance LC with ESI quadrupole TOF‐MS (UPLC–ESI‐Q‐TOF‐MS) and automated MetaboLynx analysis was used to rapidly separate and identify the chemical constituents of Danggui San, a traditional Chinese medical formula. The analysis was performed on a Waters UPLC BEH C₁₈ column using a gradient elution system. A hyphenated ESI and Q‐TOF analyzer was used for the determination of the accurate mass of the protonated or deprotonated molecule and fragment ions in both positive and negative modes. Based on retention times, accurate mass, and the mass spectrometric fragmentation characteristics, a total of 47 compounds distributed over the chemical groups of phthalides, flavonoids, monoterpene glycosides, sesquiterpenoids, phenolics, and alkaloids, were simultaneously separated within 18 min and identified or tentatively elucidated in Danggui San for the first time. UPLC–ESI‐Q‐TOF‐MS analysis revealed the complexity of the chemical composition of this formula. The method developed is rapid, accurate, reliable, and highly sensitive to characterize the chemical constituents of Danggui San.     

A Rapid and Sensitive Assay for Determining the Main Components in Processed Fructus Corni by UPLC�CQ-TOF-MS

Fructus Corni is the dried sarcocarp of Cornus officinalis Sieb. et Zucc., and has been widely used as an important traditional Chinese medicine, which is used to nourish liver and kidney conditions and to astringe semen as well as to solidify collapse. Precise structural identification of the main compounds in processed Fructus Corni has been established using ultra performance liquid chromatography (UPLC) combined with quadrupole-time-of-flight-mass spectrometry (Q-TOF-MS), and BEH C₁₈ column for direct analysis of total water extracts. Mass spectrometry was performed in reflective time-of-flight using electron spraying ionization in negative mode. The MS data for candidates, containing accurate mass for both precursors and their fragment ions, were acquired selectively. Based on the MS data, the mass spectrometric fingerprint (MSFP) for candidates, consisting of chemical formula and dissociation pattern, was determined. By this method, eight iridoid glycosides and seven other compounds were identified or tentatively identified. Therefore, MS combined with selective enrichment provided a powerful means for analyzing main compounds in processed Fructus Corni. LC?CQ-TOF-MS based chemical profiling is a rapid and powerful approach for holistic quality evaluation of processed Fructus Corni, and should also be useful for the global quality investigation of decoctions derived from other herbal medicines.     

UPLC-Q-TOF/MS for Analysis of the Metabolites of Flavone Glycosides from Scutellaria baicalensis Georgi by Human Fecal Flora in Vitro

The active ingredients of Scutellaria baicalensis Georgi, a valuable traditional Chinese medicine, are polyhydroxyflavones, namely baicalin, scutellarin and wogonoside. However, information about the metabolic routes, metabolites and even more the effect of chemical structure on the stability of the three has been limited. In this article, the three natural compounds were incubated with human fecal flora, respectively, and highly sensitive and specific ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry implementing the MetaboLynx? software method was used for the drug metabolism study. The chromatographic separation was performed on a 1.7-μm particle size Syncronis C₁₈ column using a gradient elution system. The components in the extract were identified and confirmed according to the mass spectrometric fragmentation mechanisms, MS/MS fragment ions and relevant literature by means of electrospray ionization mass spectrometry in negative ion mode. With this method, a total of ten metabolites were identified based on MS and MS/MS data. The results indicated that hydrogenation, methylation and deglycosylation were the major metabolic pathways of the three flavone glycosides in vitro, and the metabolic stability was closely related to the chemical structure. This study will be helpful for fully understanding the impact of intestinal bacteria on these active components. Furthermore, this work demonstrated the potential of the ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry approach with MetaboLynx for quite rapid, simple, reliable and automated identification of metabolites of natural products.     

High-Throughput Structural Elucidation of Lignans in Flaxseed by High-Performance Liquid Chromatography Coupled with Electrospray Ionization Mass Spectrometry

Methods based on high-performance liquid chromatography coupled with mass spectrometry and quadrupole time-of-flight mass spectrometry were developed for the rapid identification of lignans in flaxseed. The lignans were extracted from dried flaxseed extract power by ultrasonic extraction. The fragment ions were further investigated for structural elucidation and the MS/MS spectra provided an abundance of structural information for the identification. A total of eleven lignans were investigated and three were new compounds. This research demonstrates the rapid characterization of bioactive constituents in plant extracts while avoiding tedious and difficult separation and purification steps.     

Identification of metabolites of Danggui Buxue Tang in rat urine by liquid chromatography coupled with electrospray ionization time‐of‐flight mass spectrometry

A method coupling liquid chromatography with electrospray ionization time‐of‐flight mass spectrometry (LC/ESI‐TOF/MS) has been developed for rapid and sensitive analysis of rat urinary metabolite profile of Danggui Buxue Tang (DBT), a well‐known Chinese herbal formula. After oral administration of DBT, urine samples were collected during 0–24 h, and then pretreated by solid‐phase extraction. A total of 68 compounds including 13 parent compounds and 55 metabolites were detected in the drug‐containing urines compared with blank urines. The total analytical time was less than 20 min. Metabolites of DBT were identified using dynamic adjustment of the fragmentor voltage to produce structure‐relevant fragment ions. By using this approach, the mass accuracy of precursor and fragment ions was typically within ±5 ppm of the theoretical values, and enabled the identification of 43 metabolites including 27 isoflavanoid and 16 phthalide metabolites. Our results indicated that glucuronidation and sulfation were the major metabolic pathways of isoflavonoids, while glutathione conjugation, glucuronidation and sulfation were the main metabolic pathways of phthalides. No saponin‐related metabolites were detected. The results of the present study provided important structural information relating to the metabolism of DBT. Furthermore, this work demonstrated the potential of the LC/ESI‐TOF/MS approach for identification of metabolites from Chinese herbal medicines in urine. Copyright © 2009 John Wiley & Sons, Ltd.     

UPLC-Q-TOF/MS for Analysis of the Metabolites of Flavone Glycosides from Scutellaria baicalensis Georgi by Human Fecal Flora in Vitro

The active ingredients of Scutellaria baicalensis Georgi, a valuable traditional Chinese medicine, are polyhydroxyflavones, namely baicalin, scutellarin and wogonoside. However, information about the metabolic routes, metabolites and even more the effect of chemical structure on the stability of the three has been limited. In this article, the three natural compounds were incubated with human fecal flora, respectively, and highly sensitive and specific ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry implementing the MetaboLynx™ software method was used for the drug metabolism study. The chromatographic separation was performed on a 1.7-μm particle size Syncronis C₁₈ column using a gradient elution system. The components in the extract were identified and confirmed according to the mass spectrometric fragmentation mechanisms, MS/MS fragment ions and relevant literature by means of electrospray ionization mass spectrometry in negative ion mode. With this method, a total of ten metabolites were identified based on MS and MS/MS data. The results indicated that hydrogenation, methylation and deglycosylation were the major metabolic pathways of the three flavone glycosides in vitro, and the metabolic stability was closely related to the chemical structure. This study will be helpful for fully understanding the impact of intestinal bacteria on these active components. Furthermore, this work demonstrated the potential of the ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry approach with MetaboLynx for quite rapid, simple, reliable and automated identification of metabolites of natural products.

     

Rapid and global detection and characterization of the constituents in ShengMai San by ultra-performance liquid chromatography-high-definition mass spectrometry

An ultra-performance liquid chromatography-high definition mass spectrometry (UPLC-HDMS) method was developed for detection and characterization of the chemical constituents in ShengMai San (SMS), a traditional Chinese medical formula (TCMF). The full-scan LC-MS/MS data sets combined with extra mass were acquired within 14 min using UPLC-HDMS in the MS(E) mode in a single injection. As a result, 92 compounds were identified by comparing the accurate mass and fragments information with that of the authentic standards as well as by MS analysis and the correlative references data. These constituents included ginsenosides, lignans, steroidal saponins and homoisoflavanones. Among them, 25-hydroxyginsenosides were discovered in SMS for the first time. Compare with the previous studies, our research detected more compounds and presented more rapid by applying UPLC-HDMS. It is concluded that a rapid and effective method has been established based on UPLC-HDMS with the utilization of MS(E) , which shows high sensitivity and resolution that is suitable for identifying the constituents of SMS, and this method could be applied to other TCMF.     

Determination of Metabolism of Neohesperidin by Human Intestinal Bacteria by UPLC-Q-TOF/MS

Neohesperidin as the major isoflavonoid in Aurantii Fructus has been investigated intensively. However, the route and metabolites of neohesperidin by human intestinal bacteria are not well understood and its metabolites may accumulate and exert physiological effects. In this work, 5 strains including Clostridium sp.8, Bacteroides sp.15, Bacillus sp.46, and Enterobacter sp.41-1 and sp.73 were isolated and their abilities to convert neohesperidin were studied. Human fecal microflora were prepared from a healthy Chinese woman and then anaerobically incubated with neohesperidin sample at 37 °C for 48 h. A rapid and simple liquid–liquid extraction method was used for sample pretreatment. A highly sensitive and specific ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry with MetaboLynx? method has been developed for the analysis of neohesperidin and related metabolites in the reaction samples. The chromatographic separation was performed on a 1.7 μm particle size Syncronis C₁₈ column using gradient elution system. The components in the extract were identified and confirmed according to the mass spectrometric fragmention mechanisms, MS/MS fragment ions and relevant literature by means of electrospray ionization mass spectrometry in negative ion mode. With this method, a total of five metabolites were detected, the results indicated that hydrolysis, dehydroxylation, demethylation and acetylation were the major metabolism of neohesperidin. The present study provided important information about the metabolism of neohesperidin which will be helpful for fully understanding the impact of the intestinal bacteria on this active component. Furthermore, this work demonstrated the potential of the ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry approach using MetaboLynx? for quite rapid, simple, reliable and automated identification of metabolites of natural product.     

High‐speed separation and characterization of major constituents in Radix Paeoniae Rubra by fast high‐performance liquid chromatography coupled with diode‐array detection and time‐of‐flight mass spectrometry

A fast high‐performance liquid chromatography (HPLC) method coupled with diode‐array detection (DAD) and electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOFMS) has been developed for rapid separation and sensitive identification of major constituents in Radix Paeoniae Rubra (RPR). The total analysis time on a short column packed with 1.8‐µm porous particles was about 20 min without a loss in resolution, six times faster than the performance of a conventional column analysis (115 min). The MS fragmentation behavior and structural characterization of major compounds in RPR were investigated here for the first time. The targets were rapidly screened from RPR matrix using a narrow mass window of 0.01 Da to restructure extracted ion chromatograms. Accurate mass measurements (less than 5 ppm error) for both the deprotonated molecule and characteristic fragment ions represent reliable identification criteria for these compounds in complex matrices with similar if not even better performance compared with tandem mass spectrometry. A total of 26 components were screened and identified in RPR including 11 monoterpene glycosides, 11 galloyl glucoses and 4 other phenolic compounds. From the point of time savings, resolving power, accurate mass measurement capability and full spectral sensitivity, the established fast HPLC/DAD/TOFMS method turns out to be a highly useful technique to identify constituents in complex herbal medicines. Copyright © 2008 John Wiley & Sons, Ltd.     

Ultra-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry for Rapid Determination of the Metabolites of Baicalin Produced by Human Intestinal Bacteria

Baicalin, mainly isolated from Scutllaria baicalensis, has been reported to possess a wide range of biological activities. However, the information about the metabolic route and metabolites of baicalin was limited to the role of the human intestinal bacterial mixture. In this paper, four strains of bacteria including Bacteroides sp. 33 and 56, and Veillonella sp. 23 and 71 were isolated from human intestinal bacterial mixture and studied for their abilities to convert baicalin to different metabolites. A highly sensitive and specific ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) method combined with mass defect filtering (MDF) provides high throughput capabilities for drug metabolism study. The chromatographic separation was performed on a 1.7 µm particle size C 18 column using gradient elution system. The components in the extract were identified and confirmed according to the mass spectrometric fragmentation mechanisms, MS/MS fragment ions and relevant literature by means of electrospray ionization mass spectrometry in negative ion mode. With this method, a total of 4 metabolites were identified based on MS and MS/MS data. The results indicated that hydrogenation, methylation, and deglycosylation were the major metabolic pathways of baicalin in vitro. The present study provides important information about the metabolism of baicalin which will be helpful for fully understanding the impact of the intestinal bacteria on this active component. Furthermore, this work demonstrated the potential of the ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry approach for a rapid, simple, reliable, and automated identification of metabolites of natural products.