A 2‐D‐HPLC‐CE platform coupled to ESI‐TOF‐MS to characterize the phenolic fraction in olive oil

A 2‐D‐HPLC/CE method was developed to separate and characterize more in depth the phenolic fraction of olive oil samples. The method involves the use of semi‐preparative HPLC (C18 column 250×10 mm, 5 μm) as a first dimension of separation to isolate phenolic fractions from commercial extra‐virgin olive oils and CE coupled to TOF‐MS (CE‐TOF‐MS) as a second dimension, to analyze the composition of the isolated fractions. Using this method, a large number of compounds were tentatively identified, some of them by first time, based on the information concerning high mass accuracy and the isotopic pattern provided by TOF‐MS analyzer together with the chemical knowledge and the behavior of the compounds in HPLC and CE. From these results it can be concluded that 2‐D‐HPLC‐CE‐MS provides enough resolving power to separate hundreds of compounds from highly complex samples, such as olive oil. Furthermore, in this paper, the isolated phenolic fractions have been used for two specific applications: quantification of some components of extra‐virgin olive oil samples in terms of pure fractions, and in vitro studies of its anti‐carcinogenic capacity.     

Variations in chemical fingerprints and major flavonoid contents from the leaves of thirty‐one accessions of Hibiscus sabdariffa L.

The leaves of Hibiscus sabdariffa L. have been used as traditional folk medicines for treating high blood pressure and fever. There are many accessions of H. sabdariffa L. throughout the world. To assess the chemical variations of 31 different accessions of H. sabdariffa L., fingerprinting analysis and quantitation of major flavonoids were performed by high‐performance liquid chromatography (HPLC). The HPLC method was validated for linearity, sensitivity, precision, repeatability and accuracy. A quadrupole‐time‐of‐flight mass spectrometry (Q‐TOF‐MS) was applied for the characterization of major compounds. A total of 9 compounds were identified, including 6 flavonoids and 3 phenolic acids. In the fingerprint analysis, similarity analysis (SA) and principal component analysis (PCA) were used to differentiate the 31 accessions of H. sabdariffa L. Based on the results of PCA and SA, the samples No. 15 and 19 appeared much different from the main group. The total content of five flavonoids varied greatly among different accessions, ranging from 3.35 to 23.30 mg/g. Rutin was found to be the dominant compound and the content of rutin could contribute to chemical variations among different accessions. This study was helpful to understand the chemical variations between different accessions of H. sabdariffa L., which could be used for quality control. © 2015 The Authors Biomedical Chromatography Published by John Wiley & Sons Ltd.     

Identification of chemical constituents in Rhizoma Paridis Saponins and their oral administration in rat plasma by UPLC/Q‐TOF/MS

On‐line ultra‐performance liquid chromatography (UPLC) coupled with diode‐array detection (UPLC/DAD) and electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF‐MS) were used for separation, identification and structural analyses of saponins in Rhizoma Paridis saponins (RPS) and rat plasma after oral administration of RPS. Thirty steroidal saponins in RPS were identified by comparing their retention time, accurate mass measurement and positive and negative mass spectrometry data with that of reference compounds. The UPLC/Q‐TOF‐MS method was proved to be rapid and efficient in that 30 steroidal saponins, including three kinds of saponins (prototype, pennogenyl and diosgenyl saponins) were tentatively characterized within 6 min. After oral administration of RPS, 21 original saponins were absorbed in RPS‐treated rat plasma. Our results indicated that UPLC/Q‐TOF‐MS is a rapid and effective tool for identification of a series of saponins at trace level. Copyright © 2010 John Wiley & Sons, Ltd.     

Quality assessment of Cortex Phellodendri by high‐performance liquid chromatography coupled with electrospray ionization mass spectrometry

A simple method based on liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (LC‐DAD‐ESI‐MS) was developed for the quality assessment of Cortex Phellodendri (CP), which was mainly derived from two species of Phellodendron chinense Schneid and Phellodendron amurense Rupr. Total 41 compounds, including 14 phenols, 24 alkaloids and three liminoidal triterpenes were identified or tentatively characterized from the 75% methanol extract of CP samples by online ESI‐MSⁿ fragmentation and UV spectra analysis. Among them, two phenols and six alkaloids were simultaneously quantified using HPLC‐DAD method. The validated HPLC‐DAD method showed a good linearity, precision, repeatability and accuracy for the quantification of eight marker compounds. Furthermore, the plausible fragmentation pathway of the representative compounds were proposed in the present study. The differences of the chemical constituents content and the comprehensive HPLC profiles between the two CP species using LC‐DAD‐ESI‐MS method are reported for the first time, indicating that the CP drugs from different resources should be used separately in the clinic. Copyright © 2009 John Wiley & Sons, Ltd.     

Qualitative analysis of Psoraleae Fructus by HPLC‐DAD/TOF‐MS fingerprint and quantitative analysis of multiple components by single marker

A variety of bioactive substances may account for the recognized efficacy and wide clinical application of Psoraleae Fructus in China. A high‐performance liquid chromatography–diode array detector (HPLC‐DAD) fingerprint method was developed to present the comprehensive phytochemical profile of the crude drug. Thirteen major compounds were separated and identified by HPLC coupled with time‐of‐flight mass spectrometry (HPLC/TOF‐MS), namely psoralenoside (PO), isopsoralenoside (IPO), psoralen (PS), isopsoralen (IPS), neobavaisoflavone (NBF), bavachin (BC), corylin (CN), bavachromene (BCM), psoralidin (PD), isobavachalcone (IBC), bacachinin (BCN), corylifol A (CA) and bakuchiol (BK). Then quantitative analysis of multiple components by single marker (QAMS) was applied in content determination of PO, IPO, PS, IPS, BC, IBC, BCN, CA and BK, with NBF as the internal standard. The calculation results indicated no significant difference from the traditional external standard method (p > 0.05, RSD < 2.62%), suggesting that QAMS is a reliable and convenient method for content determination of multiple chemical compositions, especially when there is a shortage of reference substances. In conclusion, simultaneous qualitative and quantitative analysis of Psoraleae Fructus may be fulfilled through the newly proposed method of QAMS combined with HPLC‐DAD/TOF‐MS fingerprint.     

Bioflavonoid profile of citrus juices from Greece

High‐performance liquid chromatography with confirmation by UV–visible photodiode array detector–positive electrospray ionization–mass spectrometry [HPLC‐UV–vis‐DAD‐(+ESI)‐MS] with enhanced fragmentation by appropriate adjustment of the cone voltage was used to determine bioflavonoid content of five citrus species (tangerine, sanguine, sour orange, lemon and grapefruit) cultivated in Greece which come from citrus varieties analyzed for the first time. The main groups of bioflavonoids found in the juice of the citrus species according to HPLC retention times, spectral data and literature references were O‐glycosylated flavanones and flavones, C‐glucosylated flavones, O‐glucosylated flavones, O‐C‐glucosylated flavones like saponarin and a phenolic derivative. Copyright © 2012 John Wiley & Sons, Ltd.     

Qualitative and quantitative analysis of the chemical constituents in Mahuang‐Fuzi‐Xixin decoction based on high performance liquid chromatography combined with time‐of‐flight mass spectrometry and triple quadrupole mass spectrometers

High‐performance liquid chromatography coupled with time‐of‐flight mass spectrometry (HPLC‐TOF/MS) and high‐performance liquid chromatography–triple quadrupole mass spectrometry (HPLC‐QQQ/MS/MS) were utilized to clarify the chemical constituents of Mahuang‐Fuzi‐Xixin Decoction. There are 52 compounds, including alkaloids, amino acids and organic acids were identified or tentatively characterized by their characteristic high resolution mass data by HPLC‐QQQ/MS/MS. In the subsequent quantitative analysis, 10 constituents, including methyl ephedrine, aconine, songrine, fuziline, neoline, talatisamine, chasmanine, benzoylmesaconine, benzoylaconine and benzoylhypaconine were simultaneously determined by HPLC‐QQQ/MS/MS with multiple reaction monitoring mode. Satisfactory linearity was achieved with wide linear range and fine determination coefficient (r > 0.9992). The relative standard deviations (RSD) of inter‐ and intra‐day precisions were <3%. This method was also validated by repeatability, stability and recovery with RSD <3% respectively. A highly sensitive and efficient method was established for chemical constituents studying, including identification and quantification of Mahuang‐Fuzi‐Xixin decoction.     

Identification of the absorbed components and metabolites of modified Huo Luo Xiao Ling Dan in rat plasma by UHPLC‐Q‐TOF/MS/MS

To reveal the material basis of Huo Luo Xiao Ling Dan (HLXLD), a sensitive and selective ultra‐high performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry (UHPLC‐Q‐TOF/MS) method was developed to identify the absorbed components and metabolites in rat plasma after oral administration of HLXLD. The plasma samples were pretreated by liquid–liquid extraction and separated on a Shim‐pack XR‐ODS C₁₈ column (75 × 3.0 mm, 2.2 μm) using a gradient elution program. With the optimized conditions and single sample injection of each positive or negative ion mode, a total of 109 compounds, including 78 prototype compounds and 31 metabolites, were identified or tentatively characterized. The fragmentation patterns of representative compounds were illustrated as well. The results indicated that aromatization and hydration were the main metabolic pathways of lactones and tanshinone‐related metabolites; demethylation and oxidation were the major metabolic pathways of alkaloid‐related compounds; methylation and sulfation were the main metabolic pathways of phenolic acid‐related metabolites. It is concluded the developed UHPLC‐Q‐TOF/MS method with high sensitivity and resolution is suitable for identifying and characterizing the absorbed components and metabolites of HLXLD, and the results will provide essential data for further studying the relationship between the chemical components and pharmacological activity of HLXLD.     

Simultaneous Determination Baicalin and Forsythin in Shuanghuanglian Oral Liquid by HPLC/DAD and LC‐ESI‐MS

Rapid, simple and reliable HPLC/DAD and LC‐ESI‐MS methods for the simultaneous determination of baicalin and forsythin in the traditional Chinese medicinal preparation Shuanghuanglian oral liquid were described and validated. The separation condition for HPLC/DAD was optimized using a BDS hypersil C18 column (Thermo, 2.1 × 150 mm, particle size 5 μm) by gradient elution using methanol‐0.2 % ammonium acetate as the mobile phase. The suitable detection wavelength was set at 277 nm for the quantitative analysis of baicalin and forsythin in this method. Some operational parameters of the ESI interface were optimized, negative m/z 445[M?H]^? for baicalin and negative m/z 593[M+CH₃COO]^? for forsythin, positive m/z 447[M+H]⁺ for baicalin and positive m/z 552[M+NH 4]⁺ for forsythin, respectively. These HPLC/DAD and LC‐ESI‐MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). These methods can be used as a complementary method for the commercial quality control of Shuanghuanglian oral liquid and its pharmaceutical preparations.     

Structural characterization and identification of iridoid glycosides,saponins, phenolic acids and flavonoids in Flos Lonicerae Japonicae by a fast liquid chromatography method with diode‐array detection and time‐of‐flight mass spectrometry

A fast liquid chromatography method with diode‐array detection (DAD) and time‐of‐flight mass spectrometry (TOF‐MS) has been developed for analysis of constituents in Flos Lonicerae Japonicae (FLJ), a traditional Chinese medicine derived from the flower bud of Lonicera japonica. The chromatographic analytical time decreased to 25 min without sacrificing resolution using a column packed with 1.8‐µm porous particles (4.6 × 50 mm), three times faster than the performance of conventional 5.0‐µm columns (4.6 × 150 mm). Four major groups of compounds previously isolated from FLJ were structurally characterized by DAD‐TOF‐MS: iridoid glycosides showed maximum UV absorption at 240 nm; phenolic acids at 217, 242, and 326 nm; flavonoids at 255 and 355 nm; while saponins had no absorption. In electrospray ionization (ESI)‐TOF‐MS experiments, elimination of a glucose unit (162 Da), and successive losses of H₂O, CH₃OH and CO, were generally observed in iridoid glycosides; saponins were characterized by a series of identical aglycone ions; phenolic acids typically generated a base peak at [M–H–caffeoyl]^? by loss of a caffeic acid unit (162 Da) and several marked quinic acid moiety ions; cleavage of the glycosidic bond (loss of 162 or 308 Da), subsequent losses of H₂O, CO, RDA and C‐ring fragmentation were the most possible fragmentation pathways for flavonoids. By accurate mass measurements within 4 ppm error for each molecular ion and subsequent fragment ions, as well as the ‘full mass spectral’ information of TOF‐MS, a total of 41 compounds including 13 iridoid glycosides, 11 phenolic acids, 7 saponins, and 10 flavonoids were identified in a methanolic extract of FLJ. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of the chemical constituents of the different processed products of Anemarrhena asphodeloides Rhizomes by high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

In this work, high‐performance liquid chromatography (HPLC) coupled with a hybrid quadrupole time of‐flight mass spectrometry (Q‐TOF‐MS/MS) was used to study chemical compositions of different processed products of Rhizoma Anemarrhenae (RA). A Grace Alltima^TM C₁₈ column (250 × 4.6 mm, 5 µm) was used for separation. Mobile phase consisted of 0.1% formic acid and acetonitrile, using gradient elution. ESI‐MS data was acquired in both positive and negative mode. The experiment was established on the basis of a series of reference substances (two xanthone and seven saponins) to qualitatively identify the chemical compounds of different processed products of RA by MS analysis. There was no difference in the type of chemical constituents between different processed products of RA. A total of 25 compounds were identified, including four xanthones, 21 steroidal saponins and eight pairs of isomers. Copyright © 2015 John Wiley & Sons, Ltd.     

Fusaricidins from Paenibacillus polymyxa M‐1, a family of lipohexapeptides of unusual complexity—a mass spectrometric study

Paenibacillus polymyxa are rhizobacteria with a high potential to produce natural compounds of biotechnological and medical interest. Main products of P . polymyxa are fusaricidins, a large family of antifungal lipopeptides with a 15‐guanidino‐3‐hydroxypentadecanoic acid (GHPD) as fatty acid side chain. We use the P . polymyxa strain M‐1 as a model organism for the exploration of the biosynthetic potential of these rhizobacteria. Using matrix‐assisted laser‐desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) about 40 new fusaricidins were detected which were fractionated by reversed‐phase (rp) HPLC. Their structure was determined by MALDI‐LIFT‐TOF/TOF fragment analysis. The dominant fragment in the product ion spectra of fusaricidins appeared at m /z 256.3, 284.3 and 312.4, respectively, indicating variations in their fatty acid part. Two new subfamilies of fusaricidins were introduced which contain guanidino‐3‐hydroxyhepta‐ and nonadecanoic acid as fatty acid constituents. Apparently, the end‐standing guanidine group is not modified as shown by direct infusion nano‐electrospray ionization mass spectrometry (nano‐ESI MS). The results of this study suggest that advanced mass spectrometry is the method of choice for investigating natural compounds of unusual diversity, like fusaricidins. Copyright © 2016 John Wiley & Sons, Ltd.     

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.     

The profiling and identification of the metabolites of 8‐prenylkaempferol and a study on their distribution in rats by high‐performance liquid chromatography with diode array detection combined with electrospray ionization ion trap time‐of‐flight multistage mass spectrometry

8‐Prenylkaempferol is a prenylflavonoid that has various bioactivities and benefits for human health. A high‐performance liquid chromatography with a diode array detector combined with electrospray ionization ion trap time‐of‐flight multistage mass spectrometry (HPLC‐DAD‐ESI‐IT‐TOF‐MSⁿ) method was established to profile and identify the metabolites of 8‐prenylkaempferol in rat in vivo and in vitro, and to study the distribution of these metabolites in rats for the first time. A total of 38 metabolites were detected and tentatively identified, 30 of which were identified as new compounds. The new in vivo metabolic reactions in rats of prenylflavonoids of isomerization, polymerization, sulfation, amino acid conjugation, vitamin C conjugation and other known metabolic reactions were found in the metabolism of 8‐prenylkaempferol. The numbers of detected metabolites in feces, urine, plasma, small intestine, stomach, kidneys, liver, heart, lungs, spleen and hepatic S9 fraction were 31, 19, 1, 20, 13, 8, 7, 3, 3, 1 and 11, respectively. This indicated that small intestine and stomach were the major organs in which the 8‐prenylkaempferol metabolites were distributed. Furthermore, 16 metabolites were determined to have bioactivities based on the literature and ‘PharmMapper’ analysis. These findings are useful for better comprehension of the effective forms, target organs and pharmacological actions of 8‐prenylkaempferol. Moreover, they provide a reference for the study of the metabolism and distribution of prenylflavonoid aglycone compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Rapid identification of primary constituents in parotoid gland secretions of the Australian cane toad using HPLC/MS‐Q‐TOF

Toad parotoid gland secretion or toad venom has in recent years been increasingly shown to possess potentially beneficial pharmacological effects; this speculation has drawn much interest centred on elucidating the chemical basis of its multimodal effects. For this purpose, we explored the use of a rapid and accurate analysis method for systemic investigation of the parotoid gland chemistry, when extracted from Australian cane toads. Full‐scan data of cane toad venom extract was acquired using high‐performance liquid chromatography coupled with a hybrid quadrupole–time of flight mass spectrometry system (HPLC/MS‐Q‐TOF), with multiple ionization sources (ESI and APCI) in positive and negative mixed modes. By measuring the exact mass differences between the theoretical and measured mass of each assumed compound, we confirmed the presence of 12 key constituents. The present results demonstrate that the use of HPLC/MS‐Q‐TOF with multiple ionization sources delivers exemplary selectivity and sensitivity, allowing for the rapid and accurate identification of constituents within cane toad venom. This paves the way for this technique to be used in future routine screening of components within the genus Bufo and for key analytes too, then reliably assessed for any purported beneficial (clinic) properties. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Screening free radical scavengers in Xiexin Tang by HPLC‐ABTS‐DAD‐Q‐TOF/MS

Xiexin Tang (XXT) is a traditional Chinese medicine (TCM) that has been used in herbal clinics for more than 1800 years. Many studies have shown that XXT has therapeutic effects on patients with arteriosclerosis owing to its antioxidant activity. However, there is little information about the relationship between the chemical composition of XXT and its antioxidant activity. In this study, the HPLC‐ABTS‐DAD‐Q‐TOF/MS method, which can simultaneously identify individual components and rapidly screen for antioxidant compounds, was used to screen and identify antioxidant components in XXT. The 15 compounds identified were gluco‐syringic acid, adenine, gallic acid, biflorin, cularine, 6‐C ‐arabinose‐8‐C ‐glucose‐chrysin, 6‐C ‐glucose‐8‐C ‐arabinose–chrysin, baicalin, rhein‐8‐O‐β ‐d ‐glucopyranoside, coptisine, epiberberine, jatrorrhizine, norwogonin, 5,7,2′‐trihydroxy‐6‐ methoxyflavone and baicalein. In addition, the data showed that the antioxidant activity of peaks 4, 6, and 11 was lower in XXT than in its constituent herbs, while the activity of peaks 1, 2, 3, 5, 7, 8, 10, 12, 13, 14 and 15 was higher in XXT. Compound 5 had the strongest antioxidant activity in XXT, while compound 1 showed the strongest antioxidant activity among its constituent herb. The differences between antioxidant activities of major components of XXT and those of its constituent herbs might be due to the interaction of crude drugs that changes the solubility of active components during the decoction process. The results show that the HPLC‐ABTS‐DAD‐Q‐TOF/MS method can successfully combine on‐line mass spectrometry with activity detection system. It is a useful tool for the rapid detection and identification of antioxidants, and for quantitative analysis of individual antioxidants in complex mixtures such as plant extracts. Furthermore, this method does not require extensive extract purification and fraction collection.