Array Detection-electrospray Ionization Tandem Mass Research Articles

Prunus lusitanica L. Fruits as a Novel Source of Bioactive Compounds with Antioxidant Potential: Exploring the Unknown.

Prunus lusitanica L., also known as Portuguese laurel or locally known as ‘azereiro’, is a rare species with ornamental and ecological value. Only two studies regarding the bioactivity and chemical composition of its leaves were reported to date. Thus, the present study aims to qualitatively and quantitatively evaluate the phenolic profile, through HPLC-PAD-ESI-MS/MS (high-performance liquid chromatography–photodiode array detection–electrospray ionization tandem mass spectrometry), as well as the radical scavenging capacity, through ABTS (2,2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) and DPPH (2,2-diphenyl-1 picrylhydrazyl), and the reducing power (FRAP, ferric reducing antioxidant power) assays, of P. lusitanica fruits during a 4-year study. In total, 28 compounds were identified and quantified in the fruits, including 21 hydroxycinnamic acids (60.3%); 2 flavan-3-ols (27.9%), 2 anthocyanins (10.5%), 2 flavonols (1.0%), and 1 secoiridoid (0.3%). High antioxidant capacity was observed, with ABTS values ranging from 7.88 to 10.69 mmol TE (Trolox equivalents)/100 g fw (fresh weight), DPPH values from 5.18 to 8.17 mmol TE/100 g fw, and FRAP values from 8.76 to 11.76 mmol TE/100 g fw. According to these results, it can be concluded that these are rich sources of phenolic compounds with very promising antioxidant capacity and, therefore, with potential applications in the food and/or phytopharmaceutical sectors.

The Influence of Ripeness on the Phenolic Content, Antioxidant and Antimicrobial Activities of Pumpkins (Cucurbita moschata Duchesne)

Cucurbita moschata Duchesne (Cucurbitaceae) is a plant food highly appreciated for the content of nutrients and bioactive compounds, including polyphenols and carotenoids, which contribute to its antioxidant and antimicrobial capacities. The purpose of this study was to identify phenolic acids and flavonoids of Cucurbita moschata Duchesne using high-performance liquid chromatography–diode array detection–electrospray ionization tandem mass spectrometry (HPLC–DAD–ESI-MS) at different ripening stages (young, mature, ripened) and determine its antioxidant and antimicrobial activities. According to the results, phenolic acids and flavonoids were dependent on the maturity stage. The mature fruits contain the highest total phenolic and flavonoids contents (97.4 mg GAE. 100 g−1 and 28.6 mg QE. 100 g−1).A total of 33 compounds were identified. Syringic acid was the most abundant compound (37%), followed by cinnamic acid (12%) and protocatechuic acid (11%). Polyphenol extract of the mature fruits showed the highest antioxidant activity when measured by DPPH (0.065 μmol TE/g) and ABTS (0.074 μmol TE/g) assays. In the antimicrobial assay, the second stage of ripening had the highest antibacterial activity. Staphylococcus aureus was the most sensitive strain with an inhibition zone of 12 mm and a MIC of 0.75 mg L−1. The lowest inhibition zone was obtained with Salmonella typhimurium (5 mm), and the MIC value was 10 mg L−1.

Phenolic compounds profile of different berry parts from novel Vitis vinifera L. red grape genotypes and Tempranillo using HPLC-DAD-ESI-MS/MS: A varietal differentiation tool

The detailed phenolic composition of different berry parts from two novel V. vinifera L. red grape genotypes (Moribel and Tinto Fragoso), together the well-known Tempranillo, was established using high performance liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS/MS) over two consecutive vintages (2016 and 2017). More than 50 phenolic compounds were identified and quantified: 25 anthocyanins, 17 flavonols, 7 hydroxycinnamic acid derivatives, 2 stilbenes, and several flavan-3-ols. As far as we know, some anthocyanin and flavonol dihexosides were reported for the first time in V. vinifera L. grapes. Application of Principal Component Analysis (PCA) to experimental data showed a good separation of the novel grape genotypes and Tempranillo according to the phenolic profile of skins and seeds, mainly based on the proportion of trisubstituted anthocyanin derivatives, flavonols and flavan-3-ols, being a useful tool to differentiate these grape varieties.

Identification and evaluation of antioxidant components in the flowers of five Chimonanthus species

Chimonanthus Lindl. has seven family members with Ch. praecox blooming in winter and others in autumn. The plants are widely used in landscaping, fragrance industry, as well as production of medicinal material because of their flower fragrance. Both Chimonanthus flowers and leaves contain abundant secondary metabolites. But it is unclear whether the flowers from different species have distinct ingredients and antioxidant activity. To address the question, we evaluated the main compositions of the flowers from five Chimonanthus species and analyzed the antioxidant activity of the compositions. The results indicated that flavonoids and coumarins are the two major types of substances with antioxidant capacity in Chimonanthus flowers. Totally nine antioxidative compounds, including scopolin and scopoletin, were identified based on the high performance liquid chromatography-photo diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD/ESI–MS). All the antioxidant compounds were confirmed by pre-column 2, 2-diphenyl-1- picryhydrazyl (DPPH) assay. Rutin (highest in content), isoquercitrin and quercetin appeared stronger scavenging DPPH radical ability than the other five compounds, mainly due to their ortho-dihydroxyl groups. Among the five Chimonanthus species, Ch. praecox flower is most abundant in secondary metabolites and shows most potent antioxidant activity. The other four flowers contain the same types of ingredients with Ch. praecox, but exhibit lower antioxidant activity. The results obtained from this comprehensive analysis provide a theoretical basis for the utilization of the plant resources of Chimonanthus genus.

Determination of Phenolic Content in Different Barley Varieties and Corresponding Malts by Liquid Chromatography-diode Array Detection-Electrospray Ionization Tandem Mass Spectrometry.

A simple and reliable method for the simultaneous determination of nine phenolic compounds in barley and malted barley was established, using liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS/MS). The phenolic compounds can be easily detected with both systems, despite significant differences in sensitivity. Concentrations approximately 180-fold lower could be achieved by mass spectrometry analysis compared to diode array detection, especially for the flavan-3-ols (+)-catechin and (−)-epicatechin, which have poor absorptivity in the UV region. Malt samples were characterized by higher phenolic content comparing to corresponding barley varieties, revealing a significant increase of the levels of (+)-catechin and (−)-epicatechin during the malting process. Moreover, the industrial malting is responsible for modification on the phenolic profile from barley to malt, namely on the synthesis or release of sinapinic acid and epicatechin. Accordingly, the selection of the malting parameters, as well as the barley variety plays an important role when considering the quality and antioxidant stability of beer.

MICROWAVE-ASSISTED HYDROLYZATION OF THE FLAVONOIDS IN EPIMEDIUM KOREAMUM NAKAI

In the present paper, icariside I has been prepared by hydrolyzing the flavonoids extract of Epimedium Koreamum Nakai in acid solution. Two heating methods, reflux and microwave irradiation, were used in the hydrolysis reaction and hydrolyzed products were analyzed by high performance liquid chromatography-diode array detection-electrospray ionization tandem mass spectometry (HPLC-DAD-ESI-MSn). The four flavonoids in the extracts have been identified as epimedin A, epimedin B, epimedin C, and icariin, and the hydrolysis product was icariside I. Icariside I has been evaluated by comparing with the peak areas of HPLC-DAD spectra obtained using different heating methods. The experimental results demonstrate that the advantage of microwave irradiation over conventional reflux is validated, and the hydrolysis time is dramatically reduced, and the yield of icariside I is effectively improved. Also, optimum conditions for microwave hydrolysis are established. As a result, the optimum yield of icariside I can be reached under the following conditions: the reaction temperature is 70°C; the microwave irradiation power is 600 W; and the microwave irradiation time is 10 min. The optimum yield by this method was (300.46 ± 0.41 µg), which was 2.3 times more than the classical way (128 ± 0.86 µg). In view of the efficiency, energy saving, and environmental protection in the procedures of microwave-assisted hydrolyzation, this method will have a bright future in the industrial application.

Simultaneous Screening and Identifying Four Categories of Particular Flavonoids in the Leaves of Murraya exotica L. by HPLC-DAD-ESI-MS-MS

Polymethoxylated flavonoids (PMFs), the particular flavonoid subclass in which all or almost all hydroxyls are capped by methylation, have high oral bioavailability and various activities. A sensitive high-performance liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS-MS) method was established to screen and identify the PMFs in the leaves of Murraya exotica. Eight PMF standards, including two polymethoxylated flavones, two polymethoxylated flavanones, two polymethoxylated chalcones and two PMF glycosides, were first to be analyzed in positive mode by collision-induced dissociation MS-MS. On the basis of the ESI-MS, characterizations were deduced, and in the results of the extracted ion chromatogram MS-MS experiment, 26 PMFs, including 18 flavones, five flavanones or chalcones and three PMF glycosides, were screened out from the complex extract of the leaves of M. exotica. Among them, 24 PMFs were hydroxylated polymethoxyflavonoids, whereas the rest were all permethoxylated PMFs. This was the first systematic report on the presence of PMFs in the leaves of M. exotica. The results indicated that the established analytical method could be adopted as a rapid, effective technique for the structural characterization of PMFs from the complex extracts of traditional Chinese medicines.

Characterization and simultaneous quantification of multiple constituents in Aurantii Fructus Immaturus extracts by HPLC-DAD-ESI-MS/MS

AimTo develop a sensitive and accurate analytical method for the profiling, identification and simultaneous quantification of the chemical constituents in Aurantii Fructus Immaturus extracts. MethodsA high performance liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-MS/MS) method was established for the characterization and quantification of multi-constituents in Aurantii Fructus Immaturus extracts. Chromatographic separation was achieved on a C18 column using a detailed gradient program with 2% formic acid and acetonitrile as the mobile phase. Mass spectrometry was in the negative mode for identification, and a diode array detector was set at 283 nm for quantification. ResultsEighteen compounds were identified, or tentatively identified, by comparing their retention times, UV and MS spectra with those of authentic compounds or literature data, including 12 flavonoids, 5 coumarins and a limonoid in Aurantii Fructus Immaturus extracts. Twelve of the major compounds were determined simultaneously by HPLC-DAD. The fragmentation pathways of the authentic references were also investigated for the structural identification of the constituents in the extracts. There have been no reports on the simultaneous and comprehensive analysis of both the major and minor compounds in Aurantii Fructus Immaturus extracts. ConclusionsThe method validation indicated that the developed analysis method could be employed as a rapid, effective technique for the structural characterization and simultaneous quantification of multi-constituents in this botanical extract. This work is expected to provide comprehensive information for the quality evaluation of Aurantii Fructus Immaturus extracts, which should be a valuable support for the further study and development of this raw material and its related medicinal products.

Screening and quantitative analysis of antioxidants in the fruits of Livistona chinensis R. Br using HPLC-DAD–ESI/MS coupled with pre-column DPPH assay

In this study, a high performance liquid chromatography-photo diode array detection–electrospray ionization tandem mass spectrometry (HPLC-DAD/ESI-MS) with pre-column DPPH assay is developed for screening the antioxidant components in the fruits of Livistona chinensis R. Br. Accordingly, six antioxidative flavonoids are identified as orientin, isoorientin, vitexin, isovitexin, isorhamnetin-3-O-glucoside and tricin in methanolic extract of L. chinensis fruits, based on their mass spectra and fragmentation patterns. To the best of our knowledge, orientin, isoorientin, isovitexin and isorhamnetin-3-O-glucoside were found firstly in this plant. The free radical scavenging activity of the six antioxidants found is further examined by off-line DPPH assay. The results indicated that the free radical scavenging activity of orientin and isoorientin are stronger than those of two antioxidative drugs, vitamin C and baicalin. In addition, an HPLC-DAD method is firstly established for simultaneous determination of the six antioxidants in L. chinensis fruits. Tricin was found to be the major component in L. chinensis fruits.

Comparison of Two Species of Notopterygium by High-Performance Liquid Chromatography—Photodiode Array Detection—Electrospray Ionization Tandem Mass Spectrometry

Notopterygium incisum Ting ex H.T. Chang (N. incisum) and Notopterygium forbesii Boiss (N. forbesii) are two medicinal species of Qianghuo (a well-known traditional herbal medicine in China) that are widely used in clinical prescriptions for the treatment of colds and rheumatism. To compare the chemical constituents of these two plant materials, the phenolic constituents and coumarins of these two species were comprehensively and systematically analyzed by high-performance liquid chromatography-photodiode array detection-electrospray ionization tandem mass spectrometry (HPLC/DAD/ESI-MS(n)) for the first time. A total of 25 compounds (nine phenolic compounds and 16 coumarins) were detected in the methanol extracts. These compounds were separated on a C18 column and identified or tentatively characterized on the basis of their UV spectra and MS fragmentation behavior. In contrast to previous reports, we found that these two plant species possess very different coumarin patterns. O-Demethylfuropinnarin (18), phenethylferulate (19), notopterol (20), and isoimperatorin (22) were the predominant constituents of N. incisum, whereas nodakenin (6), 6-O-trans-feruloylnodakenin (12), p-hydroxypenethylanisate (16) and isoimperatorin (22) were the major constituents of N. forbesii. O-Demethylfuropinnarin (18), phenethylferulate (19) and notopterol (20) were only detected in N. incisum and can be regarded as useful taxonomic markers for differentiating these two plant species. Considering the marked differences in the main chemical constituents of N. incisum and N. forbesii, the biological activities of these two species should be further investigated and compared to ensure consistency and efficacy in the pharmaceutical applications of these materials.

Simultaneous qualitative and quantitative determination of major polymethoxylated flavonoids in the leaves of Murraya paniculata by RRLC-DAD-ESI-MSn

A rapid-resolution liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry (RRLC-DAD-ESI-MSn) method was established to identify and quantify the major bioactive constituents in the leaves of Murraya paniculata (L.) Jack. Seven of the main PMFs were determined by the RRLC method with detection wavelength at 330 nm in 30 minutes. The total PMFs in the samples ranged from 12.688 mg g−1 to 54.997 mg g−1. Meanwhile, sixteen PMFs could be identified from the complex extract of the leaves of M. paniculata by the RRLC-ESI-MS/MS method. The developed RRLC method was validated in terms of good linearity, precision and accuracy. The RRLC-ESI-MS/MS method was simple and useful for rapid identification of principal constituents in M. paniculata.

Determination of galacturonic acid content in pectin from fruit juices by liquid chromatographydiode array detection-electrospray ionization tandem mass spectrometry

Abstract A reversed-phase high-performance liquid chromatographic (HPLC) method is applied for the determination of galacturonic acid (GA) of pectins in different commercial fruit juices. The separation was carried out on a C18 column using precolumn derivatization with p-aminobenzoic acid (p-ABA) and UV detection at 304 nm. The identification of GA was confirmed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) in positive ion mode. The concentration of GA in the samples analyzed ranged from 12.9 ± 0.5 to 49.4 ± 0.5 mgGA L−1. Amongst the samples analyzed, mango juice was found to be richest in GA content, and therefore a good source of pectins. Detection and quantification limits of the described methodology were 1.2 and 3.9 mg L−1, respectively. Quantitative GA recoveries in the beverages had a range between 90 and 98%. The results showed that the HPLC method proposed was precise and suitable for the identification and quantification of GA in commercial fruit juices.

Rapid Identification of two Species of Peucedanum by High-Performance Liquid Chromatography-Diode Array Detection-Electrospray Ionization Tandem Mass Spectrometry

The fragmentation behaviors of the angular- and linear-type coumarins from Peucedanum praeruptorum Dunn and P. decursivum (Miq.) Maxim were simultaneously investigated by HPLC-DAD/ESI-MS(n). For more structural identification, the fragment ions were analyzed and some possible fragmentation pathways were proposed. Different positions and numbers of the substituent also led to different fragment behaviors. Two types of coumarins from P. praeruptorum and P. decursivum were structurally elucidated by these techniques. In addition, UV spectra were applied to support the MS analysis. This is the first time that the two types of coumarins from herbal extracts have been differentiated by HPLC-DAD/ESI-MS(n). The method further illustrated the importance of the ESI-MS(n) technique in the identification of different types of coumarins and was applied for the rapid differentiation of the two herbs.

Chromatographic fingerprint analysis and characterization of furocoumarins in the roots of Angelica dahurica by HPLC/DAD/ESI-MS n technique

A high performance liquid chromatography–diode array detection–electrospray ionization tandem mass spectrometry (HPLC/DAD/ESI-MS n ) method was used for the chromatographic fingerprint analysis and characterization of furocoumarins in the roots of Angelica dahurica for the first time. Nine “common peaks” were identified by comparing with the retention time, UV and MS spectra of reference furocoumarins. The software “Similarity Evaluation System for Chromatographic Fingerprint of TCM” was used to evaluate the similarities of 13 batches of Baizhi samples collected from Henan, Zhejiang, Sichuan and Anhui provinces of China. The results indicated that the samples from different batches had similar HPLC fingerprints, and the method could be applied for the quality control of the roots of Angelica dahurica. In addition, a total of 20 furocoumarins were identified or tentatively characterized by HPLC/DAD/ESI-MS n technique, and their fragmentation patterns in an electrospray ion trap mass spectrometer were also summarized.

Identification of major constituents in the traditional Chinese medicine “QI-SHEN-YI-QI” dropping pill by high-performance liquid chromatography coupled with diode array detection-electrospray ionization tandem mass spectrometry

An HPLC–DAD–ESI-MS n method was developed for simultaneous analysis of major chemical constituents in “QI-SHEN-YI-QI” dropping pill, a traditional Chinese medicine (TCM) widely used for treating cardiovascular diseases. The chromatographic separation was performed on an intertsil ODS-3 C 18 column (4.6 mm × 250 mm, 5 μm), whilst water with 0.05% acetic acid and acetonitrile were used as mobile phase. On the basis of the characteristic UV absorption profile, the information of molecular weight, and structure provided by ESI-MS n , 31 constituents derived from Astragalus membranaceus, Radix Salviae Miltiorrhizae, and Panax notoginseng, were detected and 20 of them were identified in this study. The proposed method contributes to the quality control of “QI-SHEN-YI-QI” dropping pill.

Simultaneous analysis of coumarins and secoiridoids in Cortex Fraxini by high-performance liquid chromatography–diode array detection–electrospray ionization tandem mass spectrometry

A high-performance liquid chromatography–diode array UV detection–electrospray ionization tandem mass spectrometry (HPLC–DAD–ESI-MS) method was developed and validated for the simultaneous analysis of seven major constituents in Cortex Fraxini, including esculin, esculetin, fraxetin, fraxin, escuside, oleuropein and ligustroside. The contents of the seven constituents were determined by using HPLC–DAD, and the chemical structures of these constituents were identified by using HPLC–DAD–ESI-MS method. The separation was performed on an Agilent Zorbax SB-C 18 column (250 mm × 4.6 mm i.d., 5 μm) with gradient elution of acetonitrile and 0.3% aqueous acetic acid within 40 min. Detection was performed at 254 nm. The calibration curves showed good linearity ( r 2 > 0.9992). The limits of detection (LOD) ranged from 1.07 to 3.19 ng/ml and limits of quantification (LOQ) ranged from 2.79 to 12.75 ng/ml, respectively. The intra- and inter-day precision was less than 5% and the accuracy was ranging from 96.49% to 103.55%. The recovery of the assay was in the range of 97.61–104.36%. The method was successfully applied to the quantification of the seven constituents in different samples of Cortex Fraxini. The results indicated that the developed method could be considered to be a simple, rapid and reliable method for the quality evaluation of Cortex Fraxini.

Fingerprint analysis of the fruits of Cnidium monnieri extract by high-performance liquid chromatography–diode array detection–electrospray ionization tandem mass spectrometry

A method incorporating high-performance liquid chromatography (HPLC) with electrospray ionization (ESI) and tandem mass spectrometry (MS), with parallel analysis by HPLC with UV detection using a diode-array detector (DAD) was developed for the qualitative characterization of coumarin and chromone constituents in the fruits of Cnidium monnieri. The chromatographic separations were performed on a Diamonsil™ C18 column (4.6 mm × 200 mm, 5 μm) with water with 50 mM ammonium acetate and 2% acetic acid (A) and acetonitrile (B) as the mobile phase. According to the characteristic UV spectra, the information of molecular weight and structure provided by ESI–MS/MS, 13 coumarin and 7 chromone components were detected and identified. This method is rapid and reliable for identification of the constituents in the complex herbal system, and the fragmentation patterns proposed could be extended to the similar compounds.