Detection of flavonoids from Spinacia oleracea leaves using HPLC-ESI-QTOF-MS/MS and UPLC-QqQLIT-MS/MS techniques.

Spinacia oleracea L. (Spinach) is a leafy vegetable which is considered to have a high nutritional value. Flavonoids in spinach were reported to act as antimutagenic property. Rapid detection of these flavonoids in Spinach was achieved by using HPLC-ESI-QTOF-MS/MS. Thirty six compounds were tentatively identified based on their retention times, accurate mass and MS/MS spectra. The fragmentation patterns of known compounds were applied to elucidate the structure of their corresponding derivatives having the same basic skeleton. Out of thirty six peaks, three peaks were assigned as patuletin and six peaks were assigned as spinacetin derivatives. Twelve compounds were first time identified following the fragmentation pattern of known compounds. Five of the identified compounds i.e., spinacetin, 5,3',4'-trihydroxy-3-methoxy-6,7-methylenedioxyflavone, protocatechuic acid, ferulic acid and coumaric acid were simultaneously quantified in spinach leaves by a validated UPLC-ESI-MS/MS method under MRM mode.

Development of Ultra Performance Liquid Chromatography Tandem Mass Spectrometry Method for Simultaneous Identification and Quantitation of Potential Osteogenic Phytochemicals in Butea monosperma.

An ultra performance liquid chromatography coupled with hybrid triple-quadrupole linear ion trap tandem mass spectrometry (UPLC-ESI-QqQLIT-MS-MS) method in multiple reaction monitoring mode was developed for identification and simultaneous determination of potential osteogenic compounds in ethanol extracts of different plant parts of Butea monosperma collected from different geographical regions. The chromatographic separation was carried out on an Acquity UPLC CSH C18 column (1.7 µm, 2.1 × 100 mm) with 0.1% (v/v) formic acid in water and methanol as mobile phase under gradient conditions in 8 min. The developed method was validated according to the guidelines of international conference on harmonization. The correlation coefficients of all the calibration curves were ≥0.9995 and recoveries ranged from 95.2 to 105.8% (RSD ≤ 1.95%). Relative standard deviations of intra-day, inter-day precisions and stability were ≤1.74, 1.84 and 2.8%, respectively. The quantitative results showed remarkable differences in the content of all potential osteogenic compounds in different parts of the plant as well as samples from different geographical regions. Quantitative variations studied from principal component analysis indicated tentative markers for B. monosperma cultivars which can discriminate sample of different geographical regions.

Simultaneous quantitative determination of bioactive terpene indole alkaloids in ethanolic extracts of Catharanthus roseus (L.) G. Don by ultra high performance liquid chromatography-tandem mass spectrometry.

A rapid, sensitive and reproducible method using ultra-high-performance liquid chromatography coupled with electrospray ionization hybrid triple quadrupole-linear ion trap mass spectrometry (UHPLC-ESI-QqQLIT-MS/MS) in multiple reaction monitoring (MRM) mode was developed and validated for simultaneous quantitation of anticancer (vincristine, vinblastine, vindesine), antihypertensive (ajmaline, ajmalicine, reserpine), aphrodisiac (yohimbine), sedative (serpentine) agents, dietary supplement (vinpocetine, yohimbine) and precursor of vinblastine (vindoline) from crude extracts of Catharanthus roseus. The precursor to product ion transitions for these compounds were observed at m/z 327 → 144, 355 → 144, 754 → 355, 353 → 144, 349 → 317, 825 → 225, 811 → 224, 458 → 188, 351 → 280 and 609 → 195, respectively in positive ionization mode. Chromatographic separation of all targeted TIAs was performed on ACQUITY UPLC BEH™ C18 column (1.7 µm, 2.1 mm × 50 mm). The calibration curves were linear within the concentration range 0.5-1000 ng/mL and correlation coefficients (R2) were closer to 1. Limit of detection (LOD) and limit of quantitation (LOQ) ranged from 0.039-0.583 ng/mL and 0.118-1.767 ng/mL, respectively. The intra-day (0.23-2.71% RSD) and inter-day (0.40-2.90% RSD) precision, stability (0.69-3.45% RSD) and recovery (99.63-104.30% ±â€¯%RSD ≤ 3.03%) were acceptable indicating good accuracy of the developed method. The method was successfully applied in ethanolic extracts of 39 samples of C. roseus parts (leaf, stem and root) collected from five different locations in India. Serpentine was detected as one of the most abundant TIA. Principal component analysis (PCA) was able to successfully discriminate among C. roseus samples on the basis of content of targeted TIAs.

Identification, characterization and distribution of terpene indole alkaloids in ethanolic extracts of Catharanthus roseus using high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry and the study of their geographical variation.

RATIONALE: Catharanthus roseus is a well-known dicotyledonous medicinal plant containing diverse classes of bioactive terpene indole alkaloids (TIAs), in particular the anticancer agents vinblastine and vincristine. In view of the commercial importance of these compounds there is an urgent need to develop an accurate and reliable method for the screening of TIAs from C. roseus. METHODS: A method for the separation and characterization of these compounds was developed using high-performance liquid chromatography coupled with positive electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC/ESI-QTOF-MS/MS). Chromatographic separation of TIAs was carried out using a Thermo Betasil C8 column (250 mm × 4.5 mm, 5 µm) at 25°C using 0.1% formic acid in water and acetonitrile. RESULTS: Diagnostic fragmentation pathways for vinpocetine, vindesine, catharanthine, vinblastine, vindoline and vincristine were established on the basis of their product ions. A total of 72 TIAs were detected of which 11 were unambiguously identified by comparison with their standards, and the remaining 61 were tentatively identified. The geographical distribution of the TIAs in ethanolic extracts of 30 samples of C. roseus collected from five states of India was studied using principal component analysis (PCA). CONCLUSIONS: The developed analytical method together with diagnostic fragment patterns were used to rapidly and effectively identify targeted and untargeted TIAs in C. roseus. A PCA study of the results obtained was used to discriminate among the C. roseus samples.

Development of a UHPLC-MS/MS method for the quantitation of bioactive compounds in Phyllanthus species and its herbal formulations.

Phyllanthus species are extensively used in traditional medicines for the treatment of hepatic diseases due to their bioactive hypophyllanthin and phyllanthin. This work describes the development and validation of an ultra high performance liquid chromatography with tandem mass spectrometry method in polarity switching multiple reaction monitoring mode for the simultaneous detection and quantitation of 23 compounds using ultra-performance liquid chromatography coupled with electrospray-hybrid triple quadrupole-linear ion trap mass spectrometer. The validated parameters showed good linearity (R2  ≥ 0.996), limit of detection (0.05-1.62 ng/mL), limit of quantitation (0.15-4.95 ng/mL), precisions (intra-day: RSD ≤ 2.11%), (inter-day: RSD ≤ 2.91%), stability (RSD ≤ 2.56%) and overall recovery (98.22-104.48%; RSD ≤ 2.93%). The validated method was successfully applied in ethanolic extracts of P. amarus, P. niruri, P. emblica, P. fraternus, fractions of P. amarus and their herbal formulations for quantitation. The maximum content of hypophyllanthin (29.40 mg/g) and phyllanthin (56.60 mg/g) was detected in ethyl acetate fraction of P. amarus. The total content of 23 compounds was abundant in the ethanolic extract of P. emblica fruit. Principal component analysis was used to differentiate the selected Phyllanthus species and their herbal formulations. The results indicated that the present method could be used for quality control of Phyllanthus species and its herbal formulations.

Chemometric Based Identification and Validation of Specific Chemical Markers for Geographical, Seasonal and Gender Variations in Tinospora cordifolia Stem using HPLC-ESI-QTOF-MS Analysis.

INTRODUCTION: Tinospora cordifolia is a widely distributed medicinal plant used in various traditional and commercial Ayurvedic formulations. Due to the wide use of this plant it is important to know the extent of variability in the metabolite profile resulting from geographical location, season and gender. OBJECTIVE: To develop a statistical approach based on phytochemical markers for confident prediction of variations in metabolic profile and cytotoxicity due to geographical, seasonal and gender difference in T. cordifolia stem. METHODS: A HPLC-ESI-QTOF-MS method was used for the metabolite profiling of T. cordifolia stem. The data were analysed using chemometric methods including Student's t-test, ANOVA, FA/PCA and ROC curve analysis and validated for the identification of chemical variations. The bioactivity of selected samples was also tested using a cell cytotoxicity assay to assess the functional aspect of the phytochemical variability. RESULTS: The chemometric approach applied here identified marker ions for geographical locations (m/z 294.1139 and 445.2136), seasons (m/z 344.1482, 359.1501, and 373.1305) and gender (m/z 257.1380) with 100% statistical sensitivity and specificity. An in vitro cytotoxicity evaluation revealed that male T. cordifolia stem was the most effective in inhibiting the growth of cancerous cell lines. CONCLUSIONS: The developed and validated chemometric approach identified the analytical markers for phytochemical variations in unknown T. cordifolia stem samples from male or female plants and samples collected from different geographical locations and seasons. The results are supported by comparative cytotoxic activity data. Copyright © 2017 John Wiley & Sons, Ltd.

Simultaneous determination of pyrroquinazoline alkaloids and flavonoids in Adhatoda beddomei and Adhatoda vasica and their marketed herbal formulations using ultra-high-performance liquid chromatography coupled with triple quadrupole linear ion trap mass spectrometry.

Adhatoda beddomei and Adhatoda vasica leaf, known as 'Vasaka' and/or 'Vasa' in Ayurveda and 'Malabar nut' in English, is an official drug in the Indian Pharmacopoeia. The medicinal properties of these plants are due to the presence of pyrroquinazoline alkaloids. An UHPLC-ESI/MS/MS method in both positive and negative electrospray ionization in multiple-reaction-monitoring mode was developed and validated for the estimation of alkaloids and flavonoids in Adhatoda species and their marketed herbal formulations. Chromatographic separation was achieved on an Acquity UPLC® BEH C18 -column using a gradient elution with 0.1% formic acid in water and methanol. The developed method was validated as per International Conference on Harmonization guidelines and found to be accurate with overall recovery in the range 94.2-105.0% (RSD ≤ 1.71%), precise (RSD ≤ 3.44%) and linear (R2 ≥ 0.9992) over the concentration range of 0.5-1000 ng/mL. The total content of alkaloids and flavonoids were highest in the chloroform and aqueous fraction of A. vasica leaf, respectively. The results indicated that the developed method was simple, rapid, sensitive, selective and accurate for the estimation of multiple bioactive constituents in crude mixture, and therefore could make a contribution to the quality control of Adhatoda species and its derived herbal formulations.

Analysis of isoquinoline alkaloids from Mahonia leschenaultia and Mahonia napaulensis roots using UHPLC-Orbitrap-MSn and UHPLC-QqQLIT-MS/MS.

Mahonia leschenaultia (ML) and Mahonia napaulensis (MN) are less known and unexplored medicinal plants of the family Berberidaceae. They are used by the Todas of Nilgiris in their religious and medical practices but chemically less identified. Hence, we decided to do extensive phytochemical analysis to explore the potential of these plant extracts. An ultrahigh performance electrospray tandem mass spectrometry (UHPLC-ESI-MS/MS) method was successfully developed for qualitative analysis of the bioactive components in Mahonia species using Orbitrap Velos Pro mass spectrometer. Sixteen compounds were identified by comparison of their retention times and mass spectra (MS) with authentic standards and reported literature. Multi-stage mass spectra (MS2-8) for the identification of protoberberine and aporphine alkaloids showed the sequential expulsion of all the substituents attached with their basic skeleton followed by CO loss. Eight of the identified compounds (berberine, jatrorrhizine, palmatine, magnoflorine, isocorydine, glaucine, tetrahydropalmatine and tetrahydroberberine) were simultaneously determined by another UHPLC-ESI-MS/MS method under the multiple reactions monitoring (MRM) mode quantitatively using triple quadrupole linear ion trap mass spectrometer. The analytical method was validated for 8 bioactive compounds with overall recovery in the range 98.5%-103.6% (RSD≤2.2%), precise (RSD≤2.07%) and linear (r≥0.9995) over the concentration range of 0.5-1000 ng/mL and successfully applied in ML and MN roots, which suggests the suitability of the proposed approach for the routine analysis of Mahonia species and their quality control.

Analysis of isoquinoline alkaloids from Mahonia leschenaultia and Mahonia napaulensis roots using UHPLC-Orbitrap-MSn and UHPLC-QqQLIT-MS/MS / 药物分析学报

Mahonia leschenaultia (ML) and Mahonia napaulensis (MN) are less known and unexplored medicinal plants of the family Berberidaceae. They are used by the Todas of Nilgiris in their religious and medical practices but chemically less identified. Hence, we decided to do extensive phytochemical analysis to explore the potential of these plant extracts. An ultrahigh performance electrospray tandem mass spectrometry (UHPLC–ESI–MS/MS) method was successfully developed for qualitative analysis of the bioactive components in Mahonia species using Orbitrap Velos Pro mass spectrometer. Sixteen compounds were identified by comparison of their retention times and mass spectra (MS) with authentic standards and reported literature. Multi-stage mass spectra (MS2–8) for the identification of protoberberine and aporphine alkaloids showed the sequential expulsion of all the substituents attached with their basic skeleton followed by CO loss. Eight of the identified compounds (berberine, jatrorrhizine, palmatine, magnoflorine, isocorydine, glaucine, tetrahydropalmatine and tetrahydroberberine) were simultaneously determined by another UHPLC–ESI–MS/MS method under the multiple reactions monitoring (MRM) mode quantitatively using triple quadrupole linear ion trap mass spectrometer. The analytical method was validated for 8 bioactive compounds with overall recovery in the range 98.5%–103.6%(RSD≤2.2%), precise (RSD≤2.07%) and linear (r≥0.9995) over the concentration range of 0.5–1000 ng/mL and successfully applied in ML and MN roots, which suggests the suitability of the proposed approach for the routine analysis of Mahonia species and their quality control.

Simultaneous Determination of Bioactive Monoterpene Indole Alkaloids in Ethanolic Extract of Seven Rauvolfia Species using UHPLC with Hybrid Triple Quadrupole Linear Ion Trap Mass Spectrometry.

INTRODUCTION: Rauvolfia serpentina is an endangered plant species due to its over-exploitation. It has highly commercial and economic importance due to the presence of bioactive monoterpene indole alkaloids (MIAs) such as ajmaline, yohimbine, ajmalicine, serpentine and reserpine. OBJECTIVE: To develop a validated, rapid, sensitive and selective ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry (UHPLC-QqQLIT -MS/MS) method in the multiple reaction monitoring (MRM) mode for simultaneous determination of bioactive MIAs in ethanolic extract of seven Rauvolfia species and herbal formulations. METHODS: The separation of MIAs was achieved on an ACQUITY UPLC BEH™ C18 column (1.7 µm, 2.1 mm × 50 mm) using a gradient mobile phase (0.1% aqueous formic acid and acetonitrile) at flow rate 0.3 µL/min in 7 min. The validated method showed good linearity (r(2)  ≥ 0.9999), limit of detection (LOD) (0.06-0.15 ng/mL), limit of quantitation (LOQ) (0.18-0.44 ng/mL), precisions [intraday: relative standard deviation (RSD) ≤ 2.24%, interday: RSD ≤ 2.74%], stability (RSD ≤ 1.53%) and overall recovery (RSD ≤ 2.23%). RESULTS: The validated method was applied to quantitate MIAs. Root of Rauvolfia vomitoria showed a high content of ajmaline (48.43 mg/g), serpentine (87.77 mg/g) whereas high quantities of yohimbine (100.21 mg/g) and ajmalicine (120.51 mg/g) were detected in R. tetraphylla. High content of reserpine was detected in R. micrantha (35.18 mg/g) and R. serpentina (32.38 mg/g). CONCLUSION: The encouraging results of this study may lead to easy selection of suitable Rauvolfia species according to the abundance of MIAs. Copyright © 2016 John Wiley & Sons, Ltd.

Profiling of Gallic and Ellagic Acid Derivatives in Different Plant Parts of Terminalia arjuna by HPLC-ESI-QTOF-MS/MS.

Terminalia arjuna is a medicinal plant used in ethnomedicine and the codified traditional medicine. A number of active constituents are reported, but there is no information on the whole range of gallic and ellagic acid derivatives present in this plant A rapid and sensitive analytical method was developed using reverse phase high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS/MS) for qualitative analysis to determine the array of bioactive phytochemicals and their variations in different plant parts viz. bark, unripe fruit, ripe fruit, leaf, root and stem. Separation was performed on a Thermo Betasil C8 column (250 mm x 4.5 mm, 5 µm) with a mobile phase consisted of 0.1% formic acid aqueous solution and acetonitrile at a flow rate of 0.5 mL/min in 55 min. A wide range of constituents of T. arjuna were characterized and broadly grouped as 27 gallic acid and 52 ellagic acid derivatives.

Distribution and Discrimination Study of Bioactive Compounds from Berberis species using HPLC-ESI-QTOF-MS/MS with Principle Component Analysis.

Berberis specieshave been used extensively as raw material in various herbal medicines. In this work, a fast aid sensitive method based on reversed-phase high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandemmass spectrometry (HPLC-ESI-QTOF- MS/MS) was used to assist the distribution and discrimination study of bioactive compounds from Berberis species such as B. aristata, B. asiatica, B. chitria, B.jaeschkeana, B; koehneana, B. lyceum, B. petiolaris and B. pseudoumbellata. The HPLC separation was performed on a Thermo Betasil C8 column (250 mm x 4.5 mm; 5µ) using gradient elution with a mobile phase of 0.1% aqueous solution of formic acid (v/v) and acetonitrile. The high resolution (HR) and collision induced dissociation (CID) mass spectrometry experiments were conducted in order to provide molecular-mass information and MS/MS fragmentation patterns of the compounds for structural elucidation. A total of 59 compounds were tentatively identified and characterized including three acids, forty-one alkaloids and twelve flavonoids using seventeen reference standards for authentication. Principle component analysis (PCA) was also applied to discriminate eight Berberis species according to their plant parts i.e., leaf, stem and root. The highest total content of identified compounds was detected in the root of all the Berberis species wherein alkaloids were predominant.

Structural characterization of monoterpene indole alkaloids in ethanolic extracts of Rauwolfia species by liquid chromatography with quadrupole time-of-flight mass spectrometry.

Rauwolfia species (Apocynaceae) are medicinal plants well known worldwide due to its potent bioactive monoterpene indole alkaloids (MIAs) such as reserpine, ajmalicine, ajmaline, serpentine and yohimbine. Reserpine, ajmalicine and ajmaline are powerful antihypertensive, tranquilizing agents used in hypertension. Yohimbine is an aphrodisiac used in dietary supplements. As there is no report on the comparative and comprehensive phytochemical investigation of the roots of Rauwolfia species, we have developed an efficient and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for ethanolic root extract of Rauwolfia species to elucidate the fragmentation pathways for dereplication of bioactive MIAs using high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-QTOF-MS/MS) in positive ion mode. We identified and established diagnostic fragment ions and fragmentation pathways using reserpine, ajmalicine, ajmaline, serpentine and yohimbine. The MS/MS spectra of reserpine, ajmalicine, and ajmaline showed C-ring-cleavage whereas E-ring cleavage was observed in serpentine via Retro Diels Alder (RDA). A total of 47 bioactive MIAs were identified and characterized on the basis of their molecular formula, exact mass measurements and MS/MS analysis. Reserpine, ajmalicine, ajmaline, serpentine and yohimbine were unambiguously identified by comparison with their authentic standards and other 42 MIAs were tentatively identified and characterized from the roots of Rauwolfia hookeri, Rauwolfia micrantha, Rauwolfia serpentina, Rauwolfia verticillata, Rauwolfia tetraphylla and Rauwolfia vomitoria. Application of LC-MS followed by principal component analysis (PCA) has been successfully used to discriminate among six Rauwolfia species.

Identification, characterization and distribution of monoterpene indole alkaloids in Rauwolfia species by Orbitrap Velos Pro mass spectrometer.

Monoterpene indole alkaloids (MIAs) are medicinally important class of compounds abundant in the roots of Rauwolfia species (Apocynaceae). MIAs such as yohimbine (aphrodisiac agent) and reserpine (antihypertensive, tranquilizer) are the official drugs included in Model List of Essential Drugs of World Health Organization (WHO). Therefore, we have attempt to identify and characterize the MIAs in the crude extracts of six Rauwolfia species using ultrahigh-performance liquid chromatography coupled with Orbitrap Velos Pro hybrid mass spectrometer. The identity of the MIAs were construed using the high resolution tandem mass spectrometry (HRMS/MS) spectra of standard compounds 'yohimbine' and 'reserpine' in higher energy collisional dissociation (HCD) and collision-induced dissociation (CID) modes. The diagnostic fragment ions found in HCD mode was highly affected by variation of normalized collision energy (NCE) and gave few product ions ('C-F') while CID produced intense and more diagnostic product ions ('A-F'). Consequently, CID-MS/MS mode provided significantly more structural information about basic skeleton and therefore the recommended mode for analysis of MIAs. Furthermore, six diagnostic fragmentation pathways were established by multi-stage mass analysis (MS(n) (n=5)) analysis which gave information regarding the substitution. Fragment ions 'A-F' revealed the number and position of substituents on indole and terpene moieties. The proposed diagnostic fragmentation pathways have been successfully applied for identification and characterization of MIAs in crude root extracts of six Rauwolfia species. Ten bioactive reserpine class of MIAs were tentatively identified and characterized on the basis of chromatographic and mass spectrometric features as well as HRMS/MS an MS(n) (n=4) analysis.

Analysis of phytochemical variations in dioecious Tinospora cordifolia stems using HPLC/QTOF MS/MS and UPLC/QqQLIT -MS/MS.

INTRODUCTION: The stem of dioecious Tinospora cordifolia (Menispermaceae) is a commonly used traditional Ayurvedic medicine in India having several therapeutic properties. OBJECTIVE: To develop and validate LC-MS methods for the identification and simultaneous quantitation of various secondary metabolites and to study metabolomic variations in the stem of male and female plants. METHODS: Ethanolic extract of stems were analysed by HPLC/ESI-QTOF-MS/MS for rapid screening of bioactive phytochemicals. High resolution MS and MS/MS in positive ESI mode were used for structural investigation of secondary metabolites. An UPLC/ESI-QqQ(LIT) -MS/MS method in MRM mode was developed and validated for the simultaneous quantitation of five bioactive alkaloids. RESULTS: Identification and characterisation of 36 metabolites including alkaloids, sesquiterpenes and phytoecdysteroids were performed using LC-MS and MS/MS techniques. The bioactive alkaloids such as jatrorrhizine, magnoflorine, isocorydine, palmatine and tetrahydropalmatine were successfully quantified in male and female plants. The mean abundances of magnoflorine jatrorrhizine, and oblongine were significantly (P < 0.05) higher in male plants while mean abundances of tetrahydropalmatine, norcoclaurine, and reticuline were significantly (P < 0.05) higher in female plants. CONCLUSIONS: Phytochemicals in the stem of male and female Tinospora cordifolia showed significant qualitative and quantitative variations. LC-MS and MS/MS methods can be used to differentiate between male and female plants based on their chemical profiles and quantities of the marker bioactive alkaloids. This chemical composition difference was also evident during vegetative stage when there were no male and female flowers.

Structural characterization of monoterpene indole alkaloids in ethanolic extracts of Rauwolfia species by liquid chromatography with quadrupole time-of-flight mass spectrometry / 药物分析学报

Rauwolfia species (Apocynaceae) are medicinal plants well known worldwide due to its potent bioactive monoterpene indole alkaloids (MIAs) such as reserpine, ajmalicine, ajmaline, serpentine and yohimbine. Reserpine, ajmalicine and ajmaline are powerful antihypertensive, tranquilizing agents used in hypertension. Yohimbine is an aphrodisiac used in dietary supplements. As there is no report on the comparative and comprehensive phytochemical investigation of the roots of Rauwolfia species, we have developed an efficient and reliable liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for ethanolic root extract of Rauwolfia species to elucidate the fragmentation pathways for dereplication of bioactive MIAs using high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC–ESI–QTOF–MS/MS) in positive ion mode. We identified and established diagnostic fragment ions and fragmentation pathways using reserpine, ajmalicine, ajmaline, serpentine and yohimbine. The MS/MS spectra of reserpine, ajmalicine, and ajmaline showed C-ring-cleavage whereas E-ring cleavage was observed in serpentine via Retro Diels Alder (RDA). A total of 47 bioactive MIAs were identified and characterized on the basis of their molecular formula, exact mass measurements and MS/MS analysis. Reserpine, ajmalicine, ajmaline, serpentine and yohimbine were unambiguously identified by comparison with their authentic standards and other 42 MIAs were tentatively identified and characterized from the roots of Rauwolfia hookeri, Rauwolfia micrantha, Rauwolfia serpentina, Rauwolfia verticillata, Rauwolfia tetraphylla and Rauwolfia vomitoria. Application of LC–MS followed by principal component analysis (PCA) has been successfully used to discriminate among six Rauwolfia species.

Screening of tricyclic quinazoline alkaloids in the alkaloidal fraction of Adhatoda beddomei and Adhatoda vasica leaves by high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

RATIONALE: Adhatoda beddomei and Adhatoda vasica are popular Ayurvedic medicinal plants in India, belonging to the family Acanthaceae. Tricyclic quinazoline alkaloids are found to be the most abundant in these plants which are responsible for broad-spectrum medicinal properties. This study aims to seek identification and characterization of those alkaloids based on their fragmentation patterns. METHODS: A method was developed to elucidate the main fragmentation pathways of tricyclic quinazoline alkaloids in positive ion mode using high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC/ESI-QTOF-MS/MS). Chromatographic separation was carried on a Supelco Discovery HS C18 column (15 cm × 4.6 mm, 3 µm) with 0.1% formic acid aqueous solution and acetonitrile as a mobile phase. RESULTS: In full scan mass spectra, protonated molecules were observed for all the quinazoline alkaloids. Ring cleavages of the tricyclic quinazoline moiety were observed in MS(2) spectra and the characteristic ions provide valuable structural information of these alkaloids. Fragmentation pathways and fragment ion structures were proposed in two groups of quinazoline alkaloids. CONCLUSIONS: The established fragmentation patterns have been successfully used to identify 23 tricyclic quinazoline alkaloids in the alkaloidal fraction of A. beddomei and A. vasica.

Structural characterization of flavonoid C- and O-glycosides in an extract of Adhatoda vasica leaves by liquid chromatography with quadrupole time-of-flight mass spectrometry.

RATIONALE: Adhatoda vasica Nees is a well-known Ayurvedic medicinal plant, belonging to the family Acanthaceae. This study aims to seek identification and characterization of flavonoid C- and O-glycosides in the aqueous fraction of the plant leaves. METHODS: A method was developed for simultaneous characterization of flavonoids and their glycosides using high-pressure liquid chromatography with quadrupole time-of-flight mass spectrometry (HPLC/ESI-QTOF-MS/MS). The chromatographic separation was carried on an Agilent Poroshell 120 EC-C18 column (4.6 × 150 mm, 2.7 µm) operated with 0.1% formic acid aqueous solution and methanol as the mobile phase. RESULTS: The fragmentations of the studied [M-H](-) ions of C-glycosides were shown to be cross-ring cleavages of the glycoside moiety [M-H-(60/90/120)](-) whereas O-glycosides were shown to eliminate the sugar moiety (Y0 (-) or [Y0 -H](-) ) from the aglycone unit; 6-C-glycosides exhibited [M-H-18](-) , a characteristic ion, and also a higher abundance of (0,3) X6 or 8 ions in comparison to 8-C glycosides; flavonoid 6,8-di-C-glycosides exhibited cross-ring cleavages of the sugar attached to the C-6 position preferentially. CONCLUSIONS: This method was successfully applied for analysis of flavonoids and their glycosides in Adhatoda vasica leaves. A total of 29 compounds were tentatively identified including 17 C-, nine O-glycosides and three flavonoids.

Quantitative determination of isoquinoline alkaloids and chlorogenic acid in Berberis species using ultra high performance liquid chromatography with hybrid triple quadrupole linear ion trap mass spectrometry.

Berberis species are well known and used extensively as medicinal plants in traditional medicine. They have many medicinal values attributable to the presence of alkaloids having different pharmacological activities. In this study, a method was developed and validated as per international conference on harmonization guidelines using ultra high performance liquid chromatography with hybrid triple quadrupole-linear ion trap mass spectrometry operated in the multiple reaction monitoring mode for nine bioactive compounds, including protoberberine alkaloids, aporphine alkaloids and chlorogenic acid. This method was applied in different plant parts of eight Berberis species to determine variations in content of nine bioactive compounds. The separation was achieved on an ACQUITY UPLC CSH™ C18 column using a gradient mobile phase at flow rate 0.3 mL/min. Calibration curves for all the nine analytes provided optimum linear detector response (with R(2) ≥0.9989) over the concentration range of 0.5-1000 ng/mL. The precision and accuracy were within RSDs ≤2.4 and ≤2.3%, respectively. The results indicated significant variation in the total contents of the nine compounds in Berberis species.

Rapid screening for the adulterants of Berberis aristata using direct analysis in real-time mass spectrometry and principal component analysis for discrimination.

Adulteration or substitution of commercial Berberis aristata and its herbal products with inferior-quality substituents is very common. Metabolic profiling of B. aristata, along with its common adulterants/contaminants/substituents such as B. asiatica, Mahonia borealis and Coscinium fenestratum, was rapidly carried out using direct analysis in real-time mass spectrometry (DART MS) to generate the chemical fingerprints for the differentiation of these species. Phytochemical analysis showed the presence of mainly alkaloids. The identified alkaloids were berberrubine, berberine, jatrorrhizine, ketoberberine, palmatine, dihydropalmatine or 7,8-dihydro-8-hydroxyberberine, berbamine and pakistanamine. Berberine, which was mainly reported from the root and stem bark of B. aristata, was also identified in the leaf along with chlorogenic acid. The DART MS data have been subjected to principal component analysis (PCA). The resulting score plots showed clustering and clear differentiation of the species and plant parts. It is thus apparent that the technique of DART MS followed by PCA is a quick and reliable method for the direct profiling of B. aristata and its adulterant plants and plant parts. The study reports the rapid analytical method to identify the possibility of illegal adulteration/contamination/substitution in potential plant materials and herbal extracts.