Rapid Identification of Common Secondary Metabolites of Medicinal Herbs Using High-Performance Liquid Chromatography with Evaporative Light Scattering Detector in Extracts.

The discovery and identification of novel natural products of medicinal importance in the herbal medicine industry becomes a challenge. The complexity of this process can be reduced by dereplication strategies. The current study includes a method based on high-performance liquid chromatography (HPLC), using the evaporative light scattering detector (ELSD) to identify the 12 most common secondary metabolites in plant extracts. Twelve compounds including rutin, taxifolin, quercetin, apigenin, kaempferol, betulinic acid, oleanolic acid, betulin, lupeol, stigmasterol, and ß-sitosterol were analyzed simultaneously. The polarity of the compounds varied greatly from highly polar (flavonoids) to non-polar (triterpenes and sterols). This method was also tested for HPLC-DAD and HPLC-ESI-MS/MS analysis. Oleanolic acid and ursolic acid could not be separated in HPLC-ELSD analysis but were differentiated using LC-ESI-MS/MS analysis due to different fragment ions. The regression values (R2 > 0.996) showed good linearity in the range of 50-1000 µg/mL for all compounds. The range of LOD and LOQ values were 7.76-38.30 µg/mL and 23.52-116.06 µg/mL, respectively. %RSD and % trueness values of inter and intraday studies were mostly <10%. This method was applied on 10 species of medicinal plants. The dereplication strategy has the potential to facilitate and shorten the identification process of common secondary metabolites in complex plant extracts.

High-Throughput Detection of an Alkaloidal Plant Metabolome in Plant Extracts Using LC-ESI-QTOF-MS.

Plant alkaloids represent a diverse group of nitrogen-containing natural products. These compounds are considered valuable in drug discovery and development. High-throughput identification of such plant secondary metabolites in complex plant extracts is essential for drug discovery, lead optimization, and understanding the biological pathway. The present study aims to rapidly identify different classes of alkaloids in plant extracts through the liquid chromatography with electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) approach using 161 isolated and purified alkaloids. These are biologically important unique alkaloids belonging to different sub-classes such as isoquinoline, quinoline, indole, tropane, pyridine, piperidine, quinolizidine, aporphine, steroidal, and terpenoid. The majority of these are not available commercially and are known to manifest valuable biological activities. Four pools of a maximum of 50 phytostandards each were prepared, based on their log P value to minimize co-elution for rapid and cost-effective analyses. MS/MS spectra were acquired in the positive ionization mode by using their [M + H]+ and/or [M + Na]+ with both the average collisional energy (25.5-62 eV) and individual collisional energies (10, 20, 30, and 40 eV). Accurate mass, high-resolution mass spectrometry (HR-MS) data, MS/MS data, and retention times were curated for each compound. The developed LC-MS/MS method was successfully used to interrogate and fast dereplicate alkaloids in 13 medicinal plant extracts and a herbal formulation. A total of 56 alkaloids were identified based on the reference standard retention times (RTs), HR-MS spectra, and/or MS/MS spectra. The MS data have been submitted to the MetaboLights online database (MTBLS2914). The mass spectrometric and chromatographic data will be useful for the discovery of new congeners and the study of biological pathways of alkaloids in the plant kingdom.

A high-throughput method for dereplication and assessment of metabolite distribution in Salvia species using LC-MS/MS.

Dereplication of crude plant extracts through liquid chromatography-mass spectrometry is a powerful technique for the discovery of novel natural products. Unfortunately, this technique is often plagued by a low level of confidence in natural product identification. This is mainly due to the lack of extensive chromatographic and mass spectrometric optimizations that result in improper and incomplete MS/MS fragmentation data. This study proposes a solution to this problem by the optimization of chromatographic separation and mass spectrometry parameters. We report herein a direct and high-throughput strategy for natural product dereplication in five Salvia species using high-resolution ESI-QTOF-MS/MS data. In the present study, we were able to identify a total of forty-seven natural products in crude extracts of five Salvia species using MS/MS fragmentation data. In addition to dereplication of Salvia species, quantitative profiling of twenty-one bioactive constituents of the genus was also performed on an ion trap mass spectrometer. For the quantitation study, method development focused on chromatographic optimizations to achieve maximum sensitivity. The developed dereplication and quantitation strategy can be extended to develop comprehensive metabolic profiles of other plant genera and species and thus can prove useful in the field of drug discovery from plants.

Metabolite distribution and correlation studies of Ziziphus jujuba and Ziziphus nummularia using LC-ESI-MS/MS.

Ziziphus jujuba and Ziziphus nummularia are two important species of the genus Ziziphus. Both plants offer great commercial value and are consumed as food around the world and used for their medicinal values such as anti-inflammatory, antioxidant and hepatoprotective activities. Comprehensive chemical profiling of Z. jujuba and Z. nummularia was done through identification of major metabolites using HPLC-QTOF-MS/MS and quantification of eight analytes using HPLC-IonTrap-MS/MS analysis. A total of 53 compounds were identified from their high-resolution mass spectra in both positive and negative ionization modes. Among these, 52 compounds were found to be present in Z. jujuba, and 45 in Z. nummularia. Chemometric analysis was also performed to assess the distribution of identified compounds and to determine how the obtained data can be used to discriminate between the two species. Moreover, a method for the quantification of eight analytes including, 6″'-feruloylspinosin (1), apigenin (2), apigenin-7-O-glucoside (3), catechin (4), jujuboside A (5), jujuboside B (6), luteolin (7) and quercetin (8) was developed. The method expressed excellent accuracy with less than 3% error and good reproducibility with less than 4% RSD. The limit-of-detection (LOD) was also found to be very low ranging between 0.06 ng/mL to 4.10 ng/mL while limit-of-quantitation (LOQ) values were in the range of 0.17 ng/mL to 12.42 ng/mL. The analyte concentrations were found to be varying from 1.32 mg/kg to 645.76 mg/kg in both species. The developed method was used to identify and quantify marker compounds in fruit and whole plant samples of both species and in their herbal products as well. The present work is unprecedented, as there is no such extensive study targeting fruits, leaves and herbal formulations together using high-resolution techniques. The study will provide great utility in drug discovery, in taxonomical identification of these plants and to develop quality control protocols for their herbal formulations.

Sensitive Determination of C-Alkylated Flavonoids by HPLC-ESI-MS/MS Using Multiple Reaction Monitoring Approach: Pseudarthria hookeri as a Case Study.

One of the major problems with the formulation of herbal medicines is the quality control of plant material to ensure its efficacy and safety. Quality control of medicinal plants requires analysis of many bioactive compounds present in the plant. C-alkylated flavonoids are an important bioactive subclass of flavonoids. A simple, rapid, sensitive and selective method is presented here for the quantification of bioactive C-alkylated flavonoids. This is the first quantitative method for analysis of C-alkylated flavonoids based on the multiple reaction monitoring (MRM) approach so far. This study focuses on method development for quantification of bioactive C-alkylated flavonoids. Quantification of a total of five C-alkylated flavonoids was done employing the MRM approach on an HPLC-QqQ-MS instrument. LODs and LOQs for quantified flavonoids were in the range of 0.41-1.32 and 1.23-3.96 ng/mL, respectively. Linear calibration curves between 25 and 1500 ng/mL were obtained with the regression coefficients of ≥0.996. Accuracy (% bias) and precision (% RSD) of the analyses were found to be less than 5%. Developed HPLC-ESI-MS/MS can be employed as a quality control method of plant raw materials.

Metabolite Profiling and Quantitation of Cucurbitacins in Cucurbitaceae Plants by Liquid Chromatography coupled to Tandem Mass Spectrometry.

Cucurbitaceae is an important plant family because many of its species are consumed as food, and used in herbal medicines, cosmetics, etc. It comprises annual vines and is rich in various bioactive principles which include the cucurbitacins. These steroidal natural products, derived from the triterpene cucurbitane, are mainly the bitter principles of the family Cucurbitaceae. Their biological activities include anti-inflammatory, hepatoprotective, and anti-cancer activities. A total of 10 species belonging to 6 genera of the Cucurbitaceae family along with Cissampelos pareira (Menispermaceae) were included in this study. A comprehensive profiling of certain natural products was developed using HPLC-QTOF-MS/MS analysis and a distribution profile of several major natural products in this family was obtained. A total of 51 natural products were detected in both positive and negative ionization modes, based on accurate masses and fragmentation patterns. Along with this, quantitation of four bioactive cucurbitacins, found in various important plants of the Cucurbitaceae family, was carried out using multiple reaction monitoring (MRM) approach on an ion trap mass spectrometer. Cucurbitacin Q was found to be the most abundant in C. pareira, while Citrullus colocynthis contained all four cucurbitacins in abundant quantities. The developed quantitation method is simple, rapid, and reproducible.

Combining untargeted and targeted metabolomics approaches for the standardization of polyherbal formulations through UPLC-MS/MS.

INTRODUCTION: Polyherbal formulations are an integral part of various indigenous medicinal systems such as Traditional Chinese Medicine (TCM) and Ayurveda. The presence of a very large number of compounds makes the quality control of polyherbal formulations very difficult. OBJECTIVES: To overcome this problem, we have developed a comprehensive strategy for the dereplication of natural products in polyherbal formulations by using Adhatoda vasica as a case study. METHODS: The strategy is based on five major steps: the collection of plant samples from different locations to observe the effects of environmental variables; LC-ESI-MS/MS-based untargeted metabolite profiling of the plant samples to identify marker compounds using extensive chemometric analysis of the obtained data; the identification of marker compounds in polyherbal products; the isolation, purification and characterization of the marker compounds; and MRM-based quantitative analysis of the isolated marker compounds using LC-ESI-MS/MS. RESULTS: Using this strategy, we identified a total of 51 compounds in the methanolic extract of A. vasica plants from 14 accessions. Chemical fingerprinting of the plant led to the identification of characteristic peaks that were used to confirm the presence of A. vasica in complex polyherbal formulations. Four quinazoline alkaloids (marker compounds) were isolated, purified and quantified in various herbal formulations containing A. vasica. CONCLUSION: This method demonstrates a comprehensive strategy based on untargeted and targeted metabolite analysis that can be used for the standardization of complex polyherbal formulations.

Sensitive analysis of bioactive secondary metabolites in lichen species using liquid chromatography-mass spectrometry.

Lichens are a large group of valuable lower plants with unique features and diverse applications worldwide such as in medicine, cosmetics, food, and textile industries. They are also well known for their potential in observing climate and environmental monitoring. Their successful exploitations require reliable analytical methods to check and maintain quality and efficacy of the products based on them. This study focuses on the development of a sensitive and reliable quantification method for the analysis of important depsides, depsidones, dibenzofuran and monocyclic phenols inseven known and an unidentified lichen species. Multiple Reaction Monitoring (MRM) approach using UHPLC-QqQ-MS instrument was employed for the development of the quantitative method. Both LC and MS parameters were optimized to ensure maximum separation. High sensitivity, and selectivity. LODs and LOQs were found to be in the range of 2.1-71.5ng/mL and 6.3-212.9ng/mL, respectively. The accuracy (% bias) and precision (% RSD) were found to be <5% in most cases. Metabolites 1-9 were found in the range of 0.5-41429µg/g in the analysed lichen extracts. The analysis revealed that metabolites 1, 2 and 3 are the predominant ones. This method can be used for the identification and absolute quantification of secondary metabolites in lichen extracts, and herbal or consumer products based upon them.

Sensitive quantification of coixol, a potent insulin secretagogue, in Scoparia dulcis extract using high-performance liquid chromatography combined with tandem mass spectrometry and UV detection.

Diabetes is a major global health problem which requires new studies for its prevention and control. Scoparia dulcis, a herbal product, is widely used for treatment of diabetes. Recent studies demonstrate coixol as a potent and nontoxic insulin secretagog from S. dulcis. This study focuses on developing two quantitative methods of coixol in S. dulcis methanol-based extracts. Quantification of coixol was performed using high-performance liquid chromatography-tandem mass spectrometry (method 1) and high-performance liquid chromatography-ultraviolet detection (method 2) with limits of detection of 0.26 and 11.6 pg/µL, respectively, and limits of quantification of 0.78 and 35.5 pg/µL, respectively. S. dulcis is rich in coixol content with values of 255.5 ± 2.1 mg/kg (method 1) and 220.4 ± 2.9 mg/kg (method 2). Excellent linearity with determination coefficients >0.999 was achieved for calibration curves from 10 to 7500 ng/mL (method 1) and from 175 to 7500 ng/mL (method 2). Good accuracy (bias < -8.6%) and precision (RSD < 8.5%) were obtained for both methods. Thus, they can be employed to analyze coixol in plant extracts and herbal formulations.

Sensitive quantification of six steroidal lactones in Withania coagulans extract by UHPLC electrospray tandem mass spectrometry.

A method for the concurrent determination of six known steroidal lactones (syn. withanolides or withasteroids), namely withaferin A, withanolide H, withanolide K, withanolide A, withacoagulin H, and withanolide J in Withania coagulans extracts was developed. Extracts of Withania species and purified withanolides are considered among the most important natural products used for medicinal purposes. Methanolic extract of plant material was subjected to reverse phase ultra-high performance liquid chromatography (UHPLC) coupled with electrospray (JetStream ESI) triple quadrupole mass spectrometer operated in the Multiple Reaction Monitoring (MRM) mode. Satisfactory separation of withanolide component was achieved within 9 min on UHPLC runtime. The limits of detection (LOD) and the limits of quantitation (LOQs) for the six withanolides ranged between 0.040-4.80 ng/mL, and 0.13-16 ng/mL, respectively. Linear responses were attained for all six withanolides in two orders of magnitude with the linear regression coefficient values ⩾0.998. At the five QC levels inspected, the relative standard deviations (RSD) were found below 5% in most cases. The newly developed method is fast, precise, and sensitive, therefore, the method can be used for high-throughput quantification of various withanolides in W. coagulans extract, and other herbal formulations, derived from W. coagulans.

Probing of metabolites in finely powdered plant material by direct laser desorption ionization mass spectrometry.

Natural products continue to serve as an important source of novel drugs since the beginning of human history. High-throughput techniques, such as MALDI-MS, can be techniques of choice for the rapid screening of natural products in plant materials. We present here a fast and reproducible matrix-free approach for the direct detection of UV active metabolites in plant materials without any prior sample preparation. The plant material is mechanically ground to a fine powder and then sieved through different mesh sizes. The collected plant material is dispersed using 1 µL solvent on a target plate is directly exposed to Nd:YAG 335 nm laser. The strategy was optimized for the analysis of plant metabolites after study of the different factors affecting the reproducibility and effectiveness of the analysis, including particle sizes effects, types of solvents used to disperse the sample, and the part of the plant analyzed. Moreover, several plant species, known for different classes of metabolites, were screened to establish the generality of the approach. The developed approach was validated by the characterization of withaferin A and nicotine in the leaves of Withania somnifera and Nicotiana tabacum, respectively, through comparison of its MS/MS data with the standard compound. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques were used for the tissue imaging purposes. This approach can be used to directly probe small molecules in plant materials as well as in herbal and pharmaceutical formulations for fingerprinting development.

Rapid characterization and identification of steroidal alkaloids in Sarcococca coriacea using liquid chromatography coupled with electrospray ionization quadropole time-of-flight mass spectrometry.

Rapid characterization of 23 pregnane-type steroidal alkaloids was studied using a positive ion electrospray ionization quadropole time-of-flight mass spectrometry (ESI-QqTOF-MS/MS) hybrid instrument. ESI-QqTOF-MS (positive ion mode) showed the presence of the protonated molecules [M+H](+) which through low-energy collision-induced dissociation tandem mass spectrometric (CID-MS/MS) analysis showed the characteristic loss of dimethylamine moiety [M+H-45](+) followed by the sequential lossess of attached substituents. Steroidal alkaloids having tigloyl or senecioyl group at C-3 produced diagnostic fragment ions at m/z 100 and 83. Our study also demonstrates the influence of unsaturation, and number and nature of substitutents on product ion abundance and fragment ions. Moreover, the generalization of the fragmentation pattern was linked with the structural features in steroidal skeleton. This strategy was successfully applied in LC-ESI-QqTOF-MS/MS analysis of Sarcococca coriacea extract to investigate and characterize pregnane-type steroidal alkaloids in complex mixture.

Analysis and development of structure-fragmentation relationships in withanolides using an electrospray ionization quadropole time-of-flight tandem mass spectrometry hybrid instrument.

Structural elucidation and gas-phase fragmentation of ten withanolides (steroidal lactones) were studied using a positive ion electrospray ionization quadropole time-of-flight mass spectrometry (ESI-QqTOF-MS/MS) hybrid instrument. Withanolides form an important class of plant secondary metabolites, known to possess a variety of biological activities. Withanolides which possess hydroxyl groups at C-4, C-5, C-17, C-20, and C-27, and an epoxy group at C-5/C-6, were evaluated to determine the characteristic fragments and their possible pathways. ESI-QqTOF-MS (positive ion mode) showed the presence of the protonated molecules [M + H](+) . Low-energy collision-induced dissociation tandem mass spectrometric (CID-MS/MS) analysis of the protonated molecule [M + H](+) indicated multiple losses of water and the removal of the C-17-substituted lactone moiety affording the [M + H-Lac](+) product ion as the predominant pathways. However, withanolides containing a hydroxyl group at C-24 of the lactone moiety showed a different fragmentation pathway, which include the loss of steroidal part as a neutral molecule, with highly diagnostic ions at m/z 95 and 67 being generated from the cleavage of lactone moiety. Our results also determined the influence of the presence and positions of hydroxyl and epoxy groups on product ion formation and stability. Moreover, the knowledge of the fragmentation pattern was utilized in rapid identification of withanolides by the LC/MS/MS analysis of a Withania somnifera extract.