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.