Renu Pandey

Development and validation of an ultra high performance liquid chromatography electrospray ionization tandem mass spectrometry method for the simultaneous determination of selected flavonoids in Ginkgo biloba

A rapid and sensitive ultra high performance liquid chromatography electrospray ionization tandem mass spectrometry method was developed and validated for the simultaneous determination of 13 flavonoids in leaf, stem, and fruit extracts of male and female trees of Ginkgo biloba to investigate gender- and age-related variations of flavonoids content. Chromatographic separation was accomplished on an Acquity UPLC BEH C18 column (50xa0mmxa0×xa02.1 mm id, 1.7 μm) in 5 min. Quantitation was performed using negative electrospray ionization mass spectrometry in multiple reaction monitoring mode. The calibration curves of all analytes showed a good linear relationship (r(2) ≥ 0.9977) over the concentration range of 1-1000 ng/mL. The precision evaluated by an intra- and interday study showed RSDxa0≤xa01.98% and good accuracy with overall recovery in the range from 97.90 to 101.09% (RSDxa0≤xa01.67%) for all analytes. The method sensitivity expressed as the limit of quantitation was typically 0.25-3.57 ng/mL. The results showed that the total content of 13 flavonoids was higher in the leaf extract of an old male Ginkgo tree compared to young female Ginkgo trees.

Characteristic differences in metabolite profile in male and female plants of dioecious Piper betle L.

Piper betle is a dioecious pan-Asiatic plant having cultural and medicinal uses. It belongs to the family Piperaceae and is a native of the tropics although it is also cultivated in subtropical areas. Flowering in P. betle occurs only in tropical regions. Due to lack of inductive floral cycles the plant remains in its vegetative state in the subtropics. Therefore, due to lack of flowering, gender distinction cannot be made the in the subtropics. Gender distinction in P. betle in vegetative state can be made using Direct Analysis in Real Time Mass Spectroscopy (DARTMS), a robust high-throughput method. DARTMS analysis of leaf samples of two male and six female plants showed characteristic differences in the spectra between male and female plants. Semi-quantitative differences in some of the identified peaks in male and female landraces showed gender-based differences in metabolites. Cluster analysis using the peaks at m/z 151, 193, 235 and 252 showed two distinct clusters of male and female landraces. It appears that male and female plants besides having flowers of different sexes also have characteristic differences in the metabolites representing two metabolic types.

Rapid screening and quantitative determination of bioactive compounds from fruit extracts of Myristica species and their in vitro antiproliferative activity

Efficient and sensitive LC-MS/MS methods have been developed for the rapid screening and determination of bioactive compounds in different fruit parts of four Myristica species, viz., Myristica beddomeii, Myristica fragrans, Myristica fatua and Myristica malabarica. Twenty-one compounds were identified and characterized on the basis of their accurate mass and MS/MS fragmentation pattern using HPLC-QTOF-MS/MS and NMR analysis. Quantitative determination of five major bioactive compounds was performed using multiple-reaction monitoring mode with continuous polarity switching by UHPLC-QqQLIT-MS/MS. Moreover, in vitro antiproliferative activity of these Myristica species was evaluated against five human cancer cell lines A549, DLD-1, DU145, FaDu and MCF-7 using SRB assay. Seventeen phytoconstituents were identified and reported for the first time from M. beddomeii and sixteen from M. fatua. Quantification result showed highest total content of five major bioactive compounds in mace of M. fragrans. Evaluation of in vitro antiproliferative activity revealed potent activity in all investigated species except M. fragrans.

Ultra high performance liquid chromatography tandem mass spectrometry method for the simultaneous determination of multiple bioactive constituents in fruit extracts of Myristica fragrans and its marketed polyherbal formulations using a polarity switching technique

Fruits of Myristica fragrans Houtt. are the source of two valuable spices: nutmeg and mace, traditionally used for its flavoring and medicinal properties and found as an ingredient in many marketed polyherbal formulations and food products. In this study, a sensitive and efficient ultra high performance liquid chromatography electrospray ionization tandem mass spectrometry method was developed and validated for the rapid determination of 16 bioactive constituents in different parts of the fruit of M. fragrans and its marketed polyherbal formulations using a polarity switching technique. Chromatographic separation was achieved on an Aquity UPLC BEH C18 column in 9.4 min. Quantitative analysis was performed using multiple reaction monitoring mode with continuous polarity switching in a single analysis. The developed method was found to be accurate with overall recovery in the range from 95.95 to 102.07% (RSD ≤ 1.91%), precise (RSD ≤ 1.98%), and linear (r(2) ≥ 0.9992) over the concentration range of 0.1-200 ng/mL. Quantitative analysis indicated that the total content of the 16 bioactive constituents was highest in the mace of M. fragrans. Thus, this rapid and sensitive method could be utilized as a promising reference method for the quality control of M. fragrans and its marketed herbal formulations/food products.

Simultaneous quantitative determination of multiple bioactive markers in Ocimum sanctum obtained from different locations and its marketed herbal formulations using UPLC-ESI-MS/MS combined with principal component analysis.

INTRODUCTIONnOcimum sanctum L., with phenolic acids, flavonoids, propenyl phenols and terpenoids as active pharmacological constituents, is a popular medicinal herb and is present as an ingredient in many herbal formulations. Therefore, development of a reliable analytical method for simultaneous determination of the pharmacologically active constituents of O. sanctum is of high importance.nnnOBJECTIVEnTo develop and validate a new, rapid, sensitive and selective UPLC-ESI/MS/MS method for simultaneous determination of 23 bioactive markers including phenolic acids, flavonoids, propenyl phenol and terpenoid in the leaf extract and marketed herbal formulations of O. sanctum.nnnMETHODSnAn UPLC-ESI/MS/MS method using negative electrospray ionisation (ESI) in multiple-reaction-monitoring (MRM) mode was used for simultaneous determination. Chromatographic separation was achieved on an Acquity UPLC BEH C18 -column using a gradient elution with 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Principal component analysis (PCA) was applied to correlate and discriminate eight geographical collections of O. sanctum based on quantitative data of the analytes.nnnRESULTSnThe developed method was validated as per International Conference on Harmonization guidelines and found to be accurate, with overall recovery in the range 95.09-104.84% (RSDu2009≤u20091.85%), precise (RSDu2009≤u20091.98%) and linear (r(2) u2009≥u20090.9971) over the concentration range of 0.5-1000u2009ng/mL. Ursolic acid was found to be the most abundant marker in all the samples investigated, except for the marketed tablet.nnnCONCLUSIONnThe method established is simple, rapid and sensitive, hence it can be reliably utilised for the quality control of O. sanctum and derived herbal formulations.

Quality control assessment of polyherbal formulation based on a quantitative determination multimarker approach by ultra high performance liquid chromatography with tandem mass spectrometry using polarity switching combined with multivariate analysis.

An ultra high performance liquid chromatography with electrospray ionization tandem mass spectrometry method has been developed and validated for the simultaneous quantification of 28 major bioactive compounds in Mentat tablet, a complex Indian herbal medicine used in the treatment of neurological disorder and improvement of mental health. Multiple-reaction monitoring scanning was employed for quantification in positive and negative ion switching mode. The analysis was accomplished on Waters ACQUITY UPLC BEH C18 column with linear gradient elution of water/formic acid (0.1%) and acetonitrile/formic acid (0.1%) at a flow rate of 0.3 mL/min. The proposed method was validated with acceptable linearity (r2 , 0.9984-0.9999), precision (RSD, 0.22-2.11%), stability (RSD, 0.16-1.78%), and recovery (RSD ≤ 3.74%), under optimum conditions. The limits of quantitation ranged from 0.28 to 3.88 ng/mL. The method was successfully applied for simultaneous determination of 28 compounds in 20 batches of Mentat tablet. Hierarchical cluster analysis and principal component analysis were performed to evaluate the similarity and variation of the 20 samples based on the characteristics of 28 bioactive compounds. Results indicated that this method is rapid, sensitive, and reliable to show the quality of the Mentat tablets composition, hence may be used for quality control of polyherbal formulations having similar markers/raw herbs.

A rapid analytical method for characterization and simultaneous quantitative determination of phytoconstituents in Piper betle landraces using UPLC-ESI-MS/MS

A rapid ultra performance liquid chromatography electrospray ionisation tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed and validated for identification and characterization of phytoconstituents with simultaneous quantitative determination of three major bioactive phenolics namely allylpyrocatechol-3,4-diacetate, eugenyl acetate and eugenol from thirteen landraces of Piper betle leaf extracts. Among the 29 phytoconstituents detected, 19 were identified and characterized based on their fragmentation pattern obtained via MS/MS by means of collision-induced-dissociation (CID) and by comparison of their retention time with authentic standards or by previously reported literature. The proposed method was fully validated in terms of linearity, LOD, LOQ, precision, stability and recovery. All calibration curves showed a good linear relationship (r2 ≥ 0.9981). The precision evaluated by an intra- and inter-day study showed RSD ≤ 1.0% and good accuracy with overall recovery in the range from 96.14–98.46% (RSD ≤ 1.6%) for all analytes. The quantitative results showed that there were remarkable differences in the contents of the major bioactive phenolics in all the thirteen landraces of P. betle. The developed UPLC-ESI-MS/MS method was found to be simple, precise, sensitive and accurate. Hence, it can be reliably utilized as a quality control method for P. betle or derived herbal formulations. In vitro antimicrobial activity of the crude extract of P. betle landraces was also evaluated, and all extracts tested exhibited antifungal activity but none of the extracts were found to be active against bacteria even at 500 μg mL−1 concentration.

HPLC–QTOF–MS/MS-based rapid screening of phenolics and triterpenic acids in leaf extracts of Ocimum species and their interspecies variation

ABSTRACT Species of genus Ocimum are traditionally used for their medicinal and flavoring properties. These are rich sources of essential oils and found as an ingredient in many Ayurvedic preparations and food products. Phenolics and triterpenic acids are the medicinally active compounds mainly concentrated in the leaves of Ocimum species. This study aimed to develop an efficient and reliable analytical method for the rapid screening and characterization of phenolics and triterpenic acids in the leaf extracts of 6 Ocimum species using high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC–ESI–QTOF–MS/MS). A total of 50 compounds were identified and characterized on the basis of their accurate MS and MS/MS information, out of which 23 compounds were confirmed by authentic standards. Identified compounds include 28 flavonoids, 4 propenyl phenol derivatives, 2 triterpenic acids, 11 phenolic acids, and 5 phenolic acid esters. The developed method was applied to study the interspecies variation of identified compounds. Significant variation in the distribution of identified phenolics and triterpenic acids was observed among studied Ocimum species. Hence, the established method provides an effective and reliable tool for screening and characterization of phytoconstituents in Ocimum species. GRAPHICAL ABSTRACT

A rapid and highly sensitive method for simultaneous determination of bioactive constituents in leaf extracts of six Ocimum species using ultra high performance liquid chromatography-hybrid linear ion trap triple quadrupole mass spectrometry

Ocimum species have tremendous value in pharmaceutical, perfumery, food processing and cosmetic industries, also in traditional rituals and medicines. These are a rich source of terpenoids and phenolic compounds. Therefore, determination of these bioactive constituents is significant for quality evaluation of Ocimum species. In this study, we have developed and validated a rapid and highly sensitive method for simultaneous determination of sixteen bioactive constituents in the leaf extracts of six Ocimum species using ultra high performance liquid chromatography-hybrid linear ion trap triple quadrupole mass spectrometry (UHPLC-QqQLIT-MS/MS). The developed method is applied in the leaf extracts of six Ocimum species to investigate variations in the content of sixteen bioactive constituents. Quantitative analysis was performed by UHPLC-QqQLIT-MS/MS operating under multiple reaction monitoring mode in negative electrospray ionization. Chromatographic separation was accomplished on an Acquity UPLC BEH C18 column using a gradient elution of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The calibration curves of all sixteen analytes showed good linearity (r2 ≥ 0.9989) over the concentration range of 0.5–500 ng mL−1. The intra- and inter-day precisions and accuracy were within RSDs ≤ 1.95% and ≤ 1.68%, respectively. The results indicated that ursolic acid and rosmarinic acid were the major constituents in almost all the investigated Ocimum species except for O. americanum. All the results obtained from this study demonstrated that the developed method is rapid, sensitive and efficient for the quantification of multiple constituents. Therefore, it could be reliably utilized for the quality control and authenticity establishment of Ocimum species.

Major bioactive phenolics in Bergenia species from the Indian Himalayan region: Method development, validation and quantitative estimation using UHPLC-QqQLIT-MS/MS

Bergenia species are important medicinal plants used in indigenous systems of medicine for their antilithiatic and diuretic properties. An ultra-high performance liquid chromatography coupled to hybrid linear ion trap triple quadrupole mass spectrometry (UHPLC-QqQLIT-MS/MS) method has been developed and validated for the estimation of quantitative variation of eight major bioactive phenolics in the rhizomes (150 samples) of four species of this herb, Bergenia (B. ciliata, B. ligulata, B. purpurascens and B. stracheyi). Chromatographic separation was obtained on a Waters ACQUITY UPLCTM BEH (ethylene bridged hybrid) C18 column with a mobile phase consisting of 0.1% (v/v) formic acid aqueous solution and acetonitrile under a gradient elution manner. A hybrid linear ion trap triple quadrupole mass spectrometer was operated in negative electrospray ionization mode with multiple reactions monitoring for detection and quantification of the eight compounds. The validated method demonstrated good linearity (r2 ≥ 0.9991), precision (RSD ≤ 1.87%) and accuracy (95.16–102.11%, RSD ≤ 1.83%) for all reference analytes. The quantitative results revealed that B. ligulata contains the highest amount of the major active marker-bergenin. The results also suggest that sensitive UHPLC-QqQLIT-MS/MS method, a sensitive, accurate and convenient one, could be helpful in identification of potential accession(s), rapid quality control and establishing authenticity of Bergenia species as raw material for pharmaceutical industries.