Mutant Profilin1 transgenic mice recapitulate cardinal features of motor neuron disease.

The recent identification of profilin1 mutations in 25 familial ALS cases has linked altered function of this cytoskeleton-regulating protein to the pathogenesis of motor neuron disease. To investigate the pathological role of mutant profilin1 in motor neuron disease, we generated transgenic lines of mice expressing human profilin1 with a mutation at position 118 (hPFN1G118V). One of the mouse lines expressing high levels of mutant human PFN1 protein in the brain and spinal cord exhibited many key clinical and pathological features consistent with human ALS disease. These include loss of lower (ventral horn) and upper motor neurons (corticospinal motor neurons in layer V), mutant profilin1 aggregation, abnormally ubiquitinated proteins, reduced choline acetyltransferase (ChAT) enzyme expression, fragmented mitochondria, glial cell activation, muscle atrophy, weight loss, and reduced survival. Our investigations of actin dynamics and axonal integrity suggest that mutant PFN1 protein is associated with an abnormally low filamentous/globular (F/G)-actin ratio that may be the underlying cause of severe damage to ventral root axons resulting in a Wallerian-like degeneration. These observations indicate that our novel profilin1 mutant mouse line may provide a new ALS model with the opportunity to gain unique perspectives into mechanisms of neurodegeneration that contribute to ALS pathogenesis.

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.

Profilin1 biology and its mutation, actin(g) in disease.

Profilins were discovered in the 1970s and were extensively studied for their significant physiological roles. Profilin1 is the most prominent isoform and has drawn special attention due to its role in the cytoskeleton, cell signaling, and its link to conditions such as cancer and vascular hypertrophy. Recently, multiple mutations in the profilin1 gene were linked to amyotrophic lateral sclerosis (ALS). In this review, we will discuss the physiological and pathological roles of profilin1. We will further highlight the cytoskeletal function and dysfunction caused by profilin1 dysregulation. Finally, we will discuss the implications of mutant profilin1 in various diseases with an emphasis on its contribution to the pathogenesis of ALS.

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.

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.

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 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.

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.

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.

Molecular dynamics guided design of tocoflexol: a new radioprotectant tocotrienol with enhanced bioavailability.

There is a pressing need to develop safe and effective radioprotector/radiomitigator agents for use in accidental or terrorist-initiated radiological emergencies. Naturally occurring vitamin E family constituents, termed tocols, that include the tocotrienols, are known to have radiation-protection properties. These agents, which work through multiple mechanisms, are promising radioprotectant agents having minimal toxicity. Although α-tocopherol (AT) is the most commonly studied form of vitamin E, the tocotrienols are more potent than AT in providing radioprotection and radiomitigation. Unfortunately, despite their very significant radioprotectant activity, tocotrienols have very short plasma half-lives and require dosing at very high levels to achieve necessary therapeutic benefits. Thus, it would be highly desirable to develop new vitamin E analogues with improved pharmacokinetic properties, specifically increased elimination half-life and increased area under the plasma level versus time curve. The short elimination half-life of the tocotrienols is related to their low affinity for the α-tocopherol transfer protein (ATTP), the protein responsible for maintaining the plasma level of the tocols. Tocotrienols have less affinity for ATTP than does AT, and thus have a longer residence time in the liver, putting them at higher risk for metabolism and biliary excretion. We hypothesized that the low-binding affinity of tocotrienols to ATTP is due to the relatively more rigid tail structure of the tocotrienols in comparison with that of the tocopherols. Therefore, compounds with a more flexible tail would have better binding to ATTP and consequently would have longer elimination half-life and, consequently, an increased exposure to drug, as measured by area under the plasma drug level versus time curve (AUC). This represents an enhanced residence of drug in the systemic circulation. Based on this hypothesis, we developed a new class of vitamin E analogues, the tocoflexols, which maintain the superior bioactivity of the tocotrienols with the potential to achieve the longer half-life and larger AUC of the tocopherols.

Study of Vulvovaginal Atrophy and Genitourinary Syndrome of Menopause and Its Impact on the Quality of Life of Postmenopausal Women in Central India.

Background Urogenital health is a necessary part of health for all women, especially in the postmenopausal age group. We suspected that the increased incidence of vulvovaginal atrophy (VVA) had some or other effects on the quality of life of older women. So, we aimed to study VVA/genitourinary syndrome of menopause (GSM) and its impact on the quality of life of postmenopausal women in Central India. Despite its significant prevalence and detrimental impact on women's health, VVA/GSM is underdiagnosed and undertreated. In view of the feminization of aging, VVA management is becoming increasingly crucial. This study contributes to postmenopausal women's understanding that keeping their urogenital and sexual longevity is a critical step toward healthy living and gender equality. Given its relationship with urogynecological conditions, this study will help to evaluate both subjectively and objectively the incidence of symptoms related to VVA and its effects on the quality of life of postmenopausal women. This will eventually help to understand the need to address this issue while making postmenopausal women health-related policies. Potential remedies to overcome the obstacles currently preventing patient-HCP interactions addressing sexual health include providing communication tools to facilitate the "uncomfortable" conversation, educating women, and providing enough training for healthcare professionals. Methods The current study was conducted at a rural tertiary healthcare center in Central India and is a cross-sectional study. The study population taken into consideration were all the postmenopausal women between the age group 45 and 75 years with at least one vulvovaginal symptom attending the Outpatient Department (OPD). The total study sample size was 100 women. Further study was conducted by interview method using a questionnaire by the principal investigator. Data was gathered with the help of a pretested questionnaire in the patient's language. Symptoms related to GSM were studied by the vaginal symptom Bothersomeness Scale. Further, a gynecological clinical examination for the confirmation of VVA was carried out, which included a gynecological physical examination. The Vaginal Health Index (VHI) was calculated for each female using the score scale. Assessment of the quality of life of postmenopausal females using the Day-to-Day Impact of Vaginal Aging (DIVA) Questionnaire was performed. Results The majority of females (34%) who presented with the symptoms were in the category of 55-60 years followed by 22% in the age group of 61-65 years. The most common symptoms experienced by females were vaginal dryness (77%) followed by vaginal discharge (74%). Our study confirmed that 79% of the total females included in the study have a VHI score of less than 15, i.e., they suffer from VVA, thus presenting our incidence at 79%. Conclusion According to the surveys discussed in this research, a significant portion of postmenopausal women have symptoms linked to VVA that have a negative impact on their quality of life, including their sexual relationships and self-esteem.

Synthesis of copper-reduced graphene oxide nanomaterials using glucose and study of its antibacterial and anticancer activities.

In this work, glucose-capped copper nanoparticles decorated reduced graphene oxide nanomaterial are synthesized at 100 °C and 200 °C via chemical reduction method and studied for their antibacterial and anticancer activities. Synthesized nanomaterials were characterized using x-ray diffraction, Fourier-transform infrared, transmission electron microscope, and RAMAN. It is observed in transmission electron microscopy and selected area electron diffraction studies that copper nanoparticles deposited onto reduced graphene oxide are smaller than nanoparticles generated in the absence of reduced graphene oxide. Also, the size of copper nanoparticles synthesized at 200 °C is smaller than at 100 °C. Results suggest that Cu/Glu/rGO synthesized at both temperatures showed significant antibacterial activity againstEscherichia coliandBacillus anthracis,similarly, showed significant cell death in cancer cell lines [Cal33 and HCT-116 p53 (+/+)]. Interestingly, the nanomaterials were seen to be more effective against the cancer cell lines harboring aggregating mutant p53. Tumors with aggregating mutants of p53 are difficult to treat hence, Cu/Glu/rGO can be promising therapeutic agents against these difficult cancers. However, the antibacterial and anticancer activity of Cu/Glu/rGO synthesized at 100 °C where Cu2O form is obtained was found to be more effective compared to Cu/Glu/rGO synthesized at 200 °C where Cu form is obtained. Though fine-tuning of the material may be required for its commercial applications.

Access to indole- and pyrrole-fused diketopiperazines via tandem Ugi-4CR/intramolecular cyclization and its regioselective ring-opening by intermolecular transamidation.

An efficient approach for the synthesis of indole- and pyrrole-fused diketopiperazines has been developed. This protocol involves the Ugi four-component reaction (U-4CR) followed by an intramolecular cyclization of the Ugi products at room temperature to afford the desired products in good to excellent yields. In addition, it is interesting to report the subsequent regioselective ring-opening of diketopiperazine unit occurring via an intermolecular transamidation reaction under mild condition, resulting in the formation of highly functionalized indole-2-carboxamides and pyrrole-2-carboxamides.

Bell's Palsy: A Review.

Bell's palsy, also known as "acute facial palsy of unknown cause", is a common cranial neuropathy leading to facial muscle paresis or complete paralysis characteristically on one side, occurring suddenly and may progress over 48 hours. It results from facial nerve dysfunction due to trauma or inflammation of the 7th cranial nerve or facial nerve or its branches along its course, primarily in the bony canal. Both sexes are equally affected, and though no age is immune, its incidence rises with increasing age. The risk is high in diabetics, hypertensives, women who are pregnant, obese, and people with upper respiratory tract infections. It is considered chiefly idiopathic and is diagnosed by the exclusion of other causes. Bell's palsy can cause physical and psychological complications and negatively impact patients and their relatives. Thus, early diagnosis and quick cause determination are prime roles in proper treatment. However, the exact etiology of Bell's palsy is unknown, affecting its treatment. Still, determining probable causative and risk factors is critical for employing a targeted treatment approach and requires a comprehensive examination and a complete history. Although the majority of patients recover spontaneously in less than three weeks even if they are not treated. But there is always a risk of residual paresis after treatment or recovery, which may require medical help. This review aims to furnish the most thorough understanding of Bell's palsy, focusing on anatomy, etiology, clinical features, diagnosis, clinical consequences,and preferred therapy approaches.

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.

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.

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.

Identification and characterization of phenolics and terpenoids from ethanolic extracts of Phyllanthus species by HPLC-ESI-QTOF-MS/MS.

Phyllanthus species plants are a rich source of phenolics and widely used due to their medicinal properties. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed using high-pressure liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-QTOF-MS/MS) for the identification and characterization of quercetin, kaempferol, ellagic acid and their derivatives in ethanolic extracts of Phyllanthus species. The chromatographic separation was carried out on Thermo Betasil C8 column (250 mm×4.5 mm, 5 µm) using 0.1% formic acid in water and 0.1% formic acid in methanol as the mobile phase. The identification of diagnostic fragment ions and optimization of collision energies were carried out using 21 reference standards. Totally 51 compounds were identified which include 21 compounds identified and characterized unambiguously by comparison with their authentic standards and the remaining 30 were tentatively identified and characterized in ethanolic extracts of P. emblica, P. fraternus, P. amarus and P. niruri.