Performance Liquid Chromatography-electrospray Tandem Mass Research Articles

Bioguided isolation, identification and bioactivity evaluation of anti-fatigue constituents from Schizophyllum commune

This study aims to clarify the specific anti-fatigue components of Schizophyllum commune (S.commune) and analyze its potential anti-fatigue mechanism. The main anti-fatigue active ingredient of S.commune was locked in n-butanol extract (SPE-n) by activity evaluation. Twelve compounds were identified by high performance liquid chromatography-electrospray tandem mass spectrometry (LC–ESI–MS/MS). The anti-fatigue effect of morusin is the most predominant among these 12 ingredients. The determination of biochemical indices showed that morusin could increase liver glycogen reserves, improve the activity of antioxidant enzymes in liver, and reduce reactive oxygen species (ROS) content in muscle tissue, thereby reducing myocyte damage. Further studies revealed that morusin could reduce the level of oxidative stress by activating Nrf2/HO-1 pathway, thus alleviating the fatigue of mice caused by exhaustive exercise. The current findings provide a theoretical basis for the development of natural anti-fatigue functional food.

Mineral composition, principal polyphenolic components, and evaluation of the anti-inflammatory, analgesic, and antioxidant properties of Cytisus villosus Pourr leaf extracts

Abstract Cytisus villosus Pourr. (C. villosus) is a medicinal plant belonging to the Fabaceae family, which grows in the Mediterranean area. It is used in traditional medicine against diseases related to inflammation. The objective of the present study was to identify the mineral and polyphenolic composition as well as to evaluate some biological properties including antioxidant, anti-inflammatory, and analgesic activities of C. villosus leaf aqueous extract. The chemical constituents were identified and quantified using ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) methods. The antioxidant properties of C. villosus leaves were tested using reducing power (RP), 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assays. The anti-inflammatory potency was evaluated in vitro and in vivo using the albumin denaturation test and the carrageenan test, respectively. Furthermore, the analgesic effect was performed in vivo using tail flick, acetic acid-induced contortion, and plantar tests. Mineralogical analysis revealed that potassium and calcium were the most abundant minerals. The analysis and quantification of the phytochemical composition using UPLC-ESI-MS/MS showed that quinic acid (57.478 ± 1.72 mg/kg) was the major compound of the aqueous extract, followed by salicylic acid (17.38 ± 0.2 mg/kg), isoquercetin (16.895 ± 1.01 mg/kg), and gallic acid (15.914 ± 1.51 mg/kg). The extracts showed potent antioxidant activity for all tests used. The highest antioxidant activity was recorded for the DPPH, ABTS and RP methods, with an IC50 of 3.94 ± 0.09, 2.88 ± 0.07, and 1.94 ± 0.10 μg/mL, respectively. Additionally, using the most frequent analgesic assays, the aqueous extract at a dose of 500 mg/kg exhibited a potent analgesic activity. Notably, an interesting inhibition of albumin denaturation was recorded with an IC50 of 383.94 μg/mL, corroborating the in vivo test. Overall, the results presented here may represent a scientific basis for the traditional use of C. villosus in the treatment of inflammation-related diseases.

Application of a three dimensional polyethyleneimine functionalized graphene oxide aerogel as an adsorbent for the determination of phytohormones in ginseng.

Aerogels have attracted considerable attention in sample pretreatment for their outstanding properties, such as the unique porous structure, large surface area and abundant modifiable active sites. The present research reports a three-dimensional interconnected porous network aerogel (PEI-AGO) manufactured based on graphene oxide (GO), polyethyleneimine (PEI) and agar as basic materials through a vacuum freeze-drying treatment. The PEI-AGO aerogel exhibits great potential as a solid phase extraction adsorbent for the selective purification of six endogenous plant hormones in conjunction with high performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS). Several factors affecting the extraction efficiency were investigated. Under the optimized extraction conditions, a wide linear range of 0.5-100 ng mL-1 with a good linearity (r > 0.9934) was observed. Low limits of detection (LODs) and limits of quantification (LOQs) were obtained in the range of 0.032-0.155 ng mL-1 and 0.107-0.518 ng mL-1, respectively. Furthermore, the relative recoveries for spiked ginseng samples exhibited remarkable consistency, ranging from 90.2% to 117.6%, with a relative standard deviation (RSD) of ≤9.4% (n = 3). In summary, PEI-AGO has proven to be an effective adsorbent for the pretreatment and enrichment of phytohormones which can be used for the determination of trace endogenous acidic plant hormones in ginseng leaves.

Rapid and ultra-trace levels analysis of 33 antibiotics in water by on-line solid-phase extraction with ultra-performance liquid chromatography-tandem mass spectrometry

A method was developed for the determination of 33 antibiotics belonging to 4 different antibiotic groups, including sulfonamides (16), fluoroquinolones (12), macrolides (1), and tetracyclines (4) in water samples using on-line solid-phase extraction-ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (on-line SPE-UPLC-MS/MS). The enrichment and analysis conditions were optimized for the determination of trace concentrations (nanogram per liter). Aliquots of the water samples (5 mL) were filtered through a membrane and enriched on an on-line polymeric column with hydrophilic–lipophilic balance (HLB). The analyte was eluted by the mobile phase during on-line SPE and separated on an Acquity BEH130 column, detected by tandem mass spectrometry, and quantified using an external standard method. The optimization of the analytical methods was discussed, which included optimization of pH of the sample, filtration membrane, Na2EDTA, chromatographic column, formic acid and aqueous ammonia in mobile phase. The detection limit for all test compounds by this method was in the range of 0.2–1.5 ng/L, with recoveries of 76.6–118%. The precision of the method, as indicated by the relative standard deviation, was 2.4–7.9%. Results of analysis of surface water samples demonstrated the ability of the proposed method to analyze ultra-trace levels of antibiotics, without the need for complex manual pretreatment.

Simultaneous Analyses of 5 Omega Fatty Acids on Dried Serum Spot using Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

Omega fatty acids play an important role as biomarkers of chronic inflammatory diseases such as asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, and inflammatory bowel disease. The purpose of our study was to develop a new analytical method for the detection of omega fatty acids from dried serum spots. We developed an ultrahigh performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS)-based method in the multiple reaction monitoring (MRM) mode with negative ionization. The detected MRM transitions for five omega fatty acids are as follow: α-linolenic acid (m/z=277.20→277.20), eicosapentaenoic acid (m/z=301.10→257.30), docosahexaenoic acid (m/z=327.00→283.15), arachidonic acid (m/z=303.20→259.25), and docosapentaenoic acid (m/z= 329.10→285.25). The calibration curve showed an excellent linearity within 0.1-2.5 μg/mL range (R2=0.9947~0.9994). The analytical methods howed excellent sensitivity (LOD: 0.005-0.01 μg/mL, LOQ: 0.03-0.1 μg/mL), which would allow for the targeted analyses of omega fatty acids. The recoveries of the omega fatty acid from the dried serum spots were 92.6%- 110.4% (RSD: ±0.2%-9.6%) and the retention time was within 5 min. This improved method offers rapid and sensitive quantification of omega fatty acids on dried serum spots using a small volume of serum and is expected to be applied to various biological specimens.

Determination of SK7324, a new class of hepatitis C virus inhibitor, in rat plasma by UPLC-MS/MS and its application to the pharmacokinetic study of SK7324.

In this study, a sensitive method for quantitation of the unique small-molecule inhibitor of hepatitis C virus SK7324 in rat plasma has been established using ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI/MS/MS). SK7324 and internal standard (tramadol) in plasma sample was extracted using acetonitrile. A centrifuged upper layer was then evaporated and reconstituted with the mobile phase of 0.5% formic acid-acetonitrile (35:65, v/v). The reconstituted samples were injected into a C18 reversed-phase column. Using MS/MS in the multiple reaction monitoring (MRM) mode, SK7324 and tramadol were detected without severe interference from rat plasma matrix. Detection of SK7324 in rat plasma by the UPLC-ESI/MS/MS method was accurate and precise with a quantitation limit of 1.0 ng/mL. The validation, reproducibility, stability, and recovery of the method were evaluated. The method has been successfully applied to pharmacokinetic studies of SK7324 in rat plasma. Pharmacokinetic parameters of SK7324 were evaluated after intravenous (i.v.; at doses of 5 mg/kg) and oral (p.o.; at doses of 10 mg/kg) administration of SK7324 in rats. After p.o. administration (10 mg/kg) of SK7324, F (Fraction absorbed) value was approximately 87.1%.

High-Resolution Metabolomics of 50 Neurotransmitters and Tryptophan Metabolites in Feces, Serum, and Brain Tissues Using UHPLC-ESI-Q Exactive Mass Spectrometry

Recent evidence indicates that tryptophan metabolites and neurotransmitters are potential mediators of the microbiome–gut–brain interaction. Here, a high-resolution ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) assay was developed and validated for quantifying 50 neurotransmitters, tryptophan metabolites, and bacterial indole derivatives in mouse serum, feces, and brain. The lower limit of quantitation for the 50 compounds ranged from 0.5 to 100 nmol/L, and sample preparation procedures were adapted for individual compounds to allow quantitation within linearity of the assay with a correlation coefficient >0.99. Reproducibility was tested by intra- and interday precision and accuracy of analysis: intra- and interday precision at the lower limit of quantitation was less than 20% for all compounds, with over two-thirds of the compounds achieving an interday precision below 10%, while the interday accuracy at the lower limit of quantitation ranged from 82.3 to 128.0% for all compounds. The analyte recovery was assessed based on sample-spiked stable-isotope-labeling standards, illustrating a need to consider matrix-specific recovery discrepancies when performing interorgan comparison. Carryover was evaluated by intermittent solvent blank injection. The assay was successfully applied to determining the concentration profiles of neurotransmitter and tryptophan metabolites in serum, feces, and brain of conventionally raised specific pathogen-free (SPF) C57BL/6 mice. Our method may serve as a useful analytical resource for investigating the roles of tryptophan metabolism and neurotransmitter signaling in host–microbiota interaction.

A novel isotope dilution UHPLC-ESI-MS/MS method for the quantification of 3-monochloropropane-1,2-diol in Caco-2 cell transport and receiving buffers

ABSTRACT A routine, selective and sensitive ultra-high performance liquid chromatography-electrospray ionisation tandem triple quadrupole mass spectrometry (UHPLC-ESI-MS/MS) method was developed and validated for the quantification of 3-monochloropropane-1,2-diol (3-MCPD) in Caco-2 cell transport buffer (FaSSIF-V2, the second version of a fasted state simulated intestinal fluid) and receiving buffer (HBSS, Hank’s balanced salt solution). The method involves measuring deuterated 3-MCPD (3-MCPD-d5) as internal standard (IS) during the entire analytical procedure to obtain precise and accurate results. The separation was performed on a Poroshell 120 HILIC column (2.7 µm, 3.0 × 50 mm) at a flow rate of 0.3 mL/min using water (containing 0.025% acetic acid) and acetonitrile (containing 0.025% acetic acid) as the mobile phases. Mass spectrometric detection was operated in dynamic multiple reaction monitoring (dMRM) in negative ion mode. The method exhibited high sensitivity. The limits of detection (LOD) for 3-MCPD in FaSSIF-V2 and HBSS were 0.012 and 0.014 µmol/L, and the limits of quantification (LOQ) were 0.039 and 0.045 µmol/L, respectively. Satisfactory results were observed for linearity (R2 > 0.999), intra-day precision (RSD% <7.7% in FaSSIF-V2 and <6.6% in HBSS), inter-day precision (RSD% <5.9% in FaSSIF-V2 and <5.6% in HBSS), accuracy (% error within ± 10%), and sample stability (RSD% <7.7% and % error within ± 10%). The method has been successfully applied to quantify 3-MCPD in Caco-2 cell transport and receiving buffers. The results were in good agreement with those obtained with gas chromatography-tandem mass spectrometry (GC-MS).

Simultaneous determination of formononetin, biochanin A and their active metabolites in human breast milk, saliva and urine using salting-out assisted liquid-liquid extraction and ultra high performance liquid chromatography-electrospray ionization tandem mass spectrum

Isoflavonoid phytoestrogens, referred as “dietary estrogens” are widely distributed in the plant kingdom. Formononetin, biochanin A and their active metabolites daidzein and genistein are known to be the most potent among other isoflavonoid phytoestrogens. Thus there is a growing need to determine accurately their concentration in different biological fluids. In the present work, a sensitive analytical method was developed for the quantitative determination of these compounds in human breast milk, saliva and urine. The glycoside conjugates of these compounds were enzymatically hydrolysis prior to salting-out assisted liquid-liquid extraction. Quantitative analysis was done by ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The obtained results showed high correlation coefficients (r2 > 0.998) for the linear range established for formononetine, biochanin A, daidzein and genistein. The limits of detection (LODs) and low limits of quantitation (LLOQs) were in the ranges of 0.05–1.0 ng/mL and 1.0–4.0 ng/mL for all analytes in human biological fluids, respectively. The average recoveries ranged from 83.29% to 115.24% for the analytes with relative standard deviation (n = 5) values from 1.84% to 9.75% in samples. Both intra-day and inter-day precisions and accuracy were found to be within 12.53% and ± 12.92% respectively. Under different conditions of stability, the concentrations for four isoflavonoid phytoestrogens deviated within ±12.87% of norminal values. The developed method was successfully validated and applied to human breast milk, saliva and urine. The average concentrations of daidzein and genistein found in breast milk, saliva and urine samples ranged from 0 to 104.2 µg/kg, 18.17 to 786.0 µg/kg, 0 to 10974 µg/kg, respectively. Their presence in breast milk samples shows exposure of breast-fed baby to isoflavones. It also allows for the rapid screening of human biological fluids when testing for formononetin, biochanin A, daidzein and genistein production status in human.

Saliva as a non-invasive tool for monitoring oxidative stress in swimmers athletes performing a VO2max cycle ergometer test

Biomarkers of oxidative stress are generally measured in blood and its derivatives. However, the invasiveness of blood collection makes the monitoring of such chemicals during exercise not feasible. Saliva analysis is an interesting approach in sport medicine because the collection procedure is easy-to-use and does not require specially-trained personnel. These features guarantee the collection of multiple samples from the same subject in a short span of time, thus allowing the monitoring of the subject before, during and after physical tests, training or competitions. The aim of this work was to evaluate the possibility of following the changes in the concentration of some oxidative stress markers in saliva samples taken over time by athletes under exercise. To this purpose, ketones (i.e. acetone, 2-butanone and 2-pentanone), aldehydes (i.e. propanal, butanal, and hexanal), α,β-unsaturated aldehydes (i.e. acrolein and methacrolein) and di-carbonyls (i.e. glyoxal and methylglyoxal) were derivatized with 2,4-dinitrophenylhydrazine, and determined by ultra-high performance liquid chromatography coupled to diode array detector. Prostaglandin E2, F2/E2-isoprostanes, F2-dihomo-isoprostanes, F4-neuroprostanes, and F2-dihomo-isofuranes were also determined by a reliable analytical procedure that combines micro-extraction by packed sorbent and ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry. Overall the validation process showed that the methods have limits of detection in the range of units of ppb for carbonyls and tens to hundreds of ppt for isoprostanes and prostanoids, very good quantitative recoveries (90–110%) and intra- and inter-day precision lower than 15%. The proof of applicability of the proposed analytical approach was investigated by monitoring the selected markers of oxidative stress in ten swimmers performing a VO2max cycle ergo meter test. The results highlighted a clear increase of salivary by-products of oxidative stress during exercise, whereas a sharp decrease, approaching baseline values, of these compounds was observed in the recovery phase. This study opens up a new approach in the evaluation of oxidative stress and its relation to aerobic activity.

Cytokinins Are Abundant and Widespread Among Insect Species.

Cytokinins (CKs) are a class of compounds that have long been thought to be exclusively plant growth regulators. Interestingly, some species of phytopathogenic bacteria and fungi have been shown to, and gall-inducing insects have been hypothesized to, produce CKs and use them to manipulate their host plants. We used high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-MS/MS) to examine concentrations of a wide range of CKs in 17 species of phytophagous insects, including gall- and non-gall-inducing species from all six orders of Insecta that contain species known to induce galls: Thysanoptera, Hemiptera, Lepidoptera, Coleoptera, Diptera, and Hymenoptera. We found CKs in all six orders of insects, and they were not associated exclusively with gall-inducing species. We detected 24 different CK analytes, varying in their chemical structure and biological activity. Isoprenoid precursor nucleotide and riboside forms of trans-zeatin (tZ) and isopentenyladenine (iP) were most abundant and widespread across the surveyed insect species. Notably, the observed concentrations of CKs often markedly exceeded those reported in plants suggesting that insects are synthesizing CKs rather than obtaining them from the host plant via tissue consumption, compound sequestration, and bioaccumulation. These findings support insect-derived CKs as means for gall-inducing insects to manipulate their host plant to facilitate cell proliferation, and for both gall- and non-gall-inducing insects to modify nutrient flux and plant defenses during herbivory. Furthermore, wide distribution of CKs across phytophagous insects, including non-gall-inducing species, suggests that insect-borne CKs could be involved in manipulation of source-sink mechanisms of nutrient allocation to sustain the feeding site and altering plant defensive responses, rather than solely gall induction. Given the absence of any evidence for genes in the de novo CK biosynthesis pathway in insects, we postulate that the tRNA-ipt pathway is responsible for CK production. However, the unusually high concentrations of CKs in insects, and the tendency toward dominance of their CK profiles by tZ and iP suggest that the tRNA-ipt pathway functions differently and substantially more efficiently in insects than in plants.

Full-length transcriptome analysis of Coptis deltoidea and identification of putative genes involved in benzylisoquinoline alkaloids biosynthesis based on combined sequencing platforms.

The study carry out comprehensive transcriptome analysis of C. deltoidea and exploration of BIAs biosynthesis and accumulation based on UHPLC-MS/MS and combined sequencing platforms. Coptis deltoidea is an important medicinal plant with a long history of medicinal use, which is rich in benzylisoquinoline alkaloids (BIAs). In this study, Ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) and combined sequencing platforms were performed for exploration of BIAs biosynthesis, accumulation and comprehensive transcriptome analysis of C. deltoidea. By metabolism profiling, the accumulation of ten BIAs was analyzed using UHPLC-MS/MS and different contents were observed in different organs. From transcriptome sequencing result, we applied single-molecule real-time (SMRT) sequencing to C. deltoidea and generated a total of 75,438 full-length transcripts. We proposed the candidate biosynthetic pathway of tyrosine, precursor of BIAs, and identified 64 full length-transcripts encoding enzymes putatively involved in BIAs biosynthesis. RNA-Seq data indicated that the majority of genes exhibited relatively high expression level in roots. Transport of BIAs was also important for their accumulation. Here, 9 ABC transporters and 2 MATE transporters highly homologous to known alkaloid transporters related with BIAs transport in roots and rhizomes were identified. These findings based on the combined sequencing platforms provide valuable genetic information for C. deltoidea and the results of transcriptome combined with metabolome analysis can help us better understand BIAs biosynthesis and transport in this medicinal plant. The information will be critical for further characterization of C. deltoidea transcriptome and molecular-assisted breeding for this medicinal plant with scarce resources.

Diurnal rhythmicity of endogenous phytohormones and phototropic bending capacity in potato (Solanum tuberosum L.) shoot cultures

Taking advantage of advanced high performance liquid chromatography–electrospray tandem–mass spectrometry (HPLC–ESI–MS/MS), we screened daily changes in concentrations of endogenous phytohormones of in vitro grown potato (Solanum tuberosum L. cv. Desiree) shoot cultures and checked for possible connections between the rhythmicity of endogenous phytohormones and phototropic bending capacity of the same cultures. Studies done under diurnal 16 h light and 8 h darkness (diurnal) and continuous light (CL) conditions showed prominent daily rhythmicity of endogenous phytohormone levels in both light regimes. Phototropic bending in potato is known to be rhythmic only in the diurnal, whereas in CL conditions the bending response is present but without any daily rhythmicity. For all of the studied phytohormone groups significant differences between the diurnal and CL conditions were found. Changes in the concentration of indole auxins, indole-3-acetic acid (IAA) and its catabolite 2-oxindole-3-acetic acid (OxIAA), were the most prominent. Their levels clearly alternated with level of IAA being high in diurnal and OxIAA in CL conditions. Significant concentration changes were also observed for other phytohormones such as cytokinin ribosides, salicylic acid, abscisic acid and phaseic acid. Observed changes in daily phytohormone levels indicate strong and complex involvement of diverse phytohormone groups in realization of the phototropic bending response of potato shoots.

Sensitive Determination of Four Polypeptide Antibiotic Residues in Milk Powder by High Performance Liquid Chromatography–Electrospray Tandem Mass Spectrometry

Polypeptide antibiotics abuse can lead to antibiotic residues in food products and have unwanted effects on human health. A selective, accurate, and sensitive analytical method for the simultaneous determination of four polypeptide antibiotics (gramicidin S, bacitracin, polymyxin B, and polymyxin E) in infant formula powder was developed using solid-phase extraction (SPE) combined with high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). The samples were extracted with acidified methanol, deproteinized with acetonitrile, and degreased with n-hexane before SPE. After cleanup with 0.1% formic acid and methanol (3:1, v/v) in Oasis HLB cartridges, the extracts were analyzed by HPLC–MS/MS with electrospray ionization (ESI) source and time-scheduled multiple reaction monitoring (MRM). Linearity was assessed by using matrix-matched standard calibration and good determination coefficients (r2 > 0.995) were obtained. The average recoveries for blank sample at three spiked concentration levels were in the range of 82.8–101.2%. The limits of detection (LODs) and limits of quantitation (LOQs) of all analytes were in the range of 5–15 μg kg−1 and 20–50 μg kg−1, respectively. The intra-day and inter-day precisions were lower than 10%. The results of method validation demonstrated that the developed method is accurate and reliable, and it can be applied for screening and quantitation of target polypeptide antibiotics in food.

Micro-extraction by packed sorbent combined with UHPLC-ESI-MS/MS for the determination of prostanoids and isoprostanoids in dried blood spots

This work presents a reliable analytical procedure combining micro-extraction by packed sorbent (MEPS) and ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry to determine 8-iso prostaglandin F2α, 8-iso prostaglandin E2 and prostaglandin E2 in dried blood spots (DBSs). To reach this goal, we optimized a fast semi-automated MEPS procedure for the clean-up and pre-concentration of the analytes extracted from a single DBS (50 μL) by a 70:30 v/v methanol:water mixture. Limits of detection of about 20 pg mL−1, satisfactory recoveries (90–110%) and very good intra- and inter-day precisions (RSD ≤10%) were obtained for all the analytes. The innovative addition of internal standards on the filter paper before DBS sampling allowed to compensate changes in the amount of analyte during storage. Since prostanoids and isoprostanoids are biomarkers involved in the pathogenesis and progression of many diseases (e.g. ductal patency, diabetic nephropathy, and acute lung injury), our analytical method offers interesting diagnostic and prognostic opportunities in the medical field. The present method is currently used for the analysis of such biomarkers in DBSs from preterm newborns collected in the clinical setting.

Chemical profiling, antioxidant, enzyme inhibitory and molecular modelling studies on the leaves and stem bark extracts of three African medicinal plants

Africa is famous for its floral biodiversity, exploited by local people for therapeutic purposes. However, such plants need to be scrutinised scientifically for the presence of bioactive compounds and possible biological properties. This study attempts for the first time to highlight the pharmacological and phytochemical profile of extracts prepared from leaves and stem barks of three African plants (Macaranga hurifolia Beille, Sterculia tragacantha Lindl. and Zanthoxylum gilletii (De Wild.) P. G. Waterman. The extracts were tested for antioxidant and enzyme inhibitory effects. Free radical scavenging, metal chelator, reducing power and phosphomolybdenum assays were performed to evaluate antioxidant effects. To identify enzyme inhibitory effects, cholinesterases (acetylcholinesterase (AChE) and butrylcholinesterase (BChE)), tyrosinase, α-amylase and α-glucosidase were selected as target enzymes. High performance liquid chromatography-Electrospray tandem mass spectrometry (HPLC-ESI-MS) technique was also used for chemical profiling. ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assays showed that the stem barks of all three African plants were better scavenger than leaf extracts. Sterculia tragacantha was found to be a better metal chelator (64.10 ± 4.66 mg EDTAE/g) among the studied plants. All extracts exhibited good clinical enzyme inhibitory activities. The stem bark of S. tragacantha exhibited the best acetylcholinesterase activity compared to the other plants. HPLC-ESI-MS characterization showed that the most abundant compounds in stem bark were flavonoids in M. hurifolia (4.2 ± 0.2 mg/g DE), proanthocyanidins in S. tragacantha (42 ± 1 mg/g DE) and similar concentrations of phenolic acids and flavonoids in Z. gilletii (2.8–3.1 mg/g DE). Based on the biological activity, the most abundant and relevant bioactive compounds in the extracts were studied using molecular modelling approach against tyrosinase. The studied African plants showed good antioxidant and enzymatic propensities and thus can be considered as potential bioresources for future development of nutraceuticals and/or for pharmaceutical applications.

An Ultra-High Performance Liquid Chromatography-Electrospray Tandem Mass Spectrometric Method for Screening and Simultaneous Determination of Anorexic, Anxiolytic, Antidepressant, Diuretic, Laxative and Stimulant Drugs in Dietary Supplements Marketed for Weight Loss.

The consumption of dietary supplements is increasing every year all over the world and has been accompanied by an increased frequency of adulteration of these products with synthetic pharmaceuticals. Analytical methods that allow testing for the presence of synthetic drugs in dietary supplements are needed to detect such fraudulent practices. To investigate the adulteration of dietary supplements marketed for weight loss using different commercial appeals, we developed an analytical method using ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC-ESI-MS/MS) for simultaneous determination of 32 drugs, including anorexics, anxiolytics, antidepressants, diuretics, laxatives and stimulants. Separation was accomplished in 19 minutes using a Zorbax SB-C18 column and a gradient elution program with 0.05% formic acid in water/acetonitrile as a mobile phase. Limits of quantification ranged from 0.14 to 3.92 μg L-1, and accuracy ranged from 80.00 to 119.48%. A simple extraction procedure was used in the pretreatment step by dissolving the samples in 100% methanol followed by a 1000 to 10,000-fold dilution in the mobile phase and filtration through a Teflon membrane (0.2 μm). The method was applied to the screening and quantification of the drugs in 108 formulations marketed as food supplements for slimming, weight loss, thermogenics, and supplements for meal replacement. Caffeine and p-synephrine were found as stimulants in 80 samples, listed or not on the label.

Phytochemical characterization and bioactivities of five Apiaceae species: Natural sources for novel ingredients

Several species of the Apiaceae family have been employed in traditional cultures for their curative virtues. The present study focused on five Apiaceae species, (Falcaria vulgaris (FV), Smyrniopsis aucheri (SA), Smyrniopsis munzurdagensis (SM), Smyrnium cordifolium (SC), and Actinolema macrolema (AM)). The antioxidant, enzyme inhibitory (α-amylase, α-glucosidase, acetyl- and butyrylcholinesterase, lipase, and tyrosinase), antimicrobial, phytochemical, and cytotoxicity profiles of the methanol extracts of the selected Apiaceae species were determined. SC extract (35.68 mg gallic acid equivalent/g extract) possessed the highest phenolic content while the AM extract (56.79 mg rutin equivalent/g extract) had the highest flavonoid content. HPLC-ESI-MS (High performance liquid chromatography-electrospray tandem mass spectrometry) analyses showed presence of ferulic acid in all the five species. SC extract exhibited high radical scavenging (59.28 and 94.31 mg Trolox equivalent [TE]/g extract, DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid), respectively) and reducing activity (161.44 and 113.62 mg TE/g extract, for CUPRAC (cupric reducing antioxidant capacity) and FRAP (ferric reducing antioxidant power), respectively). SM extract exhibited the highest cholinesterase’s inhibitory action (3.82 and 4.76 mg galantamine equivalent/g extract, for acetyl- and butyrylcholinesterase, respectively). The extracts showed higher inhibition against α-glucosidase (7.32-11.99 mmol acarbose equivalent [ACAE]/g extract) compared to α-amylase (0.51-0.55 mmol ACAE/g extract). SC extract was the most active (137.54 mg kojic acid equivalent/g extract) tyrosinase inhibitor and FV extract (113.75 mg Orlistat equivalent/g) the best lipase inhibitor. SM extract showed potent antibacterial effect against B. cereus (MIC (minimum inhibitory concentration) 0.180 mg/mL), P. mirabilis (MIC 0.180 mg/mL), M. flavus (MIC 0.560 mg/mL), P. aeruginosa (MIC 0.275 mg/mL), and S. typhimurium (MIC 1.500 mg/mL). FV extract (MIC 0.140 mg/mL) suppressed A. fumigatus growth. Cytotoxicity was assessed on murine macrophage (RAW 264.7), human embryonic kidney (HEK 293), and human hepatocellular carcinoma (HepG2) cell lines. FV (60.3%) and SM (57.4%) showed the highest reduction on RAW 264.7 cellular viability, whereas SM (74.1%) showed toxicity against HepG2. This study supports that the Apiaceae species could be considered as promising candidates for the development of novel pharmacophores for the management of several human ailments.

Determination of JW5473 in Rat Plasma by UPLC-MS/MS and its Application to Pharmacokinetic Study of JW5473.

A sensitive method for quantitation of JW5473 in rat plasma has been established using ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI/MS/MS). Tramadol was used as an internal standard. JW5473 and internal standard in plasma sample was extracted using acetonitrile (protein precipitation). A centrifuged upper layer was then evaporated and reconstituted with the mobile phase of 0.5% formic acid-acetonitrile (40:60, v/v). The reconstituted samples were injected into a C18 reversed-phase column. Using MS/MS in the multiple reaction monitoring (MRM) mode, JW5473 and tramadol were detected without severe interference from rat plasma matrix. JW5473 produced a protonated precursor ion ([M+H]+) at m/z 432.3 and a corresponding product ion at m/z 114.4. And the internal standard produced a protonated precursor ion ([M+H]+) at m/z 264.4 and a corresponding product ion at m/z 58.1. Detection of JW5473 in human plasma by the UPLC-ESI/MS/MS method was accurate and precise with a quantitation limit of 1.0 ng/mL. The validation, reproducibility, stability, and recovery of the method were evaluated. The method has been successfully applied to pharmacokinetic studies of JW5473 in rat plasma. Pharmacokinetic parameters of JW5473 was evaluated after intravenous (i. v.; at doses of 15 mg/kg) and oral (p.o.; at doses of 30 mg/kg) administration of JW5473 in rats. After p.o. administration (30 mg/kg) of JW5473, F (Fraction absorbed) value was approximately 70.5%.

Quantification of Panax notoginseng saponins metabolites in rat plasma with in vivo gut microbiota-mediated biotransformation by HPLC-MS/MS

Panax notoginseng saponins (PNS) are the major components of Panax notoginseng, with multiple pharmacological activities but poor oral bioavailability. PNS could be metabolized by gut microbiota in vitro, while the exact role of gut microbiota of PNS metabolism in vivo remains poorly understood. In this study, pseudo germ-free rat models were constructed by using broad-spectrum antibiotics to validate the gut microbiota-mediated transformation of PNS in vivo. Moreover, a high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was developed for quantitative analysis of four metabolites of PNS, including ginsenoside F1 (GF1), ginsenoside Rh2 (GRh2), ginsenoside compound K (GCK) and protopanaxatriol (PPT). The results showed that the four metabolites could be detected in the control rat plasma, while they could not be determined in pseudo germ-free rat plasma. The results implied that PNS could not be biotransformed effectively when gut microbiota was disrupted. In conclusion, gut microbiota plays an important role in biotransformation of PNS into metabolites in vivo.