High-performance Liquid Chromatography-electrospray Ionization-mass Spectrometry Research Articles

Functional regulation of a UDP-glucosyltransferase gene (Pq3-O-UGT1) by RNA interference and overexpression in Panax quinquefolius

Ginsenosides have been thought to be the major pharmacological active ingredient in the roots of Panax quinquefolius, P. ginseng and other Panax plants. Glycosylation is the last step in the biosynthesis pathway of ginsenosides and leads to the formation of various secondary metabolites. As for P. quinquefolius, dammarenediol-II synthase (PqDS), protopanaxadiol synthase (PqD12H) and protopanaxatriol synthase (CYP6H) have been cloned and identified in the early time. Here, we cloned and identified a novel UDP-glycosyltransferase (UGT) gene from P. quinquefolius (Pq3-O-UGT1, GenBank accession no. KiR028477) for the first time, reverse transcription-PCR (RT-PCR) analysis showed a remarkable transcription increase of Pq3-O-UGT1 in the methyl jasmonate (MeJA)-treated hairy roots, in vitro enzymatic assay confirmed that Pq3-O-UGT1 catalyzed the glycosylation of protopanaxadiol to produce ginsenoside Rh2 whose chemical structure was confirmed using high performance liquid chromatography electrospray ionization mass spectrometry (HPLC/ESI-MS). Moreover, overexpression of Pq3-O-UGT1 led to increased accumulation of Pq3-O-UGT1 mRNA and a higher level of protopanaxadiol-group ginsenosides (especially for Rh2 and Rd) in transgenic hairy roots, whereas, RNA interference against Pq3-O-UGT1 in transgenic hairy roots resulted in the relative decrease of Pq3-O-UGT1 transcription and lower content of ginsenoside Rh2. Interestingly, both overexpression and RNAi upregulated the key upstream genes Pq-D12H and Pq-CYP6H, and there was no effect on the upstream gene of Pq-DS. Thus, these results indicate that Pq3-O-UGT1 is a key enzyme for the synthesis of ginsenoside Rh2 and plays a key role in the biosynthesis of protopanaxadiol-group ginsenosides in P. quinquefolius.

Anxiolytic- and antidepressant-like effects of an aqueous extract of Tanacetum parthenium L. Schultz-Bip (Asteraceae) in mice

AimTanacetum parthenium L. Schultz-Bip (Asteraceae) is widely used worldwide in traditional medicine for the treatment of convulsions and culture-bound syndromes such as susto (fear). The aim of this work was to evaluate the anxiolytic- and antidepressant-like effects of an aqueous extract of T. parthenium in behavioral paradigms in mice. The effects of T. parthenium were compared with those produced by anxiolytic and antidepressant drugs. We carried out the chemical characterization of the main constituents of T. parthenium. The involvement with the GABAergic and serotoninergic neurotransmitter systems were explored be means of synergic and antagonist experiments. Materials and methodsThe anxiolytic-like effect was evaluated using the Burying Behavior Test (BBT) and the Elevated Plus-Maze Test (PMT). The antidepressant-like effect was evaluated in the Forced Swimming Test (FST), and ambulatory activity was assessed in the Open Field Test (OFT). Employing the behavioral tests, synergism and antagonism experiments with Alprazolam, Muscimol, and Picrotoxin were carried out in the PMT. In a series of independent experiments, concomitant administration of T. parthenium and Alprazolam, Fluoxetine, or p-chlorophenylalanine were conducted in the FST.For chemical characterization, High-Performance Liquid Chromatography-Electro Spray Ionization-Mass Spectrometry (HPLC-ESI-MS) analysis was performed. ResultsT. parthenium exerts clear anxiolytic- and antidepressant-like effects in mice, without affecting the ambulatory activity of the experimental subjects. ConclusionsAnxiolytic- and antidepressant-like T. parthenium effects result, at least part from the involvement of the GABAergic system. Our results support the use of Tanacetum parthenium in traditional medicine and suggest its therapeutic potential in the comorbid anxiety and depression.

Tocopherols and Tocotrienols: an Adapted Methodology by UHPLC/MS Without Sample Pretreatment Steps

Ultra high-performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC/ESI/MS) methodology was adapted for identification and quantification of tocopherols and tocotrienols in vegetable oils with no derivatization or sample preparation steps. The UHPLC analysis was performed using a C18 column and mobile phase composed of methanol: water: ammonium hydroxide (99:1:0.1 v/v/v) and isopropanol. A single mass spectrometer with electrospray on negative mode was used as a detector for tocopherols and tocotrienols. The samples were diluted in isopropanol. The limit of quantification for tocopherols was 0.006 μg mL−1, and the linear range was 0.006 to 0.01 μg mL−1; for tocotrienols, the limit of quantification was 0.002 μg mL−1, and the linear range of analysis was 0.002 to 0.003 μg mL−1. The correlation coefficients were higher than 0.99, indicating that the method has suitable linearity. The methodology has proven to be precise, reproducible, and robust for the parameters studied.

Proanthocyanidins purified from fruit pericarp of Clausena lansium (Lour.) Skeels as efficient tyrosinase inhibitors: structure evaluation, inhibitory activity and molecular mechanism.

Fruit pericarp of Clausena lansium (Lour.) Skeels, a food waste, was selected as a raw material for proanthocyanidins. The proanthocyanidins' structures were integrally analyzed using three methods: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) and 13C nuclear magnetic resonance (NMR). The results elucidated that these compounds were composed of prodelphinidin (75%) and procyanidin (25%) with a degree of polymerization (DP) up to the 20-mers. They were proved to be remarkable, reversible and mixed competitive inhibitors of tyrosinase according to results from enzyme experiments. The IC50 values were calculated to be 23.6 ± 1.2 and 7.0 ± 0.2 μg mL-1 for the monophenolase and diphenolase activities, respectively. In addition, the proanthocyanidins had a good inhibitory effect on cell proliferation, cellular tyrosinase activity and melanin production of B16 mouse melanoma cells. Chelation between the hydroxyl group on the B ring of the proanthocyanidins and dicopper irons of the enzyme provided one of the feasible mechanisms for the inhibition on the basis of fluorescence quenching and molecular docking analyses. This research would supply the scientific basis to these compounds application in the pharmaceutical, insecticides, and preservative fields.

Determination of hexabromocyclododecanes in ambient air by high performance liquid chromatography- electrospray ionization-mass spectrometry

A high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method was developed for the determination of hexabromocyclododecanes (HBCDs) in ambient air samples. The samples were extracted by Soxhlet extractor with hexane, then purified on the composite gel column. At first, the interfering substances were rinsed with 50 mL hexane and 100 mL hexane-dichloromethane (9:1, v/v), then 180 mL hexane -dichloromethane (4:1, v/v) was used to elute the targets. The compounds were separated by gradient elution with acetonitrile-methanol-water as mobile phases on a UF-ODS column (150 mm×2.1 mm, 3.0 μm). Electrospray ionization negative ion source and selective ion monitoring (SIM) mode were adopted in MS detection. The results showed that α -HBCD, β -HBCD and γ -HBCD could be well separated, and the chromatographic peak area ratio of α -HBCD, β -HBCD and γ -HBCD to internal standard D18- γ -HBCD with their concentrations had a good linear relationship, with the correlation coefficients (R) ≥ 0.9988. The limits of detection (LODs, S/N=3) of α -HBCD, β -HBCD and γ -HBCD were 0.4, 0.5 and 0.4 μg/L, respectively. The limits of quantification (LOQs, S/N=10) were 1.4, 1.6 and 1.3 μg/L, respectively. The method detection limits (MDL) were 0.13, 0.17 and 0.13 pg/m3 (n=5), respectively. The recoveries of HBCDs spiked in the actual air samples were in the range of 74.8%-95.8%. It is demonstrated that the method has high sensitivity and good selectivity, and can meet the requirement of monitoring and analyzing HBCDs in air samples.

Optimization of Yield for Extraction of an Essential Oil from Cinnamon Using Microwave-Assisted Extraction

Sicklepod (Senna obtusifolia L.) is synonymous both with Cassia tora L. and Cassia obtusifolia L.). It is usually viewed as a noxious weed in crop fields in southeastern United States. This plant, however, has a long history of use in traditional medicine in oriental countries. It is a prolific seed producer the constituents of which include: polysaccharides, proteins, highly colored low fat content and many phenolic compounds. The phenolic components are usually described as toxic, although recent literature shows many of these to exhibit potent therapeutic properties for human health. Pursuant to this as part of our continuing interest in the plant, we have expanded our study of the seed by extracting a mixture of anthraquinone and naphthopyrone glycosides from S. obtusifolia seed obtained from North Carolina. A survey of the composition of the extract was affected using High Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (HPLC-ESI-MS) with a variety of collision induced dissociation (CID) experiments. The major constituents of the mixture produced HPLC-ESI-MSn data consistent with: chrysophanic acid tetraglycoside; rubrufusarin and toralactone di-, tri- and tetraglycosides; torachrysone ester, di-, tri-, tetra- and pentaglycosides and cassialactone tetraglycoside. The naphthopyrone glycosides and related phenolic compounds in the seed are value-added medicinal co-products to the galactomannan polysaccharides of S. obtusifolia. FT-IR spectra of the mixture corroborate the chromatographic information obtained of the mixture of anthraquinone glycosides.

Compatible Solutes in the Akinetes of the Terrestrial Cyanobacterium <i>Nostoc</i> sp. HK-01 Contribute to Its Heat Tolerance

The detailed mechanisms that facilitate the heat tolerance of terrestrial cyanobacteria have not been completely elucidated, although several reports have revealed aspects of the heat tolerance mechanisms of several other organisms. The dormant cells, called akinetes, of the terrestrial cyanobacterium Nostoc sp. HK-01 can revive after dry heat exposure at 100℃ for more than 10 h. We investigated the compatible solutes that protect the biomolecules in Nostoc sp. HK-01 akinetes using colonies containing various proportions of akinetes. We extracted the intracellular substances from each colony with 80% ethanol, which we purified with a series of analytical columns and analyzed by high-performance liquid chromatography and liquid chromatography-electrospray ionization-mass spectrometry. The compatible solutes were screened for their ability to prevent protein aggregation upon heating using the model enzyme lactate dehydrogenase. We detected an accumulation of glucosylglycerol, betaine, and glycine in akinetes. In addition, we confirmed that betaine, glycine, sucrose, and trehalose contributed to the prevention of the protein aggregation. The levels of sucrose and glycine in the colonies were approximately 1000× higher than those of glucosylglycerol, betaine, or trehalose. Our results indicated that sucrose and glycine are the main compatible solutes in the hydrophilic fractions of the cell extracts of Nostoc sp. HK-01 akinetes.

Spin-Trapping Analysis of Thermal Degradation Reaction of Poly(butylene terephthalate)

Poly(butylene terephthalate) (PBT), and its oligomer, were doped with the spin-trapping reagent, 2-methyl-2-nitrosopropane (MNP), by a solution-casting method in order to detect the short-lived radical intermediates during thermal degradation. In the case of PBT, two types of spin adduct were observed by electron spin resonance (ESR) spectroscopy upon stepwise heating from room temperature to 200 °C and during annealing at 130 °C; the ESR spectrum exhibited an isotropic three-line signal, with a hyperfine coupling constant (hfcc) aN ≈ 0.8 mT, as well as an anisotropic component. These adducts were present on the order of 10–5 mol/L. The isotropic component was assigned to arise uniquely from the benzoyl radical, •CO-ϕ. The anisotropic component was carefully investigated by ESR spectroscopy and high-performance liquid chromatography–electrospray ionization–mass spectrometry (HPLC-ESI-MS), using low-molecular-weight model compounds such as n-butyl benzoate (BB), di-n-butyl terephthalate (DBT), and deuterat...

Functional regulation of ginsenoside biosynthesis by RNA interferences of a UDP-glycosyltransferase gene in Panax ginseng and Panax quinquefolius

Panax ginseng (Asian ginseng) and Panax quinquefolius (American ginseng) have been used as medicinal and functional herbal remedies worldwide. Different properties of P. ginseng and P. quinquefolius were confirmed not only in clinical findings, but also at cellular and molecular levels. The major pharmacological ingredients of P. ginseng and P. quinquefolius are the triterpene saponins known as ginsenosides. The P. ginseng roots contain a higher ratio of ginsenoside Rg1:Rb1 than that in P. quinquefolius. In ginseng plants, various ginsenosides are synthesized via three key reactions: cyclization, hydroxylation and glycosylation. To date, several genes including dammarenediol synthase (DS), protopanaxadiol synthase and protopanaxatriol synthase have been isolated in P. ginseng and P. quinquefolius. Although some glycosyltransferase genes have been isolated and identified association with ginsenoside synthesis in P. ginseng, little is known about the glycosylation mechanism in P. quinquefolius. In this paper, we cloned and identified a UDP-glycosyltransferase gene named Pq3-O-UGT2 from P. quinquefolius (GenBank accession No. KR106207). In vitro enzymatic activity experiments biochemically confirmed that Pq3-O-UGT2 catalyzed the glycosylation of Rh2 and F2 to produce Rg3 and Rd, and the chemical structure of the products were confirmed susing high performance liquid chromatography electrospray ionization mass spectrometry (HPLC/ESI-MS). High sequence similarity between Pq3-O-UGT2 and PgUGT94Q2 indicated a close evolutionary relationship between P. ginseng and P. quinquefolius. Moreover, we established both P. ginseng and P. quinquefolius RNAi transgenic roots lines. RNA interference of Pq3-O-UGT2 and PgUGT94Q2 led to reduce levels of ginsenoside Rd, protopanaxadiol-type and total ginsenosides. Expression of key genes including protopanaxadiol and protopanaxatriol synthases was up-regulated in RNAi lines, while expression of dammarenediol synthase gene was not obviously increased. These results revealed that P. quinquefolius was more sensitive to the RNAi of Pq3-O-UGT2 and PgUGT94Q2 when compared with P. ginseng.

Determination of N-glycans in glycoproteins using chemoenzymatic labeling with Endo-M N175Q

A novel approach for the determination of N-glycans in glycoproteins has been developed based on a combination of deglycosylation, chemoenzymatic labeling, and high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). Glycoproteins were digested with PNGase F and then the released N-glycans were enriched and separated from the digesting solution using ultrafiltration units according to the molecular mass differences. The N-glycans were analyzed by HPLC/ESI-MS after chemoenzymatic labeling with Endo-M N175Q an Endo-M mutant. We have shown that the Endo-M N175Q demonstrates a noticeable transglycosidase-like activity for natural N-glycans. The productivity of Endo-M N175Q is estimated to be 2.0-fold higher than that of Endo-M. The experimental strategy was validated using glycoproteins with known glycan structures such as ovalbumin and ribonuclease B. The proposed method offers a promising advance for determining N-glycans in glycoproteins.

Pharmacokinetics and Bioequivalence of Methotrexate in Human Plasma Studied by Liquid Chromatography-Mass Spectrometry (LC-MS)

Background: Methotrexate is a folic acid antagonist that has been in clinical use for five decades. Its use in patients with brain tumors is primarily confined to primary central nervous system lymphoma (PCNSL), in which it is the cornerstone of chemotherapy. Objectives: A selective and sensitive high-performance liquid chromatography-electrospray ionization-mass spectrometry method was developed for the determination of methotrexate in human plasma. Materials and Methods: Methotrexate was extracted from plasma with acetonitrile. The mobile phase consisted of acetonitrile/water/formic acid 74:25:1 (v/v/v), and 20 µL of the sample was chromatographically analyzed using a repacked ZORBAX-XDB-ODS C18 column (2.1 × 30 mm, 3.5 microns). The mode of mass spectrometry was selected-ion monitoring (SIM). The standard curve was linear (r = 0.998) over a concentration range of 0.1 - 100.0 ng/mL, and showed suitable accuracy and precision. Results: The limit of detection (LOD) was 0.05 ng/mL. The mean (± SD) Cmax, Tmax, AUC0-t, and AUC0–∞ values after administration of the test and reference formulations, respectively, were as follows: 13.94 (± 4.36) versus 13.49 (± 3.67) ng/mL, 2.63 (± 1.45) versus 2.75 (± 1.74) hours, 122.57 (± 54.34) versus 125.94 (± 53.09) ng/mL/h, and 140.74 (± 56.69) versus 155.80 (± 65.11) ng/mL/h. The mean (± SD) t1/2 was 5.32 (± 2.01) hours for the test formulation and 5.34 (± 2.13) hours for the reference formulation. No statistical differences were shown for Cmax or the area under the plasma concentration-time curve for the test and reference tablets. The calculated 90% confidence intervals, based on ANOVA analysis for the mean test/reference ratios of Cmax, AUC0-∞, and AUC0-th of methotrexate, were in the bioequivalence range (96% - 101%). Conclusions: The developed LC-MS method is quick, easy, stable, and precise for the partition, assignment, pharmacokinetic, and bioavailability evaluation of methotrexate in healthy Iranian adult male volunteers.

Distribution of glucosinolates in camelina seed fractions by HPLC‐ESI‐MS/MS

High glucosinolate concentrations were detected and identified in camelina (Camelina sativa L. Crantz.) seed fractions using reversed phase high performance liquid chromatography‐electrospray ionization‐mass spectrometry (HPLC‐ESI‐MS) in multiple reaction monitoring mode. Total glucosinolate quantitation was performed using proton nuclear magnetic resonance spectrometry using N,N‐dimethylformamide as an internal standard. Individual glucosinolate concentrations were determined by analysis on extracted ion chromatograms generated by a MS detector. Distribution of glucosinolates in C. sativa seed fractions during pressing is reported.Practical applications: HPLC‐ESI‐MS and HPLC‐ESI‐MS/MS using monolithic HPLC columns were used to detect, quantify, and identify the three major glucosinolates in Camelina sativa L. and fractions of C. sativa produced by processing. Quantitative extracted ion MS chromatograms afforded excellent quantitation of individual glucosinolates. The use of monolithic HPLC columns enabled rapid analysis of these samples.Schematic of a proposed dehulling system for Camelina sativa seed.

Extraction and Separation of Active Ingredients in Schisandra chinensis (Turcz.) Baill and the Study of their Antifungal Effects

Schisandra chinensis extracts (SEs) have traditionally been used as an oriental medicine for the treatment of various human diseases, however, their further application in the biocontrol of plant disease remains poorly understood. This study was conducted to develop eco-friendly botanical pesticides from extracts of S. chinensis and assess whether they could play a key role in plant disease defense. Concentrated active fractions (SE-I, SE-II, and SE-III) were obtained from S. chinensis via specific extraction and separation. Then, lignan-like substances, such as Schisanhenol B, were detected via High-Performance Liquid Chromatography-ElectroSpray Ionization-Mass Spectrometry (HPLC-ESI-MS) analyses of the active fractions. Moreover, the results from biological tests on colony growth inhibition and spore germination indicated that SE-I, SE-II, and SE-III could inhibit hyphal growth and spore generation of three important plant pathogenic fungi (Monilinia fructicola, Fusarium oxysporum, and Botryosphaeria dothidea). The study of the mechanisms of resistant fungi revealed that the oxidation resistance system, including reactive oxygen species (ROS), malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD), was activated. The expression of genes related to defense, such as pathogenesis-related protein (PR4), α-farnesene synthase (AFS), polyphenol oxidase (PPO), and phenylalanine ammonia lyase (PAL) were shown to be up-regulated after treatment with SEs, which suggested an increase in apple immunity and that fruits were induced to effectively defend against the infection of pathogenic fungi (B. dothidea). This study revealed that SEs and their lignans represent promising resources for the development of safe, effective, and multi-targeted agents against pathogenic fungi.

Preparation of Methylated Products of A-type Procyanidin Trimers in Cinnamon Bark and Their Protective Effects on Pancreatic β-Cell.

Polyphenols are partial metabolized to methylated conjugations in vivo, and then could modify bioavailability and bioactivity related to the uptake of parent compounds. Our previous studies have found that the antidiabetic effects of cinnamon barks are mainly related to polyphenol components, particularly A-type procyanidin trimer cinnamtannin-1 (CT1). It is necessary to understand the antidiabetic activity of methylations of CT1, nevertheless, sufficient amounts of methylated CT1 are difficult to obtain from metabolites in vivo. In this study, O-methyl derivatives of CT1 were prepared through one-pot methyl iodide reaction and isolation via column chromatography and RP-HPLC semipreparation. The structures of O-methyl substituents were determined through NMR (Nuclear Magnetic Resonance) and HPLC-ESI-MS (High-performance liquid chromatography-electrospray ionization-mass spectrometry). Five purified O-methyl substituents and 2 isomers of CT1 were obtained. Their protective effects on a palmitic acid-induced pancreatic β-cell apoptosis model were then evaluated. Results showed that the protective effects on pancreatic β-cell of O-methyl substituents were weaker than those of CT1. The results suggested that the methylation of catechol groups could be a relevant factor contributing to the decline of protective effects on pancreatic β-cell of CT1 via obstructing quinone intermediate formation and affecting antioxidant abilities. The antidiabetic effects of O-methyl derivatives of CT1 should be further determined by other antidiabetic models.

Simultaneous determination of nine bioactive compounds in Yijin-tang via high-performance liquid chromatography and liquid chromatography-electrospray ionization-mass spectrometry

BackgroundYijin-tang (YJ) has been used traditionally for the treatment of cardiovascular conditions, nausea, vomiting, gastroduodenal ulcers, and chronic gastritis. In this study, a simple and sensitive high-performance liquid chromatography (HPLC) method was developed for the quantitation of nine bioactive compounds in YJ: homogentisic acid, liquiritin, naringin, hesperidin, neohesperidin, liquiritigenin, glycyrrhizin, 6-gingerol, and pachymic acid. MethodsChromatographic separation of the analytes was achieved on an RS Tech C18 column (4.6mm × 250mm, 5μm) using a mobile phase composed of water containing 0.1% (v/v) trifluoroacetic acid (TFA) and acetonitrile with a gradient elution at a flow rate of 1.0mL/min. ResultsCalibration curves for all analytes showed good linearity (R2 ≥ 0.9995). Lower limits of detection and lower limits of quantification were in the ranges of 0.03–0.17μg/mL and 0.09–0.43μg/mL, respectively. Relative standard deviations (RSDs; %) for intra- and interday assays were < 3%. The recovery of components ranged from 98.09% to 103.78%, with RSDs (%) values ranging from 0.10% to 2.59%. ConclusionThis validated HPLC method was applied to qualitative and quantitative analyses of nine bioactive compounds in YJ and fermented YJ, and may be a useful tool for the quality control of YJ.

HepG2 cells biospecific extraction and HPLC-ESI-MS analysis for screening potential antiatherosclerotic active components in Bupeuri radix

HepG2 cells biospecific extraction method and high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) analysis was proposed for screening of potential antiatherosclerotic active components in Bupeuri radix, a well-known Traditional Chinese Medicine (TCM). The hypothesis suggested that when cells are incubated together with the extracts of TCM, the potential bioactive components in the TCM should selectively combine with the receptor or channel of HepG2 cells, then the eluate which contained biospecific component binding to HepG2 cells was identified using HPLC-ESI-MS analysis. The potential bioactive components of Bupeuri radix were investigated using the proposed approach. Five compounds in the saikosaponins of Bupeuri radix were detected as these components selectively combined with HepG2 cells, among these compounds, two potentially bioactive compounds namely saikosaponin b1 and saikosaponin b2 (SSb2) were identified by comparing with the chromatography of the standard sample and analysis of the structural clearance characterization of MS. Then SSb2 was used to assess the uptake of DiI-high density lipoprotein (HDL) in HepG2 cells for antiatherosclerotic activity. The results have showed that SSb2, with indicated concentrations (5, 15, 25, and 40μM) could remarkably uptake dioctadecylindocarbocyanine labeled- (DiI) -HDL in HepG2 cells (Vs control group, *P<0.01). In conclusion, the application of HepG2 biospecific extraction coupled with HPLC-ESI-MS analysis is a rapid, convenient, and reliable method for screening potential bioactive components in TCM and SSb2 may be a valuable novel drug agent for the treatment of atherosclerosis.

Proanthocyanidins Extracted from Rhododendron pulchrum Leaves as Source of Tyrosinase Inhibitors: Structure, Activity, and Mechanism.

The objective of this study was to assess the structure, anti-tyrosinase activity, and mechanism of proanthocyanidins extracted from Rhododendron pulchrum leaves. Results obtained from mass spectra of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) revealed that proanthocyanidins were complex mixtures of procyanidins, prodelphinidins, propelargonidins, and their derivatives, among which procyanidins were the main components. The anti-tyrosinase analysis results indicated that the mixtures were reversible and mixed competitive inhibitors of tyrosinase. Interactions between proanthocyanidins with substrate (L-tyrosine and 3,4-dihydroxyphenylalanine) and with copper ions were the important molecular mechanisms for explaining their efficient inhibition. This research would provide scientific evidence for the use of R. pulchrum leaf proanthocyanidins as new novel tyrosinase inhibitors.

Submerged fermentation production and characterization of intracellular triterpenoids from Ganoderma lucidum using HPLC-ESI-MS.

As the main bioactive metabolites of Ganoderma lucidum, triterpenoids have various pharmacological effects. In this paper, the nutritional requirements and culture conditions of a submerged culture of G. lucidum were optimized using the response surface methodology; maximum mycelia biomass and intracellular triterpenoid production reached 1.87 g/100 ml and 93.21 mg/100 ml, respectively, for a culture consisting of wort 4.10% (0.041 g/ml) and yeast extract 1.89% (0.0189 g/ml), pH 5.40. For the first time, we established that wort, which is cheap and abundant, can replace the more commonly used glucose as the sole source of carbohydrate. Using high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS), 10 major ganoderic acids were tentatively identified based on the predominant fragmentation pathways with the elimination of H2O and CO2, as well as cleavage of the D-ring.

Studies on intestinal transport of ginsenoside compatibility with Veratrum nigrum via Caco-2 cell monolayer model coupled with UPLC-ESI-MS method

The Caco-2 cells have been recognized as effective tools to be applied to imitating the drug absorption in human intestine for the transport of drug. Herein, Caco-2 cell monolayer model was used to study the transport of the ginsenoside compatibility with Veratrum nigrum in different proportions. A specific high performance liquid chromatography-electrospray ionization-mass spectrometry(HPLC-ESI-MS) method was developed for the semiquantitative determination of ginsenoside in intestinal transport with Dioscin as an internal standard. For the Caco-2 model constructed, two influencing factors were investigated, including time and concentration. The results suggest that the absorption of ginsenoside Re, Rg1, Rb1, Rc, Rb2 and Rd are time- and concentration-dependent and the excretions of Rb1, Rc, Rb2 and Rd have a relatronship with some transport proteins. The bioavailability of the ginsenosides has reduced compared to the single Panax ginseng extract when compatibility with a certain amount of Veratrum nigrum.

Modified HPLC-ESI-MS Method for Glycated Hemoglobin Quantification Based on the IFCC Reference Measurement Procedure and Its Application for Quantitative Analyses in Clinical Laboratories of China.

The level of glycated hemoglobin (HbA1c ) has been recognized as an important indicator of long-term glycemic control. However, the HbA1c measurement is not currently included as a diagnostic determinant in China. Current study aims to assess a candidate modified International Federation of Clinical Chemistry reference method for the forthcoming standardization of HbA1c measurements in China. The HbA1c concentration was measured using a modified high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method. The modified method replaces the propylcyanide column with a C18 reversed-phase column, which has a lower cost and is more commonly used in China, and uses 0.1% (26.5 mmol/l) formic acid instead of trifluoroacetic acid. Moreover, in order to minimize matrix interference and reduce the running time, a solid-phase extraction was employed. The discrepancies between HbA1c measurements using conventional methods and the HPLC-ESI-MS method were clarified in clinical samples from healthy people and diabetic patients. Corresponding samples were distributed to 89 hospitals in Beijing for external quality assessment. The linearity, reliability, and accuracy of the modified HPLC-ESI-MS method with a shortened running time of 6 min were successfully validated. Out of 89 hospitals evaluated, the relative biases of HbA1c concentrations were < 8% for 74 hospitals and < 5% for 60 hospitals. Compared with other conventional methods, HbA1c concentrations determined by HPLC methods were similar to the values obtained from the current HPLC-ESI-MS method. The HPLC-ESI-MS method represents an improvement over existing methods and provides a simple, stable, and rapid HbA1c measurement with strong signal intensities and reduced ion suppression.