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

Catalytic reduction of 4,4′-(2,2,2-trichloroethane-1,1-diyl)bis(chlorobenzene) (DDT) with nickel(I) salen electrogenerated at vitreous carbon cathodes in dimethylformamide

Cyclic voltammetry, controlled-potential (bulk) electrolysis, gas chromatography, gas chromatography–mass spectrometry, and high-performance liquid chromatography–electrospray ionization–mass spectrometry have been utilized to investigate the catalytic reduction of 4,4′-(2,2,2-trichloroethane-1,1-diyl)bis(chlorobenzene) (DDT) by nickel(I) salen electrogenerated at a carbon cathode in dimethylformamide (DMF) containing 0.050M tetramethylammonium tetrafluoroborate (TMABF4). Cyclic voltammograms for reduction of nickel(II) salen in the presence of DDT provide evidence for catalytic reduction such as enhanced cathodic current for the nickel(II) salen–nickel(I) salen redox couple and a decrease in the anodic current associated with oxidation of nickel(I) salen. Bulk electrolysis of nickel(II) salen–DDT solutions at reticulated vitreous carbon cathodes leads to a mixture of products that includes 1,1-diphenylethene, 1-chloro-4-(1-phenylvinyl)benzene, 4,4′-(ethene-1,1-diyl)bis(chlorobenzene) (DDNU), 4,4′-(2-chloroethene-1,1-diyl)bis(chlorobenzene) (DDMU), 4,4′-(2-chloroethane-1,1-diyl)bis(chlorobenzene) (DDMS), 4,4′-(2,2-dichloroethane-1,1-diyl)bis(chlorobenzene) (DDD), and 4,4′-(2,2-dichloroethene-1,1-diyl)bis(chlorobenzene) (DDE). In addition, we have detected adducts formed from a fragment of DMF and radical intermediates arising from reduction of DDT, along with nickel salen species for which the imino (CN) bond of the ligand is modified with different intermediates derived from catalytic reduction of DDT. A mechanistic scheme is proposed to account for the formation of products.

Characterization of alkyl thiosulfinate in Allium hookeri root using HPLC-ESI-MS

Allicin produced by alliinase system of Allium hookeri was evaluated via high performance liquid chromatography (HPLC). Allicin contents of A. hookeri were 56.6±3.5 μg per g of fresh root and 12.7±3.2 μg per g of fresh stem. These values were relatively low as compared with garlic. HPLC-electrospray ionization-mass spectrometry analyses showed A. hookeri root extract contained ten alkyl thiosulfinates, and the chemical structures were characterized by MS/MS analyses.

Preventive effects of Morus alba L. anthocyanins on diabetes in Zucker diabetic fatty rats

The mulberry plant (Morus alba L.) contains abundant anthocyanins (ANCs), which are natural antioxidants. The aim of this study was to determine the ANC composition of Thai Morus alba L. fruits and to assess the effect of an ANC extract on blood glucose and insulin levels in male leptin receptor-deficient Zucker diabetic fatty (ZDF) rats. The major components of the ANC extract were identified by high-performance liquid chromatography-electrospray ionization-mass spectrometry. ZDF and lean rats were treated with 125 or 250 mg ANCs/kg body weight, or 1% carboxymethylcellulose (CMC) twice daily for 5 weeks. Neither ANC dose had an effect on body weight. Following 5 weeks of treatment, glucose levels were observed to increase from 105.5±8.7 to 396.25±21 mg/dl (P<0.0001) in the CMC-treated ZDF rats; however, the glucose levels were significantly lower in the rats treated with 125 or 250 mg/kg ANCs (228.25±45 and 131.75±10 mg/dl, respectively; P<0.001 versus CMC). The administration of 250 mg/kg ANCs normalized glucose levels in the ZDF rats towards those of the lean littermates. Insulin levels were decreased significantly in the ZDF rats treated with CMC or 125 mg/kg ANCs (P<0.0001), but not in the rats treated with 250 mg/kg ANCs. Histologically, 250 mg/kg ANCs was observed to prevent islet degeneration compared with the islets in CMC-treated rats. This study, demonstrated that ANCs extracted from Morus alba L. were well tolerated and exhibited effective anti-diabetic properties in ZDF rats. ANCs represent a promising class of therapeutic compounds that may be useful in the prevention of type 2 diabetes.

Physicochemical and Functional Comparability Between the Proposed Biosimilar Rituximab GP2013 and Originator Rituximab

BackgroundRegulatory approval for a biosimilar product is provided on the basis of its comparability to an originator product. A thorough physicochemical and functional comparability exercise is a key element in demonstrating biosimilarity. Here we report the characterization of a proposed biosimilar rituximab (GP2013) and originator rituximab.ObjectiveTo compare GP2013 with originator rituximab using an extensive array of routine analytical and extended characterization methods.MethodsPrimary and higher order protein structures were analyzed using a variety of methods that included high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS), peptide mapping with UV and MS detection, circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, hydrogen deuterium exchange (HDX) MS, 1D 1H nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and differential scanning calorimetry (DSC). Charge and amino acid modifications were assessed using cation exchange chromatography (CEX) and peptide mapping using reversed-phase (RP) HPLC. Boronate affinity chromatography was used to determine the relative amount of glycation. Glycans were identified and quantified after 2-aminobenzamide (2-AB) labeling and separation using normal phase HPLC with fluorescence and MS detection, respectively. Glycan site occupancy was determined using reducing capillary electrophoresis with sodium dodecyl sulfate (CE-SDS). Size heterogeneity was determined using reducing and non-reducing CE-SDS, size exclusion chromatography (SEC) and asymmetric flow field flow fractionation (AF4). Biological characterization included a series of bioassays (in vitro target binding, antibody-dependent cell-mediated cytotoxicity [ADCC], complement-dependent cytotoxicity [CDC] and apoptosis) and surface plasmon resonance (SPR) Fc receptor binding assays.ResultsIntact mass analysis of GP2013 and the heavy and light chains using RP HPLC–ESI–MS revealed the expected molecular mass of rituximab. The amino acid sequence was shown to be identical between GP2013 and the originator rituximab. Further sequence confirmation using RP-HPLC-UV/MS peptide mapping showed non-distinguishable chromatograms for Lys-C digested GP2013 and originator rituximab. The higher order structure of GP2013 was shown to be indistinguishable from originator rituximab using a large panel of redundant and orthogonal methods. GP2013 and originator rituximab were comparable with regard to charge variants, specific amino acid modifications and the glycan pattern. GP2013 was also shown to have similar purity, aggregate and particle levels when compared with the originator. Functionally, and by using a comprehensive set of bioassays and binding assays covering a broad range of rituximab’s functional activities, GP2013 could not be distinguished from originator rituximab.ConclusionGP2013 was shown to be physicochemically highly similar to originator rituximab at the level of primary and higher order structure, post-translational modifications and size variants. An extensive functional characterization package indicated that GP2013 has the same biological properties as originator rituximab.

Association of high levels of α-defensins and S100A proteins with Candida mannan detection in bronchoalveolar lavage fluid of preterm neonates

Candida mannan (Mn) detection in bronchoalveolar lavage fluid (BALF) was shown to be useful for earlier identification and preemptive therapy targeting in preterm infants at high risk of invasive Candida infection. We investigated whether early detection of Candida Mn in BALF is associated with the presence of some neutrophilic products, as markers of prenatal infection/inflammation. BALF specimens were collected during the first 48 h of life from mechanically ventilated preterm newborns. Samples were analyzed by high-performance liquid chromatography-electrospray ionization-mass spectrometry. The relative amounts of α-defensins 1-4 and S100A proteins were measured by extracted ion current peak area. Absolute and differential white cell counts in BALF were obtained. Mn antigen concentrations were determined by the Platelia Candida antigen kit. Twenty-five studied neonates were divided into two groups: Mn-positive group and Mn-negative group. Levels of α-defensins 1-4 and S100A12 were significantly higher in the Mn-positive group than in the Mn-negative group. Moreover, positive significant correlations between the absolute number of neutrophils and the levels of α-defensins 1-4 and S100A8 were observed. The detection of Mn antigen in BALF of preterm infants is consistent with evidence of an innate immune response in their lungs as demonstrated by higher levels of α-defensins and S100A proteins.

Genomic DNA in human blastocoele fluid

IVF often requires embryo cryopreservation through vitrification. During the vitrification process, the embryos can be collapsed by withdrawing the blastocoele fluid. The metabolomic profile of blastocoele fluid has been recently investigated by high-performance liquid chromatography-electrospray ionization-mass spectrometry to provide metabolite information that can help estimations of implantation efficiency. However, the presence of embryo DNA in blastocoele fluid has not been reported to date. This study shows using real-time PCR that genomic DNA was present in about 90% of blastocoele fluid samples harvested during the vitrification procedure. Moreover, the potential for determining embryo sex directly from blastocoele fluid is demonstrated by amplifying the multicopy genes TSPY1 (on the Y chromosome) and TBC1D3 (on chromosome 17). This opens up the possibility of screening embryos from couples carrying an X-linked disorder to identify male embryos at high risk of disease. The application of whole-genome amplification technologies to fluid samples is also shown to be feasible, potentially allowing more comprehensive genetic tests. As proof of principle, microarray comparative genomic hybridization was attempted to confirm the sex of embryos as well as detect several aneuploidies. However, further studies are needed to validate this approach and confirm that the accuracy is sufficient for diagnostic purposes.IVF offers personal approaches to couples according to their specific infertility problems. IVF includes a preimplantation genetic diagnosis, single-embryo transfer, blastocyst transfer and optimization of uterine receptivity. These procedures require a valid embryo cryopreservation system. Vitrification is a cryopreservation method that offers superior survival and pregnancy rates performing an artificial shrinkage of a fully expanded blastocyst. This procedure can be performed with different methods, including laser-pulse, repeated micropipetting, microneedle puncture or microsuction, and there is a significant improvement in survival rates when blastocysts were collapsed. With microsuction, the blastocoele fluid is withdrawn and (typically) discarded. The aim of this study was to investigate the presence of DNA in blastocoele fluid. Recently, the metabolomic profile of blastocoele fluid was characterized, but to date, no studies have reported evidence of DNA. Here, using a real-time PCR-based approach, we showed that genomic DNA was amplifiable in about 90% blastocoele fluid samples analysed. Moreover, we demonstrated the potential for monitoring sex-linked diseases directly from blastocoele fluid, by amplifying the multicopy genes TSPY1 (on the Y chromosome) and TBC1D3 (on chromosome 17). The application of whole-genome amplification technologies to fluid samples has also shown to be feasible, potentially allowing more comprehensive genetic tests (i.e. microarray comparative genomic hybridization). However, further studies are needed to validate this approach for reliable diagnostic purposes.

A Novel 3-Sulfinopropionyl Coenzyme A (3SP-CoA) Desulfinase from Advenella mimigardefordensis Strain DPN7 T Acting as a Key Enzyme during Catabolism of 3,3′-Dithiodipropionic Acid Is a Member of the Acyl-CoA Dehydrogenase Superfamily

3-Sulfinopropionyl coenzyme A (3SP-CoA) desulfinase (AcdDPN7) is a new desulfinase that catalyzes the sulfur abstraction from 3SP-CoA in the betaproteobacterium Advenella mimigardefordensis strain DPN7(T). During investigation of a Tn5::mob-induced mutant defective in growth on 3,3'-dithiodipropionate (DTDP) and also 3-sulfinopropionate (3SP), the transposon insertion was mapped to an open reading frame with the highest homology to an acyl-CoA dehydrogenase (Acd) from Burkholderia phenoliruptrix strain BR3459a (83% identical and 91% similar amino acids). An A. mimigardefordensis Δacd mutant was generated and verified the observed phenotype of the Tn5::mob-induced mutant. For enzymatic studies, AcdDPN7 was heterologously expressed in Escherichia coli BL21(DE3)/pLysS by using pET23a::acdDPN7. The purified protein is yellow and contains a noncovalently bound flavin adenine dinucleotide (FAD) cofactor, as verified by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) analyses. Size-exclusion chromatography revealed a native molecular mass of about 173 kDa, indicating a homotetrameric structure (theoretically 179 kDa), which is in accordance with other members of the acyl-CoA dehydrogenase superfamily. In vitro assays unequivocally demonstrated that the purified enzyme converted 3SP-CoA into propionyl-CoA and sulfite (SO3(2-)). Kinetic studies of AcdDPN7 revealed a Vmax of 4.19 μmol min(-1) mg(-1), an apparent Km of 0.013 mM, and a kcat/Km of 240.8 s(-1) mM(-1) for 3SP-CoA. However, AcdDPN7 is unable to perform a dehydrogenation, which is the usual reaction catalyzed by members of the acyl-CoA dehydrogenase superfamily. Comparison to other known desulfinases showed a comparably high catalytic efficiency of AcdDPN7 and indicated a novel reaction mechanism. Hence, AcdDPN7 encodes a new desulfinase based on an acyl-CoA dehydrogenase (EC 1.3.8.x) scaffold. Concomitantly, we identified the gene product that is responsible for the final desulfination step during catabolism of 3,3'-dithiodipropionate (DTDP), a sulfur-containing precursor substrate for biosynthesis of polythioesters.

Phytochemical Compositions and Antioxidant and Anti-Inflammatory Activities of Crude Extracts fromFicus pandurataH. (Moraceae)

Background. Ficus pandurata H. (Moraceae) is widely used in traditional Chinese medicine as a healthy food condiment or a medicine for treatment of various diseases including inflammation. Objective. The purpose of the present study is to investigate the phytochemical compositions and antioxidant and anti-inflammatory activities of crude water (FPW) and ethanolic extracts (FPE) from Ficus pandurata H. Methods. Phytochemical compositions were identified by a high-performance liquid chromatography-electrospray ionization-mass spectrometry method (HPLC-ESI-MS). The antioxidant activities were evaluated by diphenylpicrylhydrazyl (DPPH) and hydroxyl radical assays, and the anti-inflammatory activities were evaluated by paw edema and levels of inflammatory mediator TNF-α and PGE2 in monosodium urate (MSU) crystal-induced rats. Results. Six compounds were identified by HPLC-MS method, and abundance of phenolics was found in FPE. The FPE showed concentration-dependent-significant scavenging of DPPH and hydroxyl radicals with IC50 values 118.4 and 192.9 μg/mL, respectively. The FPE treatment significantly inhibited the paw edema and the production of TNF-α and PGE2 in MSU crystal-induced rats. Conclusion. The FPE exerted stronger antioxidant and anti-inflammatory activities which may be attributed to its high phenolic content.

Characterisation of flavonoids in anthurium spathes and their contribution to spathe colouration

SummaryAnthurium cultivars are popular worldwide, with flowers having red, pink, coral, white, green, or brown spathes. Anthocyanins are important pigments in the anthurium spathe; however, the presence of other flavonoids has not yet been clarified. Three anthocyanins, seven flavone glycosides, and four flavonol glycosides were characterised using high–performance liquid chromatography–electrospray ionisation–mass spectrometry (HPLC–ESI–MS) in the spathe of 25 anthurium cultivars. Correlation analyses and multivariate analyses were then used to explore the relationships between flavonoid composition and spathe colour. The content of anthocyanin cyanidin 3-rutinoside (Cy3R), and the ratio of Cy3R content to pelargonidin 3-rutinoside (Pg3R) content were negatively correlated with spathe lightness (L*), and positively correlated with the chromatic component a*. The value of the chromatic component b* was positively correlated with the contents of several flavonols. This indicated that the anthocyanins Cy3R and Pg3R were the key factors responsible for the red, pink, and purple spathe colours. Flavonols and flavones accounted for the yellow hue of the spathe. This is the first report on flavonoid characterisation in the genus Anthurium using HPLC–ESI–MS. This work will be valuable for elucidating the flavonoid compositions of other anthurium spathes and their effects on the colouration of the spathe.

Analysis of two new degradation products of arsenic triglutathione in aqueous solution

Inorganic arsenicals, including arsenite (AsIII) and arsenate (AsV), are well-known human carcinogens. Recently, studies have indicated that arsenic triglutathione (As(GS)3) is unstable in an aqueous solution. The present study was designed to evaluate the degradation mechanism of As(GS)3 in an aqueous solution using high-performance liquid chromatography-electrospray ionisation mass spectrometry (HPLC-ESI-MS). Based on the fragments obtained from MS2 and MS3, we identified two new compounds: one was an isomer of glutathione (GSH), and the other was a product from the cleavage of the glutamyl of oxidised glutathione (GSSG). The isomerization of GSH resulted in the loss of its function such as detoxification of many reactive metabolites. The formation of the two new compounds affected the ratio of GSH/GSSG, and thus may affect the antioxidant and detoxification of GSH/GSSG in mammalian cells.

Isolation and determination of major anthocyanin pigments in the pericarp of P. communis L. cv. ‘Red Du Comices’ and their association with antioxidant activity

The anthocyanins in the fruit skin of Pyrus communis cv. ‘Red Du Comice’ were isolated, identified and quantified using high-performance liquid chromatography/diode array detection (HPLC/DAD) and HPLC/electrospray ionization/mass spectrometry (HPLC/ESI/MS). The individual anthocyanins were identified by comparing their mass spectral data and retention times with those of standards and published data. Cyanidin-3-galactoside was the major compound, taking up to 93% of the total anthocyanin content. The content of Cyanidin-3-galactoside reached 23.7 ± 3.2 mg 100 g-1 fresh weight, followed by the eighth peak (2.14%) and Cyanidin-3-glucoside (1.23%). Pelargonidin-3-rutinoside (pg-3-rutinoside) was identified for the first time in P. communis L. 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays showed that the extract from the fruit peel of ‘Red Du Comice’ contained anthocyanins and possessed high antioxidant capacity. Key words: Pyrus communis, anthocyanin, high-performance liquid chromatography-electrospray ionization-mass spectrometry, antioxidant activity.

Quantification of HPLC-separated peptides and proteins by spectrofluorimetric detection of native fluorescence and mass spectrometry

Due to relatively low reproducibility of the ionization and differences when using buffers as mobile phases, the quantitative analysis by electrospray ionization mass spectrometry (ESI-MS) can be often challenging. In the present study, the native fluorescence of phenylalanine, tyrosine, and tryptophan was investigated as an improvement tool for the analytical quantification of peptides and proteins by HPLC–ESI-MS. Natively fluorescent amino acids as well as peptides, proteins, and protein digests were successfully separated by HPLC, and quantified with a spectrofluorimetric detector and ESI-MS. The two detectors were connected in series and enabled the sequential measurements of the fluorescence intensities as well as the measurements of the ion signals and mass spectral characterization of separated polypeptides. Fluorescence detector provided better linearity and repeatability of quantification than mass spectrometer, and similar limits of detection for most of biomolecules analyzed. The fluorescence signal was linear over 3–4 orders of magnitude with limits of detection in picomole or high femtomole range, depending on nature and number of natively fluorescent amino acid residues present in the analyzed polypeptides. Hence, native fluorescence of phenylalanine, tyrosine, and tryptophan can be used as a label-free methodology to facilitate quantification of peptides and proteins by LC–ESI-MS.

Optimization of the Extraction of Anthocyanins from the Fruit Skin of Rhodomyrtus tomentosa (Ait.) Hassk and Identification of Anthocyanins in the Extract Using High-Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (HPLC-ESI-MS)

Anthocyanins are naturally occurring polyphenols that impart bright color to fruits, vegetables and plants. In this study, the extraction of anthocyanins from freeze-dried fruit skin of downy rose-myrtle (Rhodomyrtus tomentosa (Ait.) Hassk var. Gangren) was optimized using response surface methodology (RSM). Using 60% ethanol containing 0.1% (v/v) hydrochloric acid as extraction solvent, the optimal conditions for maximum yields of anthocyanin (4.358 ± 0.045 mg/g) were 15.7:1 (v/w) liquid to solid ratio, 64.38 °C with a 116.88 min extraction time. The results showed good fits with the proposed model for the anthocyanin extraction (R2 = 0.9944). Furthermore, the results of high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) analysis of the anthocyanins extracted from the fruit skin of downy rose-myrtle revealed the presence of five anthocyanin components, which were tentatively identified as delphinidin-3-glucoside, cyanidin-3-glucoside, peonidin-3-glucoside, petunidin-3-glucoside and malvidin-3-glucoside.

Direct and nickel(I) salen-catalyzed reduction of 1,1,2-trichloro-1,2,2-trifluoroethane (CFC-113) in dimethylformamide

Cyclic voltammetry, controlled-potential (bulk) electrolysis, gas chromatography (GC), gas chromatography–mass spectrometry (GC–MS), and high-performance liquid chromatography–electrospray ionization–mass spectrometry (HPLC–ESI–MS) have been employed to investigate the direct reduction of 1,1,2-trichloro-1,2,2-trifluoroethane (CFC-113) at a carbon cathode in dimethylformamide (DMF) containing 0.10M tetra-n-butylammonium tetrafluoroborate (TBABF4) as well as the nickel(I) salen-mediated reduction of CFC-113 in the same medium. Cyclic voltammograms for direct reduction of CFC-113 show two irreversible cathodic peaks attributable to formation of, first, 1-chloro-1,2,2-trifluoroethene (CFC-1113) and, second, trifluoroethene (HFC-1123); bulk electrolyses of CFC-113 at potentials corresponding to each of the cathodic peaks afford CFC-1113 and HFC-1123, respectively, in essentially quantitative yield. Cyclic voltammograms for reduction of nickel(II) salen in the presence of CFC-113 provide evidence for the catalytic formation of CFC-1113 and HFC-1123, and bulk electrolyses involving electrogenerated nickel(I) salen lead to mixtures of CFC-1113 and HFC-1123, although 1,1,1,2-tetrafluoroethane (HFC-134a) has been detected as a minor product. It has been determined with the aid of HPLC–ESI–MS that the salen ligand of the nickel(I) catalyst is modified during the catalytic reduction of CFC-113. A mechanistic scheme is proposed for the direct reduction of CFC-113.

NMR, HPLC-ESI-MS, and MALDI-TOF MS Analysis of Condensed Tannins from Delonix regia (Bojer ex Hook.) Raf. and Their Bioactivities

The structures of the condensed tannins isolated from leaf, fruit, and stem bark of Delonix regia (Bojer ex Hook.) Raf. have been investigated with (13)C nuclear magnetic resonance ((13)C NMR) and high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) coupled with thiolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses. The results showed that these condensed tannins from D. regia possessed structural heterogeneity in monomer units and degree of polymerization. Propelargonidin (PP) and procyanidin (PC) were found in the leaf, fruit, and stem bark of D. regia, while prodelphinidin (PD) was found only in the leaves. The polymer chain lengths of condensed tannins from leaf and fruit organs were detected to be trimers to hexadecamers but from trimers to tridecamers for stem bark. B-type linkages were present in all these compounds. Condensed tannins from different parts of D. regia can be explored as tyrosinase inhibitors and food antioxidants because of their potent antityrosinase and antioxidant activities. The inhibitor concentration leading to 50% enzyme activity (IC(50)) was estimated to be 38 ± 1, 73 ± 2, and 54 ± 1.5 μg/mL for the condensed tannins of leaf, fruit, and stem bark. Condensed tannins extracted from stem bark exhibited the highest antioxidant activity; the DPPH scavenging activity (IC(50)) and the FRAP values were 90 ± 2 μg/mL and 5.42 ± 0.09 mmol AAE/g, respectively.

Identification of the chemical constituents of bazhen decoction in rat feces by high performance liquid chromatography electro spray ionization mass spectrometry (HPLC-ESI/MS) n

A high performance liquid chromatography/mass spectrometry (HPLC/MS) was developed for the identification of rat feces in metabolites of the bazhen decoction. The metabolites were separated using gradient elution with mobile phase of 0.15% formic acid and acetonitrile. Blank and drug-containing feces samples were detected in the negative electrospray ionization, scan scope was 100 to 1200 and total ion current (TIC) chromatography was recorded. 29 major fragment ion peaks were marked in the TIC chromatogram of feces sample after the intragastric administration of bazhen decoction. According to the characteristic of fragmentation information, we identified 19 chemical constituents, including 10 prototype constituents: Paeoniflorin, Atractylenolide II, Benzoyl-paeoniflorin, Liquiritin, Pachymic acid, Ginsenoside Re, Ginsenoside Rg1, Ginsenoside Rb1, Ginsenoside Rd and Glycyrrhizic acid and 9 metabolites: Paeonimetabolin I, Aucubinine A, 20-O-β-D-glucopyranosyl-20 (S)-protopanaxadiol, 20 (S)-Protopanaxatriol, 16α-hydroxy acids perforation, 20 (S)-Protopanaxadiol, Licorice saponine, 18β-Glycyrrhetinic acid and Ginsenoside Rh1. The results show that the final metabolites in rat feces of bazhen decoction are quite valuable for the further studies on material basis of efficacy and clinical application of bazhen decoction. Key words: Bazhen decoction, high performance liquid chromatography/mass spectrometry HPLC-ESI/MS, metabolites, feces.

An MC3T3-E1 Cell Line Biomembrane Extraction and HPLC–ESI-MS n Method for Simultaneous Analysis of Potential Anti-Osteoporosis Components of Epimedium koreanum

Aerial parts of Epimedium koreanum Nakai have been used as Herba Epimedii in China. Its anti-osteoporosis effect has attracted much attention in recent years. In this study, a method involving osteoblastic cell (MC3T3-E1 cell line) extraction and high-performance liquid chromatography–electrospray ionisation–mass spectrometry (HPLC–ESI-MS n ) was developed for screening of potential anti-osteoporosis agents in E. koreanum. Four compounds identified as epimedin A, epimedin B, epimedin C and icariin were found to interact with MC3T3-E1 cells and possessed potent osteoblast-stimulating activity as evaluated by cell proliferation [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay] and differentiation [alkaline phosphatase (ALP) activity and Ca content] in vitro. The results suggest that these four flavonoids are the anti-osteoporosis constituents of Herba Epimedii and that the method combining MC3T3-E1 cell extraction with HPLC–ESI-MS n is rapid and effective in screening anti-osteoporosis agents from traditional Chinese medicines.

Online high performance liquid chromatography (HPLC)-ABTS+ based assay and HPLC-electrospray ionization mass spectrometry analysis of antioxidant phenolic compounds in Salsola komarovii

Online high performance liquid chromatography (HPLC)-2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS+) based assay system for phenolic antioxidants in Salsola komarovii extracts was applied. HPLC elute was mixed with a stabilized solution of ABTS+ reagents, and the negative peaks, indicating the antioxidant activity, were monitored by measuring the decrease in absorbance at 734 nm. HPLC with electrospray ionization mass spectrometry was used to identify the seven flavonoids and two phenolic amides in the S. komarovii extracts. The antioxidant compounds were identified as rutin, isoquercitrin, astragalin, and isorhamnetin.

High-Performance Liquid Chromatography–Electrospray Ionization-Mass Spectrometry Ligand Fishing Assay: A Method for Screening Triplex DNA Binders from Natural Plant Extracts

A novel ligand fishing assay was established to screen triplex DNA binders from complicated samples by a combination of immobilization of triplex DNA on agarose beads and high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). The biotinylated oligodeoxynucleotides were first bound to the streptavidin agarose beads and then incubated with the duplex DNA as the baits for ligand fishing. This assay was validated by the testing ligand library consisting of coralyne, ethidium bromide, vitexin, and formononetin. The binding affinities of ligands to target DNA were also obtained based on the calibration curves of ligands. Two components (berberine and palmatine) in the extract of Phellodendron chinense Schneid cortexes were fished out as triplex DNA binders by this assay, which indicated its feasibility for screening triplex DNA binders from complicated samples. This preliminary assay can be used for not only screening binders of triplex DNA from natural products extracts but also can obtain their binding affinity information.

Highly selective capture of phosphopeptides using a nano titanium dioxide–multiwalled carbon nanotube nanocomposite

In this study, a titanium dioxide–multiwalled carbon nanotube (TiO2–MWNT) nanocomposite was first used to enrich phosphopeptides as a binding agent. The TiO2–MWNT nanocomposite was synthesized by the hydrothermal reaction process and characterized by transmission electron microscopy (TEM), Fourier transform infrared (FTIR), and X-ray powder diffraction (XRD). The results demonstrated that the anatase phase TiO2 nanoparticles had been successfully linked by MWNTs. The TiO2–MWNT nanocomposite was applied as a sorbent to enrich phosphopeptides, and the results showed that the performance of the TiO2–MWNT nanocomposite was better than 5μm TiO2, as confirmed by the analysis of phosphopeptides from a tryptic digest of a standard protein (β-casein and bovine serum albumin) using high-performance liquid chromatography–electrospray ionization–mass spectrometry (HPLC–ESI–MS). The use of lactic acid in the loading buffer significantly enhances the selectivity of the TiO2–MWNT nanocomposite. This nanocomposite material was further applied to enrich the phosphopeptides in a protein digest obtained from nonfat milk successfully.