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One megastigmane derivative 1 , one methyl jasmonate glycoside derivative 2 , and two C-28 steroids with 3 β ,5 β - cis -dihydroxyl conformation 3 and 4 , together with eight known compounds 5 – 12 were isolated from the 70% ethanol extract of linseed meal ( Linum usitatissimum L). Structures of 1 – 4 were elucidated by spectroscopic methods including NMR, HRESIMS, and Mo 2 (OAc) 4 -induced CD. The absolute configuration of 1 and 3 was determined by observing their induced circular dichroism after addition of Mo 2 (OAc) 4 in DMSO. The absolute configuration of 2 was determined by NOESY experiment together with conformational analysis. The structure of 4a was corrected as 4 by an extensive analysis of its 1D and 2D NMR, in combination with the Mo 2 (OAc) 4 -induced CD in DMSO. The effect of all the isolates on nitric oxide (NO) generation by stimulated macrophages was evaluated, and none of them showed active.

RATIONALE: Impurity analysis plays an important role to guarantee the quality and safety of pharmaceuticals. However, identification of impurities remains challenging, especially for those unknown or at trace levels. We present an integrated approach to detect and characterize the trace impurities in drugs. METHODS: Based on liquid chromatography–tandem mass spectrometry (LC–MS/MS), an approach integrating automatic impurity screening method using multiple mass defect filters (MMDFs) and background subtraction (BS) was developed. This approach was used to acquire the structural and semi-quantitative information in a single sample run, and even to discover the impurity signals submerged by background and drug ions. This approach was illustrated by the comprehensive impurity analysis of levofloxacin. RESULTS: This approach was sensitive to detect impurities at the level of 0.02% with respect to levofloxacin concentration. Nineteen impurities were detected, fourteen of which were structurally characterized and eight impurities were reported for the first time. Impurity profiles of levofloxacin drug substances and degradation samples were obtained reliably. A plausible degradation pathway of levofloxacin was proposed including descarboxyl reaction under acid, piperazinyl ring cleavage degradation under light, and N-oxidation under oxidative condition. CONCLUSIONS: The generic approach integrating LC–MS/MS and an automatic impurity screening method was developed for the detection, characterization and monitoring of impurities, especially those unknown or at trace levels. This approach was demonstrated to be rapid, sensitive and automatic for impurity profiling of drugs. Copyright © 2014 John Wiley & Sons, Ltd.

Berberine (BBR) has been confirmed to show extensive bioactivities for the treatments of diabetes and hypercholesterolemia in clinic. However, there are few pharmacokinetic studies to elucidate the excretions of BBR and its metabolites. Our research studied the excretions of BBR and its metabolites in rats after oral administration (200 mg/kg). Metabolites in bile, urine, and feces were detected by liquid chromatography coupled to ion trap time-of-flight mass spectrometry; meanwhile, a validated liquid chromatography coupled with tandem mass spectrometry method was developed for their quantifications. Sixteen metabolites, including 10 Phase I and six Phase II metabolites were identified and clarified after dosing in vivo. Total recovered rate of BBR was 22.83% (19.07% of prototype and 3.76% of its metabolites) with 9.2 × 10(-6) % in bile (24 h), 0.0939% in urine (48 h), and 22.74% in feces (48 h), respectively. 83% of BBR was excreted as thalifendine (M1) from bile, whereas thalifendine (M1) and berberrubine (M2) were the major metabolites occupying 78% of urine excretion. Most of BBR and its metabolites were found in feces containing 84% of prototype. In summary, we provided excretion profiles of BBR and its metabolites after oral administration in rats in vivo.

The chiral separations of four pharmaceutical racemates which contain N -alkyl groups were satisfactorily resolved using SBE- β -CD as a chiral mobile phase additive (CMPA) in a RP-HPLC system (the resolution is 2.701 for ondansetron hydrochloride, 1.996 for sulpiride, 1.293 for clenbuterol hydrochloride and 0.816 for omeprazole). In addition, the effects of different parameters such as CD type and CD concentration were investigated. The separation mechanism arises through the combination of several potential interactions, including electrostatic interactions as well as hydrogen bonding interactions and hydrophobic inclusion interactions, which allow for the SBE- β -CD–drug complexation with strong stereoselectivity and stability. The resolution also relates to the number and location of N atoms in the enantiomers. This method will be applicable to the isolation of various types of biologically important enantiomers containing N -alkyl groups.

The enantiomers separation of eight pharmaceutical racemates collected in Chinese Pharmacopoeia 2010 (Ch.P2010), including nitrendipine, felodipine, omeprazole, praziquantel, sulpiride, clenbuterol hydrochloride, verapamil hydrochloride and chlorphenamine maleate, was performed on chiral stationary phase of amylose ramification by high performance liquid chromatography (HPLC) on Chiralpak AD-H column and Chiralpak AS-H column with the mobile phase consisted of isopropanol and n-hexane. The detection wavelength and the flow rate were set at 254 nm and 0.7 mL/min, respectively. The effects of proportion of organic additives, alcohol displacer and temperature on the separation were investigated. The results indicated that eight chiral drugs were separated on chiral stationary phase of amylase ramification in normal phase chromatographic system. The chromatographic retention and resolution of enantiomers were adjusted by factors, including the changes of the concentration of alcohol displacer in mobile phase, organic alkaline modifier and column temperature. It was shown that the resolution was improved with reducing concentration of alcohol displacer. When the concentration of organic alkaline modifier was 0.2%, the resolution and the peak shape were fairly good. Most racemates mentioned above had the best resolution at column temperature of 25 °C. The best temperature should be kept unchanged in the process of separation so as to obtain stable separation results.

An integrated approach combining data acquisition using MS(E) and multi-period product ion scan (mpMS/MS), with high-resolution characteristic extracted ion chromatograms (hcXIC) as a data mining method, was developed for in vivo drug metabolites screening and identification. This approach is illustrated by analyzing metabolites of a potential anticancer agent, 3,6,7-trimethoxyphenanthroindolizidine (CAT) in rat urine based on rapid resolution liquid chromatography combined with tandem mass spectrometry (RRLC-MS/MS). Untargeted full-scan MS(E) enabled the high-throughput acquisition of potential metabolites, and targeted mpMS/MS contributed to the sensitivity and specificity of the acquisition of molecules of interest. The data processing method hcXIC, based on the structure of CAT, was shown to be highly effective for the metabolite discovery. Through the double-filtering effect of the characteristic ion and accurate mass, conventional extracted ion chromatograms that contained a substantial number of false-positive peaks were simplified into chromatograms essentially free of endogenous interferences. As a result, 21 metabolites were detected in rat urine after oral administration of CAT. Based on the characteristic fragmentation patterns of the phenanthroindolizidine alkaloid, the structures of 9 metabolites were identified. Furthermore, the interpretation of the MS/MS spectra of these metabolites enabled the determination of demethylation position as well as the differentiation between N-oxidized and hydroxylated metabolites.

Nuclear magnetic resonance/liquid chromatography-mass spectroscopy parallel dynamic spectroscopy (NMR/LC-MS PDS) is a method aimed at the simultaneous structural identification of natural products in complex mixtures. In this study, the method is illustrated with respect to 1H NMR and rapid resolution liquid chromatography-mass spectroscopy (RRLC-MS) data, acquired from the crude extract of Anoectochilus roxburghii, which was separated into a series of fractions with the concentration of constituent dynamic variation using reversed-phase preparative chromatography. Through fraction ranges and intensity changing profiles in 1H NMR/RRLC–MS PDS spectrum, 1H NMR and the extracted ion chromatogram (XIC) signals deriving from the same individual constituent, were correlated due to the signal amplitude co-variation resulting from the concentration variation of constituents in a series of incompletely separated fractions. 1H NMR/RRLC-MS PDS was then successfully used to identify three types of natural products, including eight flavonoids, four organic acids and p-hydroxybenzaldehyde, five of which have not previously been reported in Anoectochilus roxburghii. In addition, two groups of co-eluted compounds were successfully identified. The results prove that this approach should be of benefit in the unequivocal structural determination of a variety of classes of compounds from extremely complex mixtures, such as herbs and biological samples, which will lead to improved efficiency in the identification of new potential lead compounds.

The most attractive advantage of dynamic combinatorial chemistry (DCC) is that it can screen the compound library as soon as compounds are synthesized. However, it is very difficult to analyze a dynamic combinatorial library with free probes using the state-of-the art analysis technologies. We report herein a method that uses a resin-immobilizing reversed peptide probe to screen vancomycin derivatives and provides a solution to this problem.

A norditerpene glucopyranoside with a novel carbon skeleton (1), eight new aromatic glycosides (2-9), and 25 known glycosides have been isolated from a H2O-soluble portion of an ethanolic extract of the stem bark of Fraxinus sieboldiana. Their structures were determined by spectroscopic and chemical methods. Based on analysis of the NMR data of threo- and erythro-arylglycerols in different solvents, an application of Delta delta C8-C7 values to distinguish threo-arylglycerol and erythro-arylglycerol isomers was proposed. In the in vitro assays, compound 5 displayed TNF-alpha secretion inhibitory activity with an IC50 value of 1.6 microM, compound 6 showed antioxidative activity inhibiting Fe+2-cystine-induced rat liver microsomal lipid peroxidation with an IC50 value of 0.9 microM, and plantasioside (10) showed selective activity against the human colon cancer cell line (HCT-8) with an IC50 value of 3.4 microM.

[structure: see text] Two unusual glycosidic triterpene alkaloids, machilaminosides A (1) and B (2), have been isolated from the stem barks of Machilus yaoshansis. Their structures were elucidated by detailed spectroscopic analysis. A possible biogenetic origin of 1 and 2 mediated by the coupling of 2-O-beta-D-glucopyranosyl-cucurbitacin I, respectively, with urea and adenosine was postulated. 1 and 2 showed nonselective cytotoxic activities against several human cancer cell lines as well as TNF-alpha secretion inhibitory activities.

Martentoxin is a peptide of 37 amino acid residues purified from the venom of the Chinese scorpion Buthus martensi Karch, which has been demonstrated to be an inhibitor of voltage-dependent sodium channel and voltage-dependent delayed rectifier potassium channel. To elucidate the molecular mechanism of this interaction, the structure of martentoxin was studied by 2D-NMR. The secondary structure of martentoxin consists of a triple-stranded beta-sheet connected to a alpha-helical structure. This helix encompasses 10 residues from Ser11 to Lys20. The three strands of beta-sheet probably comprise residues Gly2-Asp5, Q27-N30 and Glu33-Cys36, Cys30-Asn33 with a type I'beta turn centered on Asn31-Asn32. The results indicate that martentoxin possesses the conserved beta alpha beta beta structure of all the potassium channel toxins.

Quantitative 19 F NMR (QNMR) was developed and employed to determine the amounts of four kinds of fluoropolyphosphates (FPPs) in a mixture containing sodium monofluoro-phosphate (MFP), sodium monofluoro-dipolyphosphate (MFDPP), sodium monofluoro-tripolyphosphate (MFTPP) and sodium difluoro-tripolyphosphate (DFTPP). The amounts of these ingredients cannot be measured by high-performance liquid chromatography (HPLC) because no high-purity standard samples are available. The main parameter “delay time (d1) between two scans” of affecting the response of NMR signal was determined by measuring longitudinal relaxation time (T1) to be 25 s. By using NaF as an internal reference to measure the amount of MFP solution with known concentration and then to compare the deviation between experiment value and real value, a simple and effective approach for checking out the validity of 19 F QNMR method was carried out. Six experiments with different mole ratios of NaF/MFP over 200 times were repeated and the relative standard deviation (R.S.D.) of results is less than 2.0%, the limit of detection of 19 F QNMR can reach to 1.0 mmol/L. NaF was used as an internal reference for the quantitative analysis of FPPs mixture. The amount of each FPP in FPPs mixture obtained by 19 F QNMR was calculated and the R.S.D.s of results were less than 4.81%.

Fractionation of the ethanolic extract of the stem bark of Morus macroura resulted in the isolation of four new Diels-Alder type adducts, named guangsangons K--N (1, 2, 5, 6), together with two known compounds, mulberrofuran G (3) and K (4). Their structures were determined on the basis of spectroscopic analyses and chemical methods. Furthermore, by means of (1)H-NMR variable temperature experiments and the Cotton curves in the circular dichroism (CD) spectra, the stereochemistry of four new compounds was elucidated. The isolated new compounds showed good activity on anti-oxidant in vitro, with the inhibitory rates of MDA being from 91.8 to 100.0% at concentrations of 10(-5) mol/l.

Direct O-glycosylation of amino acids bound to TentaGel resin with a number of glycosyl trichloroacetimidate donors results in high yields. The glycosylation reaction can be easily monitored by analyzing the bead-bound amino acids with high-resolution magic angle spinning (HR-MAS) NMR. These studies pave a new way for the construction of "one-bead one-compound" O-glycopeptide libraries with standard amino acid building blocks and appropriate glycosyl trichloroacetimidate donors.

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) and liquid chromatography coupled with sequential mass spectrometry (LC/MSn) were applied to identify trace-level phenanthroindolizidine alkaloids in crude extracts from Tylophora atrofolliculata. Based on the relationship between the characteristic fragmentation reactions and the structural features of related compounds of known structure from this plant, the bioactive crude extract was analyzed in detail by positive and negative ion ESI-MSn, LC/UV-MS and LC/MSn techniques. A total of nine constituents in the crude extract were identified rapidly, including several isomers; seven of these constituents are new and two are known compounds. The structures of four of these constituents were subsequently confirmed by nuclear magnetic resonance (NMR) and accurate mass measurements using high-resolution fast-atom bombardment mass spectrometry (FAB-HRMS). Copyright © 2003 John Wiley & Sons, Ltd.

A new triterpene, 13β-methyl isomer of bauerenol acetate, named mongolenin, has been isolated from the aerial parts of Artemisia mongolica. The structure was elucidated by spectroscopic methods. Its 13C- and 1H-NMR chemical shifts have been assigned unambiguously on the basis of detailed 2D-NMR analyses.

A novel phenylpropanoid ester of sucrose, shegansu C (1), was isolated from the rhizome of Belamcanda chinensis (L.) DC. (Iridaceae) along with 18 known compounds. The structure of 1 was characterized as β-D-[3-O-(4-O-(3,4-,dimethoxycinnamoyl)-5-O-feruloyl)-caffeoyl]-fructofuranosyl(2-1′)-α-D-(3′-O-acetyl)-glucopyranoside on the basis of chemical and spectral evidence including 2DNMR studies.

Berberine (BBR) has been confirmed to have multiple bioactivities in clinic, such as cholesterol-lowering, anti-diabetes, cardiovascular protection and anti- inflammation. However, BBR's plasma level is very low; it cannot explain its pharmacological effects in patients. We consider that the in vivo distribution of BBR as well as of its bioactive metabolites might provide part of the explanation for this question. In this study, liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LC/MS(n)-IT-TOF) as well as liquid chromatography that coupled with tandem mass spectrometry (LC-MS/MS) was used for the study of tissue distribution and pharmacokinetics of BBR in rats after oral administration (200 mg/kg). The results indicated that BBR was quickly distributed in the liver, kidneys, muscle, lungs, brain, heart, pancreas and fat in a descending order of its amount. The pharmacokinetic profile indicated that BBR's level in most of studied tissues was higher (or much higher) than that in plasma 4 h after administration. BBR remained relatively stable in the tissues like liver, heart, brain, muscle, pancreas etc. Organ distribution of BBR's metabolites was also investigated paralleled with that of BBR. Thalifendine (M1), berberrubine (M2) and jatrorrhizine (M4), which the metabolites with moderate bioactivity, were easily detected in organs like the liver and kidney. For instance, M1, M2 and M4 were the major metabolites in the liver, among which the percentage of M2 was up to 65.1%; the level of AUC (0-t) (area under the concentration-time curve) for BBR or the metabolites in the liver was 10-fold or 30-fold higher than that in plasma, respectively. In summary, the organ concentration of BBR (as well as its bioactive metabolites) was higher than its concentration in the blood after oral administration. It might explain BBR's pharmacological effects on human diseases in clinic.

Two new taxane diterpenoids, 1-hydroxy-2-deacetoxytaxinine J and 7,2′-bisdeacetoxyaustrospicatine, were isolated from the barks of Taxus wallichiana together with 13 known compounds, including taxol. Their structures were elucidated and identified by spectroscopic methods. The structure of 7,2′-bisdeacetoxyaustrospicatine was further confirmed by X-ray crystallographic analysis.

Two new taxane diterpenoids, 1-hydroxy-2-deacetoxytaxinine J and 7,2′-bisdeacetoxyaustrospicatine, were isolated from the barks of Taxus wallichiana together with 13 known compounds, including taxol. Their structures were elucidated and identified by spectroscopic methods. The structure of 7,2′-bisdeacetoxyaustrospicatine was further confirmed by X-ray crystallographic analysis.

Twenty-five N-demethylvancomycin derivatives were synthesized on solid-support and their structures were determined by LC-MS/MS. Biological evaluation of these compounds indicated that bulky hydrophobic substituent on vancosamine of N-demethylvancomycin can increase antibacterial activity against vancomycin-resistant Enterococcus faecalis.

Three C-14 oxygenated taxanes were isolated from Taxus yunnanensis cell cultures.

Cytochalasin D which shows marked cytotoxic effects on multi-tumor cells was newly isolated at high content(5.28 mg/g, dry weight) from Fungus Hypocrella bambusae(B.et Br.) Sacc. Its structure was elucidated by spectroscopic methods. Two-dimensional NMR techniques were applied to make complete assignment for the 1H- and 13C-NMR chemical shifts of this compound.