Two-dimensional Nuclear Magnetic Resonance Research Articles

Isolation and identification of 3,4-seco-labdane diterpenoids from Callicarpa nudiflora and investigation of their cytotoxicity against HepG2 cells

Twenty-one previously undescribed compounds, including nineteen 3,4-seco-labdanes (nudiflopenes P–W, Y, AI–JI), one 3,4-seco-pimarane (nudiflopene X), and one labdane (nudiflopene Z), along with nine known compounds (one 3,4-seco-pimarane and eight 3,4-seco-labdanes) were isolated from the leaves of Callicarpa nudiflora Hook. Et Arn. The structures of these compounds were elucidated by high-resolution electrospray ionization mass spectrometry and one- and two-dimensional nuclear magnetic resonance spectroscopy. In addition, configurations of the isolated compounds were determined by electronic circular dichroism, DP4+ probability analysis, and single-crystal X-ray diffraction experiments. All undescribed compounds were evaluated for their cytotoxicity against HepG2 cells in vitro, among which compound 12 exhibited a moderate activity with an IC50 value of 27.8 μM.

Identification of hexahydrocannabinol (HHC), dihydro-iso-tetrahydrocannabinol (dihydro-iso-THC) and hexahydrocannabiphorol (HHCP) in electronic cigarette cartridge products.

Since 2021, products claiming to contain hexahydrocannabinol (HHC) and hexahydrocannabiphorol (HHCP), which are tetrahydrocannabinol (THC) analogs, have been distributed via the Internet. Owing to the presence of three asymmetric carbons in their structure, HHC and HHCP have multiple stereoisomers. This study aimed to identify the actual stereoisomers of HHC and HHCP isolated from electronic cigarette cartridge products using nuclear magnetic resonance (NMR) spectroscopy. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-photodiode array-mass spectrometry (LC-PDA-MS) were used for the analyses of two major peaks and one minor peak in product A and two major peaks in product B. These five compounds were isolated by silica gel column chromatography, and their structures were analyzed by 1H, 13C-NMR and various two-dimensional NMR techniques, i.e., H-H correlation spectroscopy, heteronuclear multiple quantum coherence, heteronuclear multiple-bond correlation, and nuclear Overhauser effect spectroscopy. Three compounds isolated from product A were identified as rel-(6aR,9R,10aR)-hexahydrocannabinol (11β-hexahydrocannabinol; 11β-HHC), rel-(6aR,9S,10aR)-hexahydrocannabinol (11α-hexahydrocannabinol, 11α-HHC), and a minor compound (2R,5S,6R)-dihydro-iso-tetrahydrocannabinol (dihydro-iso-THC). Meanwhile, the structural isomers of the major compound isolated from product B were identified as rel-(6aR, 9R, 10aR)-hexahydrocannabiphorol (11β-hexahydrocannabiphorol; 11β-HHCP) and rel-(6aR, 9S, 10aR)-hexahydrocannabiphorol (11α-hexahydrocannabiphorol; 11α-HHCP). The presence of both 11β-HHC and 11α-HHC in the HHC products analyzed in this study suggests that they were most likely synthesized via the reduction reaction of Δ8-THC or Δ9-THC. Dihydro-iso-THC was probably obtained as a byproduct of the synthesis of Δ8-THC or Δ9-THC from cannabidiol. Similarly, 11β-HHCP and 11α-HHCP in the HHCP product could stem from Δ9-tetrahydrocannabiphorol.

Synthesis, characterization and antiproliferative activity of platinum(II) complexes with 3-(2-pyridyl)-N1,2-methyl-1,2,4-triazoles

The present work describes the synthesis, investigation and in vitro cytotoxicity of Pt(II) complexes with 3-(2-pyridyl)-N1-methyl-1,2,4-triazole (L1) and 3-(2-pyridyl)–N2-methyl-1,2,4-triazole (L2). The crystal structures of PtL1Cl2 and PtL2Cl2 were established using X-Ray analysis, which revealed the square-planar PtN2Cl2 coordination geometry for the central metal ion. High-resolution mass spectrometry (HRMS), as well as one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy showed stability of the investigated complexes in DMF solution, in contrast to DMSO, where species were shown to partially dissociate. The cytotoxicity of the investigated compounds was determined by the colorimetric MTT assay against two human cancer cell lines, including cisplatin-sensitive human ovarian adenocarcinoma A2780 cells (IC50 = 26 ± 5 and 6.1 ± 0.6 for PtL1Cl2 and PtL2Cl2 correspondingly) and their cisplatin-resistant A2780cis analogue (IC50 = 24 ± 2 and 20 ± 3 for PtL1Cl2 and PtL2Cl2 correspondingly).

Cytotoxic effects of aporphine alkaloids from the stems and leaves of Stephania dielsiana Y.C.Wu

Phytochemical studies of the stems and leaves of Stephania dielsiana Y.C.Wu yielded two new aporphine alkaloids (1 and 5), along with six known alkaloids (2–4 and 6–8). Their structures were characterised based on analyses of spectroscopic data, including one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS). The cytotoxic activities of the isolated compounds against a small panel of tumour cell lines were assessed by MTS assay. Interestingly, compound 2 exhibited particularly strong cytotoxic activities against HepG2, MCF7 and OVCAR8 cancer cell lines, with IC50 values of 3.20 ± 0.18, 3.10 ± 0.06 and 3.40 ± 0.007 µM, respectively. Furthermore, molecular docking simulations were carried out to explore the interactions and binding mechanisms of the most active compound (compound 2) with proteins. Our results contribute to understanding the secondary metabolites produced by S. dielsiana and provide a scientific rationale for further investigations of cytotoxicity of this valuable medicinal plant.

Structural and Dynamic Behaviour of Heterocycles Derived from Ethylenediamines with Formaldehyde: 1,3,5-Triazinanes and Bis(imidazolidinyl)methanes

Formaldehyde is a simple chemical compound that is used as a building block in obtaining a wide range of products. The versatility of formaldehyde in chemical synthesis becomes evident when it is reacted with N-alkylethylenediamines. Therefore, this paper reports the structure and reactivity of a series of compounds derived from easily accessible molecules, such as formaldehyde, sodium hydrosulphide, and N-alkylethylenediamines. The 1,3,5-triazines (1a-1d) and bis(3-alkyl-imidazolidin-1-yl)methanes (2a-2d) were obtained by simple reaction conditions. Additionally, different proportions of sodium hydrosulphide and formaldehyde were used with N-benzylamine to obtain N-benzyltriazinane (3), N-benzylthiadiazinane (4) and N-benzyldithiazinane (5). All these compounds were characterized by analytical, spectroscopic, and spectrometric techniques, such as melting point, solubility, one-dimensional and two-dimensional nuclear magnetic resonance (13C, 1H, 15N, COSY, HETCOR, NOESY, COLOC), elemental analysis, high- and low-resolution mass spectrometry, among others. The structures of compounds 4 and 5 were obtained by single-crystal X-ray diffraction. The results show that small variations in the stoichiometry and the reaction conditions significantly influence the products obtained.

Identification and Isolation of α-Glucosidase Inhibitors from Siraitia grosvenorii Roots Using Bio-Affinity Ultrafiltration and Comprehensive Chromatography.

The discovery of bioactive compounds from medicinal plants has played a crucial role in drug discovery. In this study, a simple and efficient method utilizing affinity-based ultrafiltration (UF) coupled with high-performance liquid chromatography (HPLC) was developed for the rapid screening and targeted separation of α-glucosidase inhibitors from Siraitia grosvenorii roots. First, an active fraction of S. grosvenorii roots (SGR2) was prepared, and 17 potential α-glucosidase inhibitors were identified based on UF-HPLC analysis. Second, guided by UF-HPLC, a combination of MCI gel CHP-20P column chromatography, high-speed counter-current countercurrent chromatography, and preparative HPLC were conducted to isolate the compounds producing active peaks. Sixteen compounds were successfully isolated from SGR2, including two lignans and fourteen cucurbitane-type triterpenoids. The structures of the novel compounds (4, 6, 7, 8, 9, and 11) were elucidated using spectroscopic methods, including one- and two-dimensional nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. Finally, the α-glucosidase inhibitory activities of the isolated compounds were verified via enzyme inhibition assays and molecular docking analysis, all of which were found to exhibit certain inhibitory activity. Compound 14 exhibited the strongest inhibitory activity, with an IC50 value of 430.13 ± 13.33 μM, which was superior to that of acarbose (1332.50 ± 58.53 μM). The relationships between the structures of the compounds and their inhibitory activities were also investigated. Molecular docking showed that the highly active inhibitors interacted with α-glucosidase through hydrogen bonds and hydrophobic interactions. Our results demonstrate the beneficial effects of S. grosvenorii roots and their constituents on α-glucosidase inhibition.

The impact of xylan on the biosynthesis and structure of extracellular lignin produced by a Norway spruce tissue culture.

In order to develop more economic uses of lignin, greater knowledge regarding its native structure is required. This can inform the development of optimized extraction methods that preserve desired structural properties. Current extraction methods alter the polymeric structure of lignin, leading to a loss of valuable structural groups or the formation of new non-native ones. In this study, Norway spruce (Picea abies) tissue-cultured cells that produce lignin extracellularly in a suspension medium were employed. This system enables the investigation of unaltered native lignin, as no physicochemical extraction steps are required. For the first time, this culture was used to investigate the interactions between lignin and xylan, a secondary cell wall hemicellulose, and to study the importance of lignin-carbohydrate complexes (LCCs) on the polymerization and final structure of extracellular lignin (ECL). This has enabled us to study the impact of xylan on monolignol composition and structure of the final lignin polymer. We find that the addition of xylan to the solid culture medium accelerates cell growth and impacts the ratio of monolignols in the lignin. However, the presence of xylan in the lignin polymerization environment does not significantly alter the structural properties of lignin as analyzed by two-dimensional nuclear magnetic resonance (NMR) spectroscopy and size exclusion chromatography (SEC). Nevertheless, our data indicate that xylan can act as a nucleation point, leading to more rapid lignin polymerization, an important insight into biopolymer interactions during cell wall synthesis in wood. Lignin structure and interactions with a secondary cell wall hemicellulose were investigated in a model cell culture: we found that the polymerization and final structure of lignin are altered when the hemicellulose is present during cell growth and monolignol production. The physicochemical interactions between lignin and xylan partly define the extractability and utility of native lignin in high value applications, so this work has implications for lignin extraction as well as fundamental plant biology.

Spiroligomer-Based Macrocycles for Atomically Precise Membranes.

Here, we report a new class of peptidomimetic macrocycles with well-defined three-dimensional structures and low conformational flexibility. They are assembled from fused-ring spiro-ladder oligomers (spiroligomers) by modular solid-phase synthesis. Two-dimensional nuclear magnetic resonance confirms their shape persistency. Triangular macrocycles of tunable sizes assemble into membranes with atomically precise pores, which exhibit size and shape-dependent molecular sieving towards a series of structurally similar compounds. The exceptional structural diversity and stability of spiroligomer-based macrocycles will be explored for more applications.

The influence of partial chemical component removal on water state at high relative humidity and cell wall saturation state

Water sorption in wood is mostly determined by the cell wall pores and the availability of sorption sites. In this study, we partially removed hemicellulose and lignin to adjust hydroxyl accessibility and pore size distribution of the cell wall of beech (Fagus sylvatica). With the assistance of the two-dimensional time-domain nuclear magnetic resonance technique, the water state at high relative humidity (RH) regions and cell wall saturation state were investigated. Both hemicellulose removal and delignification brought newly created pore spaces, lowered the average pore size, and increased the specific surface area. At the same time, a decrease in hydroxyl accessibility was observed for the hemicellulose-removal samples, but an increase in delignified samples. Two cell wall waters (B-water and C-water) with different mobility and two cell lumen waters were categorized. At high RH regions, more water molecules accumulated in microfibril and its surrounding hemicellulose (the domain of B-water) with lower mobility after partial chemical components removal. In combination with the mobility change with the physiochemical environment of the water molecules, it may be established that the accumulation of B-water was affected more by the change in the physical environment. Furthermore, cell wall saturation may alter the matrix environment for both treated and untreated samples; following water saturation, the mobility of B-water increased in the delignified samples but decreased in the untreated and partially hemicellulose-removed samples.

An Optimized Two-Dimensional Quantitative Nuclear Magnetic Resonance Strategy for the Rapid Quantitation of Diester-Type C19-Diterpenoid Alkaloids from Aconitum carmichaelii.

With the development of nuclear magnetic resonance (NMR) spectrometers and probes, two-dimensional quantitative nuclear magnetic resonance (2D qNMR) technology with a high signal resolution and great application potential has become increasingly accessible for the quantitation of complex mixtures. However, the requirement that the relaxation recovery time be equal to at least five times T1 (longitudinal relaxation time) makes it difficult for 2D qNMR to simultaneously achieve high quantitative accuracy and high data acquisition efficiency. By comprehensively using relaxation optimization and nonuniform sampling, we successfully established an optimized 2D qNMR strategy for HSQC experiments at the half-hour level and then accurately quantified the diester-type C19-diterpenoid alkaloids in Aconitum carmichaelii. The optimized strategy had the advantages of high efficiency, high accuracy, good reproducibility, and low cost and thus could serve as a reference to optimize 2D qNMR experiments for quantitative analysis of natural products, metabolites, and other complex mixtures.

A robust t1 noise suppression method in NMR spectroscopy.

Artefacts in high-resolution multidimensional nuclear magnetic resonance (NMR) spectra, known as t1 noise, can significantly downgrade the spectral quality and remain a significant noise source, limiting the sensitivity of most two-dimensional NMR experiments. In addition to highly sensitive hardware and experimental designs, data post-processing is a relatively simple and cost-effective method for suppressing t1 noise. In this study, histograms and quantiles were used to obtain a robust estimation of noise level. We constructed a weighted matrix to suppress the t1 noise. The weighted matrix was calculated from the logistic functions, which were adaptively computed from the spectrum. The proposed method is robust and effective in both simulations and actual experiments. Further, it can maintain the quantitative relationship of the spectrogram and is suitable for various complex peak types.

Variations in the composition and structure of the lignins of oat (Avena sativa L.) straws according to variety and planting season

The differences in the composition and structure of the lignins from straws of different oat (Avena sativa L.) varieties, planted in two seasons (winter and spring), were studied in detail by different analytical techniques such as pyrolysis coupled to gas chromatography–mass spectrometry (Py-GC/MS), two-dimensional nuclear magnetic resonance (2D-NMR), derivatization followed by reductive cleavage (DFRC), and gel permeation chromatography (GPC). Overall, the analyses revealed that oat straw lignins were enriched in guaiacyl (G; 50–56 %) and syringyl (S; 39–44 %) units, with relatively lower amounts of p-hydroxyphenyl (H; 4–6 %) units. The lignins also incorporated significant quantities of p-coumarates (8–14 % of total lignin units), which are acylating the γ-OH of the lignin side chains, and predominantly over the S units. Furthermore, oat straw lignins also incorporated considerable amounts of the flavone tricin (5–12 % of total lignin units). Interestingly, this study revealed that the lignin content and composition of the oat straws vary with genotype and planting season. Since p-coumarates and tricin are high-value aromatic compounds especially attractive from a biorefinery point of view, the information disclosed here is highly relevant to plant breeding programs aimed at developing functional foods and lignin modifications for improved biorefinery applications.

A New Method for Mobility Logging Evaluation Based on Flowing Porosity in Shale Oil Reservoirs

Shale oil reservoirs differ from conventional reservoirs in several aspects, including the sedimentary model, accumulation mechanism, and reservoir characteristics, which pose significant challenges to their exploration and development. Therefore, identifying the location of optimal spots is crucial for the successful exploration and development of shale oil reservoirs. Mobility, particularly in low-permeability shale oil reservoirs with nano-scale pores, is a crucial petrophysical property that determines the development plan. However, two-dimensional nuclear magnetic resonance (2D-NMR) is expensive and has limited applicability, although it can estimate shale oil mobility. Hence, it is of great significance to find a precise method for evaluating shale oil mobility using conventional logging. In this paper, we propose a new method for assessing shale oil mobility based on free oil porosity derived from the difference in flowing porosity detected at different ranges of logging, utilizing the Maxwell conductivity model and conductivity efficiency theory. Our study shows that longitudinal-T2 (T1-T2) NMR logging can accurately evaluate the mobility of shale oil. This is demonstrated by comparing the processing results obtained from our proposed method with those from 2D-NMR and laboratory NMR experiments. The predicted results based on conventional well logs also show good agreement with experimental results, confirming the effectiveness and reliability of our new method. Our proposed method carries reference significance for evaluating shale oil reservoir quality.

The use of nitrogen-15 in microbial natural product discovery and biosynthetic characterization.

This mini-review covers the use of nitrogen-15 in bacterial and fungal natural product discovery and biosynthetic characterization from 1970 to 2022. Nitrogen is an important element in a number of bioactive and structurally intriguing natural products including alkaloids, non-ribosomal peptides, and hybrid natural products. Nitrogen-15 can be detected at natural abundance utilizing two-dimensional nuclear magnetic resonance and mass spectrometry. Additionally, it is a stable isotope that can be added to growth media for both filamentous fungi and bacteria. With stable isotope feeding, additional two-dimensional nuclear magnetic resonance and mass spectrometry strategies have become available, and there is a growing trend to use nitrogen-15 stable isotope feeding for the biosynthetic characterization of natural products. This mini-review will catalog the use of these strategies, analyze the strengths and weaknesses of the different approaches, and suggest future directions for the use of nitrogen-15 in natural product discovery and biosynthetic characterization.

Probing the Binding Mechanism of Acylated Peptides to Human Serum Albumin

Peptides represent an increasingly important class of pharmaceutical products. During the last decade or so, acylation with fatty acids has demonstrated considerable success in prolonging the circulating half-life of therapeutic peptides by exploiting the ability of fatty acids to reversibly bind to human serum albumin (HSA), thus significantly impacting their pharmacological profiles. Employing methyl-13C-labeled oleic acid or palmitic acid as probe molecules and exploiting HSA mutants designed to probe fatty acid binding, the signals in two-dimensional (2D) nuclear magnetic resonance (NMR) spectra corresponding to high-affinity fatty acid binding sites in HSA were assigned. Subsequently, using a set of selected acylated peptides, competitive displacement experiments by 2D NMR identified a primary fatty acid binding site in HSA utilized in acylated peptide binding. These results represent an important first step toward understanding the structural basis for acylated peptides binding to HSA.

Production of a New Biosurfactant by a New Yeast Species Isolated from Prunus mume Sieb. et Zucc.

Biosurfactants reduce surface and interfacial tension due to their amphiphilic properties and are an eco-friendly alternative for chemical surfactants. In this study, a new yeast strain JAF-11 that produces a biosurfactant was selected using drop collapse method, and the properties of the extracts were investigated. The nucleotide sequences of the strain were compared with closely related strains and identified based on the D1/D2 domain of the large subunit ribosomal DNA (LSU) and internal transcribed spacer (ITS) regions. Neodothiora populina CPC 39399T, the closest species with strain JAF-11, showed a sequence similarity of 97.75% for LSU and 94.27% for ITS, respectively. The result suggests that the strain JAF-11 represents a distinct species that cannot be assigned to any existing genus or species in the family Dothideaceae. Strain JAF-11 produced a biosurfactant reducing the surface tension of water from 72 mN/m to 34.5 mN/m on the sixth day of culture and the result of measuring the critical micelle concentration (CMC) by extracting the crude biosurfactant was found to be 24 mg/l. The molecular weight 502 of the purified biosurfactant was confirmed by measuring the fast atom bombardment mass spectrum. The chemical structure was analyzed by measuring 1H nuclear magnetic resonance (NMR), 13C NMR, and two-dimensional NMRs of the compound. The molecular formula was C26H46O9, and it was composed of one octanoyl group and two hexanoyl groups to myo-inositol moiety. The new biosurfactant is the first report of a compound produced by a new yeast strain, JAF-11.

Triterpenoid saponins from Camellia sinensis roots with cytotoxic and immunomodulatory effects

Camellia sinensis L. (Theaceae) leaves have been used as a beverage in both Eastern and Western cultures for a long time, while its root has not been intensively studied. In this study, seven undescribed triterpenoid saponins (1–7) and twelve known saponins (8–19) with different combinations of substituents, such as oxygenated isoprenyl substituents and sugar moieties, and lengths of sugar chains, were isolated from the C. sinensis roots. Their structures were unequivocally determined using one- and two-dimensional nuclear magnetic resonance data and acid hydrolysis analysis. Investigation of the biological activities of isolated compounds revealed that only those without functional acetyl groups exhibited cytotoxic activities against mouse and human cancer cells (B16F10) and human cervical cancer cell line (HeLa) at 50 μM. Compounds with an aldehyde group at C-23 of aglycone showed immunomodulatory activity against Th1 and Th17 cells at 10 μM. Ten compounds with biological activities from C. sinensis roots extracts, including three previously undescribed ones (3, 6, and 7), were identified.

Helicobacter pylori Modulates Heptose Metabolite Biosynthesis and Heptose-Dependent Innate Immune Host Cell Activation by Multiple Mechanisms.

Heptose metabolites including ADP-d-glycero-β-d-manno-heptose (ADP-heptose) are involved in bacterial lipopolysaccharide and cell envelope biosynthesis. Recently, heptoses were also identified to have potent proinflammatory activity on human cells as novel microbe-associated molecular patterns. The gastric pathogenic bacterium Helicobacter pylori produces heptose metabolites, which it transports into human cells through its Cag type 4 secretion system. Using H. pylori as a model, we have addressed the question of how proinflammatory ADP-heptose biosynthesis can be regulated by bacteria. We have characterized the interstrain variability and regulation of heptose biosynthesis genes and the modulation of heptose metabolite production by H. pylori, which impact cell-autonomous proinflammatory human cell activation. HldE, a central enzyme of heptose metabolite biosynthesis, showed strong sequence variability between strains and was also variably expressed between strains. Amounts of gene transcripts in the hldE gene cluster displayed intrastrain and interstrain differences, were modulated by host cell contact and the presence of the cag pathogenicity island, and were affected by carbon starvation regulator A (CsrA). We reconstituted four steps of the H. pylori lipopolysaccharide (LPS) heptose biosynthetic pathway in vitro using recombinant purified GmhA, HldE, and GmhB proteins. On the basis of one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry, the structures of major reaction products were identified as β-d-ADP-heptose and β-heptose-1-monophosphate. A proinflammatory heptose-monophosphate variant was also identified for the first time as a novel cell-active product in H. pylori bacteria. Separate purified HldE subdomains and variant HldE allowed us to uncover additional strain variation in generating heptose metabolites. IMPORTANCE Bacterial heptose metabolites, intermediates of lipopolysaccharide (LPS) biosynthesis, are novel microbe-associated molecular patterns (MAMPs) that activate proinflammatory signaling. In the gastric pathogen Helicobacter pylori, heptoses are transferred into host cells by the Cag type IV secretion system, which is also involved in carcinogenesis. Little is known about how H. pylori, which is highly strain variable, regulates heptose biosynthesis and downstream host cell activation. We report here that the regulation of proinflammatory heptose production by H. pylori is strain specific. Heptose gene cluster activity is modulated by the presence of an active cag pathogenicity island (cagPAI), contact with human cells, and the carbon starvation regulator A. Reconstitution with purified biosynthesis enzymes and purified bacterial lysates allowed us to biochemically characterize heptose pathway products, identifying a heptose-monophosphate variant as a novel proinflammatory metabolite. These findings emphasize that the bacteria use heptose biosynthesis to fine-tune inflammation and also highlight opportunities to mine the heptose biosynthesis pathway as a potential therapeutic target against infection, inflammation, and cancer.

Quantitative 2D 1H, 13C HSQC NMR Spectroscopy for the Determination of Chondroitin Sulfate and Dermatan Sulfate Content in Danaparoid Sodium.

Danaparoid sodium is a biopolymeric complex drug composed of the most abundant heparan sulfate (HS) followed in descending order by dermatan sulfate (DS) and chondroitin sulfate (CS). This composite nature explains its peculiar antithrombotic and anticoagulant properties and make it particularly advantageous when the risk of heparin-induced thrombocytopenia occurs. A specific control of the danaparoid composition is required by the Ph. Eur. The monograph includes the CS and DS limit contents and describes the method for their quantification through selective enzymatic degradations. In this study, a quantitative two-dimensional nuclear magnetic resonance (NMR) method is proposed as a new method suitable for CS and DS quantification. Statistical comparison of the results provided by the analysis of a series of danaparoid samples with both NMR and enzymatic methods highlights a small systematic difference, likely derived from lyase-resistant sequences bearing oxidized terminals. Some modified structures, whose survival to the enzymatic action was confirmed by mass spectrometry, can be detected and quantified by NMR. The proposed NMR method can serve for the determination of DS and CS contents, is an easy-to-apply method with no dependence from enzymes and standards, and provides extensive structural information on the overall glycosaminoglycans mixture.

Separation and structural characterization of unknown impurity in vancomycin by two-dimensional preparative liquid chromatography, LC-MS and NMR

Vancomycin is an effective antibiotic used for the treatment of Gram-positive bacterial infections. During the analysis of vancomycin, an unknown impurity at the level of 0.5% was detected by high-performance liquid chromatography (HPLC). To characterize the structure of the impurity, a new two-dimensional preparative liquid chromatography (2D-Prep-LC) method was developed to separate the impurity from the vancomycin sample. After further analysis including liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy, the structure of the unknown impurity was identified as a vancomycin analog in which the N-Methyl-leucine residue on the side chain is replaced by an N-methylmethionine residue. In this study, we established a reliable and efficient method for separating and identifying vancomycin impurities, which will provide a valuable contribution to the field of pharmaceutical analysis and quality control.