Gelatin Methacryloyl (GelMA) Hydrogel Scaffolds: Predicting Physical Properties Using an Experimental Design Approach.

There is a growing interest for complex in vitro environments that closely mimic the extracellular matrix and allow cells to grow in microenvironments that are closer to the one in vivo. Protein-based matrices and especially hydrogels can answer this need, thanks to their similarity with the cell microenvironment and their ease of customization. In this study, an experimental design was conducted to study the influence of synthesis parameters on the physical properties of gelatin methacryloyl (GelMA). Temperature, ratio of methacrylic anhydride over gelatin, rate of addition, and stirring speed of the reaction were studied using a Doehlert matrix. Their impact on the following parameters was analyzed: degree of substitution, mass swelling ratio, storage modulus (log(G')), and compression modulus. This study highlights that the most impactful parameter was the ratio of methacrylic anhydride over gelatin. Although, temperature affected the degree of substitution, and methacrylic anhydride addition flow rate impacted the gel's physical properties, namely, its storage modulus and compression modulus. Moreover, this experimental design proposed a theoretical model that described the variation of GelMA's physical characteristics as a function of synthesis conditions.

The Molecular Bases of the Interaction between a Saponin from the Roots of Gypsophila paniculata L. and Model Lipid Membranes.

In view of the possible medical applications of saponins, the molecular structure of a GOTCAB saponin from the roots of Gypsophila paniculata L. was determined by NMR. The biological activity of saponins may depend on the interaction with cell membranes. To obtain more insight in the mechanism of membrane-related saponin function, an experimental and theoretical study was conducted. Ternary lipid systems composed of sphingomyelin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and cholesterol were used as models of mammalian cell membranes. The membrane-saponin interaction was studied experimentally by monitoring surface pressure in the monomolecular films formed at the air-aqueous subphase interface. The behavior of GOTCAB saponin in a water box and model monolayer systems was characterized by molecular dynamics simulations. The results obtained showed that, in the systems used, cholesterol had a decisive effect on the interaction between GOTCAB and phosphocholine or sphingomyelin as well as on its location within the lipid film.

Molecular Identification of Endophytic Bacteria in Leucojum aestivum In Vitro Culture, NMR-Based Metabolomics Study and LC-MS Analysis Leading to Potential Amaryllidaceae Alkaloid Production.

In this study, endophytic bacteria belonging to the Bacillus genus were isolated from in vitro bulblets of Leucojum aestivum and their ability to produce Amaryllidaceae alkaloids was studied. Proton Nuclear Magnetic Resonance (1H NMR)-based metabolomics combined with multivariate data analysis was chosen to compare the metabolism of this plant (in vivo bulbs, in vitro bulblets) with those of the endophytic bacteria community. Primary metabolites were quantified by quantitative 1H NMR (qNMR) method. The results showed that tyrosine, one precursor of the Amaryllidaceae alkaloid biosynthesis pathway, was higher in endophytic extract compared to plant extract. In total, 22 compounds were identified including five molecules common to plant and endophyte extracts (tyrosine, isoleucine, valine, fatty acids and tyramine). In addition, endophytic extracts were analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS) for the identification of compounds in very low concentrations. Five Amaryllidaceae alkaloids were detected in the extracts of endophytic bacteria. Lycorine, previously detected by 1H NMR, was confirmed with LC-MS analysis. Tazettine, pseudolycorine, acetylpseudolycorine, 1,2-dihydro-chlidanthine were also identified by LC-MS using the positive ionization mode or by GC-MS. In addition, 11 primary metabolites were identified in the endophytic extracts such as tyramine, which was obtained by decarboxylation of tyrosine. Thus, Bacillus sp. isolated from L. aestivum bulblets synthesized some primary and specialized metabolites in common with the L.aestivum plant. These endophytic bacteria are an interesting new approach for producing the Amaryllidaceae alkaloid such as lycorine.

Direct 1H NMR evidence of spin-rotation coupling as a source of para → ortho-H2 conversion in diamagnetic solvents.

At ambient temperature, conversion from 100% enriched para-hydrogen (p-H2; singlet state) to ortho-hydrogen (o-H2; triplet state) leads necessarily to the thermodynamic equilibrium proportions: 75% of o-H2 and 25% of p-H2. When p-H2 is dissolved in a diamagnetic organic solvent, conversion is very slow and can be considered as arising from nuclear spin relaxation phenomena. A first relaxation mechanism, specific to the singlet state and involving a combination of auto-correlation and cross correlation spectral densities, can be retained: randomly fluctuating magnetic fields due to inter-molecular dipolar interactions. We demonstrate here that (i) this dipolar mechanism is not sufficient for accounting for the para→ortho conversion rate, (ii) spin-rotation interaction, an intra-molecular mechanism, behaves similarly to random-field interaction and, thus, may be involved in the singlet relaxation rate. Also, as the para→ortho conversion is monitored by proton nuclear magnetic resonance (NMR) of dissolved o-H2 (p-H2 is NMR-silent), one has to account for H2 exchange between the liquid phase and the gas phase within the NMR tube, as well as for dissolution effects. Experimental evidence of the above statements is brought here in the case of two organic solvents: acetone-d6 and carbon disulfide. The observed temperature dependence of the para→ortho conversion rate shows that spin-rotation can be the dominant contribution to the p-H2 relaxation rate in the absence of tangible dipolar interactions. Our findings shed new light on the "mysterious" mechanism of the para→ortho conversion which has been searched for several decades.

Nanoliposomes Permeability in a Microfluidic Drug Delivery Platform across a 3D Hydrogel.

Nanoliposomes are nano-sized vesicles that can be used as drug delivery carriers with the ability to encapsulate both hydrophobic and hydrophilic compounds. Moreover, their lipid compositions facilitate their internalization by cells. However, the interaction between nanoliposomes and the membrane barrier of the human body is not well-known. If cellular tests and animal testing offer a solution, their lack of physiological relevance and ethical concerns make them unsuitable to properly mimic human body complexity. Microfluidics, which allows the environment of the human body to be imitated in a controlled way, can fulfil this role. However, existing models are missing the presence of something that would mimic a basal membrane, often consisting of a simple cell layer on a polymer membrane. In this study, we investigated the diffusion of nanoliposomes in a microfluidic system and found the optimal parameters to maximize their diffusion. Then, we incorporated a custom made GelMA with a controlled degree of substitution and studied the passage of fluorescently labeled nanoliposomes through this barrier. Our results show that highly substituted GelMA was more porous than lower substitution GelMA. Overall, our work lays the foundation for the incorporation of a hydrogel mimicking a basal membrane on a drug delivery microfluidic platform.

New application of proton nuclear spin relaxation unraveling the intermolecular structural features of low-molecular-weight organogel fibers.

Proton nuclear spin relaxation has been for the first time extensively used for a structural and dynamical study of low-molecular-weight organogels. The gelator in the present study is a modified phenylalanine amino acid bearing a naphthalimide moiety. From T(1) (spin-lattice relaxation time in the laboratory frame) and T(1ρ) (spin-lattice relaxation time in the rotating frame) measurements, it is shown that the visible gelator NMR spectrum below the liquid-gel transition temperature corresponds to a so-called isotropic compartment, where gelator molecules behave as in a liquid phase but exchange rapidly with the molecules constituting the gel structure. This feature allows one to derive, from accessible parameters, information about the gel itself. Nuclear Overhauser effect spectroscopy (NOESY) experiments have been exploited in view of determining not only cross-relaxation rates but also specific longitudinal rates. The whole set of relaxation parameters (at 25 °C) leads to a correlation time of 5 ns for gelator molecules within the gel structure and 150 ps for gelator molecules in the isotropic phase. This confirms, on one hand, the flexibility of the organogel fibers and, on the other hand, the likely presence of clusters in the isotropic phase. Concerning cross-relaxation rates, a thorough theoretical investigation in multispin systems of direct and relayed correlations in a NOESY spectrum allows one to make conclusions about contacts (around 2-3 Å) not only between naphtalimide moieties of different gelator molecules but also between the phenyl ring and the naphtalimide moiety again of different gelator molecules. As a result, not only is the head-to-tail structure of amino acid columns confirmed but also the entangling of nearby columns by the naphthalimide moieties is demonstrated.

On the Observation of 14N Quadrupole Resonance Transitions in Water Proton NMR Relaxometry Dispersion Curves: The Case of a Labile NH Grouping in a Semirigid Molecular Moiety.

The electric field gradient tensor (considered here at the level of a nitrogen nucleus) can be described by two parameters: the largest element in the (X,Y,Z) principal axis system, denoted by VZZ (leading to the nuclear quadrupole coupling), and the asymmetry parameter η = (|VYY| - |VXX|)/|VZZ| with |VZZ| > |VYY| > |VXX|. The frequencies of the three nitrogen-14 nuclear quadrupole resonance (NQR) transitions depend on both parameters but, for sensitivity reasons, their determination may be especially difficult and time consuming. For a partly rigid NH grouping with a labile proton, water nuclear magnetic resonance (NMR) relaxometry curves may exhibit these three transitions (dubbed quadrupolar dips or quadrupole relaxation enhancement (QRE)), provided that the NH grouping belongs to a moiety possessing a sufficient degree of ordering. Their line shape leads to the correlation time describing mainly the motion of the NH grouping (the proton of which being in exchange with water protons), and their amplitude can be interpreted in terms of an effective NH distance. This approach is applied to a hydrogel, where separate NQR lines are observed for the different types of water existing in this system. Furthermore, the analysis of experimental data allows one to determine the nuclear quadrupole coupling in the protonated and deprotonated forms of this molecular moiety involving a labile NH grouping.

Water behavior in mesoporous materials as studied by NMR relaxometry.

Water in mesoporous materials possessing a two-dimensional hexagonal structure has been studied by the variation of its NMR longitudinal relaxation time T(1) as a function of the static magnetic field value, or equivalently of the NMR measurement frequency. This technique, dubbed relaxometry, has been applied from 5 kHz (measurement frequency) up to 400 MHz with various instruments including a variable-field spectrometer operating between 8 and 90 MHz. Moreover, the range 0-5 kHz could be investigated by transverse relaxation, T(2) denoting the corresponding relaxation time, and relaxation in the rotating frame, T(1ρ) denoting the corresponding relaxation time. Measurements of proton relaxation rates (inverse of relaxation times) have been performed with H(2)O and HOD (residual protons of heavy water) at water volumes of 80%, 60%, and 40% relative to the porous volume. Comparison between H(2)O and HOD shows clearly that, above 1 MHz where both sets of data are superposed, relaxation is purely intermolecular and due to paramagnetic relaxation (dipolar interactions of water protons with unpaired electrons of paramagnetic entities). Below 1 MHz, it is possible to subtract the intermolecular contribution (given by HOD data) from H(2)O data so that one is left with intramolecular relaxation which is solely due to water reorientational motions. The analysis of these low-frequency data (in terms of Lorentzian functions) reveals two types of water within the pores: one interacting strongly with the surface and the other corresponding to a second layer. High-frequency data, which arise from paramagnetic relaxation, exhibit again two types of water. Due to their correlation times, one type is assigned to relatively free water within the pores while the other type corresponds to bulk (interparticular) water. Their proportions, given as a function of the volume fraction, are consistent with the above assignments.

Co-assembly and multicomponent hydrogel formation upon mixing nucleobase-containing peptides.

Peptide-based hydrogels are physical gels formed through specific supramolecular self-assembling processes, leading to ordered nanostructures which constitute the water entrapping scaffold of the soft material. Thanks to the inherent properties of peptides, these hydrogels are highly considered in the biomedical domain and open new horizons in terms of application in advanced therapies and biotechnologies. The use of one, and only one, native peptide to formulate a gel is by far the most reported approach to design such materials, but suffers from several limitations, including in terms of mechanical properties. To improve peptide-based hydrogels interest and give rise to innovative properties, several strategies have been proposed in the recent years, and the development of multicomponent peptide-based hydrogels appears as a promising and relevant strategy. Indeed, mixing two or more compounds to develop new materials is a much-used approach that has proven its effectiveness in a wide variety of domains, including polymers, composites and alloys. While still limited to a handful of examples, we would like to report herein on the formulation and the comprehensive study of multicomponent hybrid DNA-nucleobase/peptide-based hydrogels using a multiscale approach based on a large panel of analytical techniques (i.e., rheometry, proton relaxometry, SAXS, electronic microscopy, infrared, circular dichroism, fluorescence, Thioflavin T assays). Among the six multicomponent systems studied, the results highlight the synergistic role of the presence of the two complementary DNA-nucleobases (i.e., adenine/thymine and guanine/cytosine) on the co-assembling process from structural (e.g., morphology of the nanoobjects) to physicochemical (e.g., kinetics of formation, fluorescence properties) and mechanical (e.g., stiffness, resistance to external stress) properties. All the data confirm the relevance of the multicomponent peptide-based approach in the design of innovative hydrogels and bring another brick in the wall of the understanding of these complex and promising systems.

Polymer functionalization through an enzymatic process: Intermediate products characterization and their grafting onto gum Arabic.

The modification of gum Arabic with ferulic acid oxidation products was performed in aqueous medium, at 30 °C and pH 7.5, in the presence of Myceliophthora thermophila laccase as biocatalyst. First, this study aimed to investigate the structures of the oxidation products of ferulic acid that could possibly be covalently grafted onto gum Arabic. HPLC analyses revealed that this reaction produced several oxidation products, whose structures were investigated using LC-MS/MS analyses (liquid chromatography-mass spectrometry with mass fragmentation analyses) and NMR experiments. The chemical structure of one intermediate reaction product was fully elucidated as the 2-(4-hydroxy-3-methoxyphenyl)-4-[(4-hydroxy-3-methoxyphenyl) methylidene] cyclobutane-1, 3-dione, called by the authors cyclobutadiferulone. Secondly, this study aimed to locate the grafting of the oxidation products onto gum Arabic by performing several NMR experiments. This study did not determine how much and specifically which oxidation products were grafted but some of them were undeniably present onto modified gum Arabic, close to the glucuronic acid C5 carbon or close to the galactose C6 carbon.

Mn-Doped Quinary Ag-In-Ga-Zn-S Quantum Dots for Dual-Modal Imaging.

Doping of transition metals within a semiconductor quantum dot (QD) has a high impact on the optical and magnetic properties of the QD. In this study, we report the synthesis of Mn2+-doped Ag-In-Ga-Zn-S (Mn:AIGZS) QDs via thermolysis of a dithiocarbamate complex of Ag+, In3+, Ga3+, and Zn2+ and of Mn(stearate)2 in oleylamine. The influence of the Mn2+ loading on the photoluminescence (PL) and magnetic properties of the dots are investigated. Mn:AIGZS QDs exhibit a diameter of ca. 2 nm, a high PL quantum yield (up to 41.3% for a 2.5% doping in Mn2+), and robust photo- and colloidal stabilities. The optical properties of Mn:AIGZS QDs are preserved upon transfer into water using the glutathione tetramethylammonium ligand. At the same time, Mn:AIGZS QDs exhibit high relaxivity (r 1 = 0.15 mM-1 s-1 and r 2 = 0.57 mM-1 s-1 at 298 K and 2.34 T), which shows their potential applicability for bimodal PL/magnetic resonance imaging (MRI) probes.

Improving and fine-tuning the properties of peptide-based hydrogels via incorporation of peptide nucleic acids.

Peptide self-assemblies have attracted intense research interest over the last few decades thanks to their implications in key biological processes (e.g., amyloid formation) and their use in biotechnological and (bio)material fields. In particular, peptide-based hydrogels have been highly considered as high potential supramolecular materials in the biomedical domain and open new horizons in terms of applications. To further understand their self-assembly mechanisms and to optimize their properties, several strategies have been proposed with the modification of the constituting amino acid chains via, per se, the introduction of d-amino acids, halogenated amino acids, pseudopeptide bonds, or other chemical moieties. In this context, we report herein on the incorporation of DNA-nucleobases into their peptide nucleic acid (PNA) forms to develop a new series of hybrid nucleopeptides. Thus, depending on the nature of the nucleobase (i.e., thymine, cytosine, adenine or guanine), the physicochemical and mechanical properties of the resulting hydrogels can be significantly improved and fine-tuned with, for instance, drastic enhancements of both the gel stiffness (up to 70-fold) and the gel resistance to external stress (up to 40-fold), and the generation of both thermo-reversible and uncommon red-edge excitation shift (REES) properties. To decipher the actual role of each PNA moiety in the self-assembly processes, the induced modifications from the molecular to the macroscopic scales are studied thanks to the multiscale approach based on a large panel of analytical techniques (i.e., rheology, NMR relaxometry, TEM, thioflavin T assays, FTIR, CD, fluorescence, NMR chemical shift index). Thus, such a strategy provides new opportunities to adapt and fit hydrogel properties to the intended ones and pushes back the limits of supramolecular materials.

Location of a metallic cation complexed in a calixarene cavity as determined by calixarene 13C spin relaxation. application to cesium and thallium complexed by p-sulfonatocalix[4]arene in water.

This study deals with the exact location of the monovalent metal cations Cs(+) and Tl(+) which are complexed by the p-sulfonatocalix[4]arene in water. This determination rests on the measurements of longitudinal relaxation times of carbon-13 not directly bonded to protons. The difference between the relaxation times of the free calixarene and of the complex definitely demonstrates that the monovalent metal cation is well inside the calixarene cavity. These features are in fact enhanced by the presence of paramagnetic species which act in a different way in the complexed form. Experimental results also show without any ambiguity that the calixarene cavity is essentially hydrophobic. Finally, it is observed that thallium is more mobile than cesium within the calixarene cavity.

Behavior of cesium and thallium cations inside a calixarene cavity as probed by nuclear spin relaxation. Evidence of cation-pi interactions in water.

We have studied the complexes formed between the p-sulfonatocalix[4]arene and cesium or thallium metal cation, first by carbon-13 longitudinal relaxation of the calixarene molecule at two values of the magnetic field B(0). From the longitudinal relaxation times of an aromatic carbon directly bonded to a proton, thus subjected essentially to the dipolar interaction with that proton, we could obtain the correlation time describing the reorientation of the CH bond. The rest of this study has demonstrated that it is also the correlation time describing the tumbling of the whole calixarene assembly. From three non-proton-bearing carbons of the aromatic cycles (thus subjected to the chemical shift anisotropy and dipolar mechanisms), we have been able to determine the variation of the chemical shift anisotropy when going from the free to the complex form of the calixarene. These variations not only provide the location of the cation inside the calixarene cavity but also constitute a direct experimental proof of the cation-pi interactions. These results are complemented by cesium and thallium relaxation measurements performed again at two values of the magnetic field B(0). An estimation of the mean distance between the cation and the calixarene protons could be obtained. These measurements have also revealed an important chemical shift anisotropy of thallium upon complexation.

Effect of the static magnetic field strength on parahydrogen induced polarization NMR spectra.

Spin polarization transfer from parahydrogen (p-H(2)) to another molecular entity is generally thought to be mediated by longitudinal spin order (represented by the operator product I(z)(A)I(z)(B), A and B being the two hydrogen nuclei which originate from p-H(2) after a hydrogenation reaction). The longitudinal spin order leads to antiphase patterns in the proton NMR spectrum. In addition to these antiphase patterns, in-phase patterns, arising from polarization differences (represented by (I(z)(A)-I(z)(B))), have been experimentally observed. A complete theory, based on a density operator treatment, has been worked out and applied to the two types of parahydrogen induced polarization experiments: PASADENA (PArahydrogen and Synthesis Allow Dramatically Enhanced Nuclear Alignment; hydrogenation reaction inside the NMR magnet) and (ALTADENA) (Adiabatic Longitudinal Transport After Dissociation Engenders Nuclear Alignment; hydrogenation reaction outside the NMR magnet). It is shown that polarization differences are always created in the case of a PASADENA experiment but that their amplitude depends critically on the ratio of the J coupling over the frequency difference between A and B. In the case of an ALTADENA experiment, if the sample is slowly transferred toward the NMR magnet, polarization differences are definitely created and their amplitude can be larger than the amplitude of the longitudinal spin order. Some test experiments demonstrate the validity of the proposed theory.

Increased synthesis of a new oleanane-type saponin in hairy roots of marigold (Calendula officinalis) after treatment with jasmonic acid.

Native plant of marigold (Calendula officinalis L.) synthesizes oleanolic acid saponins classified as glucosides or glucuronides according to the first residue in sugar chain bound to C-3 hydroxyl group. Hairy root culture, obtained by transformation with Agrobacterium rhizogenes strain 15834, exhibit a potent ability of synthesis of oleanolic acid glycosides. The HPLC profile of saponin fraction obtained from C. officinalis hairy roots treated with plant stress hormone, jasmonic acid, showed the 10-times increase of the content of one particular compound, determined by NMR and MALDI TOF as a new bisdesmoside saponin, 3-O-ß-d-glucuronopyranosyl-28-O-ß-d-galactopyranosyl-oleanolic acid. Such a diglycoside does not occur in native C. officinalis plant. It is a glucuronide, whereas in the native plant glucuronides are mainly accumulated in flowers, while glucosides are the most abundant saponins in roots. Thus, our results revealed that the pathways of saponin biosynthesis, particularly reactions of glycosylation, are altered in C. officinalis hairy root culture.

High-Relaxivity Gd(III)-Hemicryptophane Complex.

The polytopic hemicryptophane cage HC1 combining a cyclotriveratrylene (CTV) unit and a tris(2-aminoethyl)amine (tren) moiety connected by three 2-hydroxyisophthalamide linkers was synthesized in 12 steps. The resulting highly functionalized covalent host is soluble in aqueous medium and has been used to complex Gd(III) ion. The Gd(III)@HC1 complex presents promising relaxivity properties when compared to the clinically used Dotarem MRI agent.

Solution structure and backbone dynamics of the reduced form and an oxidized form of E. coli methionine sulfoxide reductase A (MsrA): structural insight of the MsrA catalytic cycle.

Methionine sulfoxide reductases (Msr) reduce methionine sulfoxide (MetSO)-containing proteins, back to methionine (Met). MsrAs are stereospecific for the S epimer whereas MsrBs reduce the R epimer of MetSO. Although structurally unrelated, the Msrs characterized so far display a similar catalytic mechanism with formation of a sulfenic intermediate on the catalytic cysteine and a concomitant release of Met, followed by formation of at least one intramolecular disulfide bond (between the catalytic and a recycling cysteine), which is then reduced by thioredoxin. In the case of the MsrA from Escherichia coli, two disulfide bonds are formed, i.e. first between the catalytic Cys51 and the recycling Cys198 and then between Cys198 and the second recycling Cys206. Three crystal structures including E. coli and Mycobacterium tuberculosis MsrAs, which, for the latter, possesses only the unique recycling Cys198, have been solved so far. In these structures, the distances between the cysteine residues involved in the catalytic mechanism are too large to allow formation of the intramolecular disulfide bonds. Here structural and dynamical NMR studies of the reduced wild-type and the oxidized (Cys51-Cys198) forms of C86S/C206S MsrA from E. coli have been carried out. The mapping of MetSO substrate-bound C51A MsrA has also been performed. The data support (1) a conformational switch occurring subsequently to sulfenic acid formation and/or Met release that would be a prerequisite to form the Cys51-Cys198 bond and, (2) a high mobility of the C-terminal part of the Cys51-Cys198 oxidized form that would favor formation of the second Cys198-Cys206 disulfide bond.

Selective HOESY experiments for stereochemical determinations.

Heteronuclear Overhauser effect spectroscopy (HOESY) is a powerful method for tracking geometrical proximities between two heteronuclei (for instance, (1)H and (13)C, as this will be the case here). The method is based on cross-relaxation arising from dipolar interactions. Sensitivity permitting, it is applied in the 2D mode yielding all spatial correlations in a single experiment. Whenever sensitivity is not sufficient, it can be applied in the one-dimensional mode by selectively inverting one particular proton. In that case, it yields, from the carbon-13 spectrum, remote spatial correlations. The method has been employed here for the discrimination between two possible E or Z isomers in a medium-size molecule.

A new liquid chromatography-high resolution Orbitrap mass spectrometry-based strategy to characterize Glucuronide Oleanane-type Triterpenoid Carboxylic Acid 3, 28-O-Bidesmosides (GOTCAB) saponins.A case study of Gypsophila glomerata Pall ex M. B. (Caryophyllaceae).

Glucuronide Oleanane-type Triterpenoid Carboxylic Acid 3, 28-Bidesmosides (GOTCAB) saponins are bioactive natural compounds spread in Caryophyllidae. The high complexity of GOTCAB occurring as closely related isobaric and positional isomers is a challenge in their separation and identification. A new liquid chromatography - high resolution Orbitrap mass spectrometry acquisition strategy would be important for the structural elucidation of GOTCAB in plant extracts. In this study, the fragmentation behaviors of GOTCAB from methanol-aqueous root extract of Gypsophila glomerata Pall ex M. B. (Caryophyllaceae) were investigated using ultra high-performance liquid chromatography (UHPLC) coupled with hybrid quadrupole-Orbitrap high resolution mass spectrometry (HRMS). A new saponin was isolated and its structure was established by 1D and 2D-NMR spectroscopic experiments as 3-O-ß-D-galactopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-ß-D-glucuronopyranosyl gypsogenin 28-O-α-L-arabinopyranosyl-(1→3)-[ß-D-xylopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→2)-ß-D-fucopyranosyl ester. On the basis of the accurate mass measurements, fragmentation patterns in MS/MS analyses and comparison with previously isolated authentic references, a total of 41 GOTCAB saponins were identified or tentatively elucidated in G. glomerata roots, including 14 pairs of isobars. Possible fragmentation pathways for three groups of GOTCAB are suggested. The group I appeared to be GOTCAB of gypsogenin with two carbohydrate chains: a branched trisaccharide at C-3 and tri- to hexa-saccharide attached to C-28 of the aglycone through a deoxyhexose residue. Saponins with monoacetylated (group II) or sulphated (group III) C-28 chain were evidenced, as well as quillaic and oleanolic acid GOTCAB. Sixteen GOTCAB were previously not described. The content of Gypsophila prosaponins, gypsogenin 3-O-glucuronide (7.4079 ±â€¯0.0723 mg/g dry weight, dw) and quillaic acid 3-O-glucuronide (4.4593 ±â€¯0.1207 mg/g dw), was determined by solid phase extraction - high-performance liquid chromatography (SPE-HPLC). In this study is presented the first systematic investigation on the fragmentation patterns and diagnostic fingerprints of the fragment ions in the MS/MS spectra of the gypsogenin -, quillaic acid - and oleanolic acid - bidesmosides. A LC-HRMS Orbitrap acquisition strategy could give an insight in the GOTCAB containing taxa.