Lars I. Nord

Separation and structural analysis of saponins in a bark extract from Quillaja saponaria Molina

Six major saponins were isolated from a bark extract from Quillaja saponaria Molina. Solid-phase extraction, followed by a two-step reversed-phase HPLC separation procedure with phosphate and ammonium acetate buffers of different pH values, was used. The compounds were characterised using NMR spectroscopy, mass spectrometry and chemical methods.

A novel method for examination of the variable contribution to computational neural network models

Abstract Computational neural networks (CNNs or, as they are commonly referred to; artificial neural networks, ANNs) have been demonstrated in a large number of applications to be useful for modeling and prediction. They suffer, however, in their conventional use, that is feed forward/back-propagation of the error, from the lack of a simple or straightforward means of interpreting the variable contribution to the models. CNNs are therefore often referred to as black box models. In this study novel algorithmic approaches to the interpretation of CNN models are proposed, examined and compared with the corresponding variable contribution in partial least squares (PLS) regression models. A sensitive analysis of the CNN models is carried out by sequentially setting each input variable to zero. In addition, to evaluate the direction of the variable contribution, the linear regression coefficients for each input variable are generated. The results of these two approaches are then combined to facilitate comparison with PLS models. CNN models for data on chiral separation, 3D-QSRR (quantitative structure–retention relationships) and SIMS (secondary ion mass spectroscopy) are used to demonstrate the feasibility of the method. For the latter two data sets, there is close agreement between the PLS and CNN models with regard to variable contribution. For the nonlinear data set for chiral separation, differences in variable contribution are revealed.

Modification and cross-linking parameters in hyaluronic acid hydrogels—Definitions and analytical methods

Definitions and methods for the quantification of degree of modification and cross-linking in cross-linked hyaluronic acid (HA) hydrogels are outlined. A novel method is presented in which the HA hydrogel is degraded by the enzyme chondroitinase AC and the digest product analyzed by size exclusion chromatography combined with electrospray ionization mass spectrometry (SEC-ESI-MS). This method allows for the determination of effective cross-linker ratio (CrR) which together with the degree of modification (MoD), determined by, e.g. (1)H NMR spectroscopy, enables the calculation of the degree of substitution (DS) and degree of cross-linking (CrD). The method, could be applicable to the major cross-linked HA hydrogels currently on the market, and is exemplified here by application to two HA hydrogels. The definitions and methods presented are important contributions in attempts to find relationships between MoD, DS and CrD to mechanical properties as well as to biocompatibility of HA hydrogels.

Prediction of liquid chromatographic retention times of steroids by three-dimensional structure descriptors and partial least squares modeling

Abstract Prediction of the retention times of steroids in two liquid chromatographic systems, i.e., straight- and reversed-phase, has been modeled by means of different quantitative structure-retention relationship (QSRR) methods. The 3D-QSRR descriptors were generated by a process including semiempirical structure optimization, structure alignments, and electronic 3D field descriptions by the GRID method or by a novel method based on summation of the electronic charges at grid points ( Q -field). The compound data set was split into a calibration set and a test set using the self-organizing feature map (SOFM) technique. The correlation between the molecular descriptors and the retention time of the compounds in the calibration set was established using the partial least squares (PLS) method. Optimal predictive models were generated by a cross-validation procedure. These models were then used to predict the retention times of the compounds in the independent test set. A further check on the validity of the models was obtained by back-projection of the most important variables of the models onto the molecular structure. In addition, the predictive capability of variable-reduced models was examined. The various 3D-QSRR models generated gave for the reversed-phase system Q prediction 2 values in the range 0.60–0.79 for the test set, the corresponding Q prediction 2 values for the straight-phase system being in the range 0.42–0.75. In the former case, variable-reduced models resulted in considerably better predictions, although these were not as good as for those models obtained by means of classical physical–chemical descriptors.

Novel acetylated triterpenoid saponins in a chromatographic fraction from Quillaja saponaria Molina

Six novel fucose 3-O-acetylated saponins, with a quillaic acid aglycone, were isolated from a bark extract from the Quillaja saponaria Molina tree. In addition, a saponin with a novel aglycone (phytolaccagenic acid) and a novel fatty acyl group [(S)-2-methylbutanoyl] for Quillaja saponins was found. The compounds were characterised using NMR spectroscopy, mass spectrometry and chemical methods.

Structural classification of acyl‐substituted Quillaja saponins by electrospray ionisation ion trap multiple‐stage mass spectrometry in combination with multivariate analysis

Thirty-eight saponins in two chromatographic fractions (QH-B and QH-C) from Quillaja saponaria Molina have been separated by a two-step high-performance liquid chromatography (HPLC) procedure and investigated by electrospray ionisation ion trap multiple-stage mass spectrometry (ESI-ITMS(n)) in positive ion mode. MS(2) and MS(3) spectra of the compounds were investigated by principal component analysis (PCA) and could be classified by partial least squares - discriminant analysis (PLS-DA) according to the structures of the oligosaccharides at C-3 and C-28 of the saponins. Four minor components with novel structures were found in a previously non-investigated fraction of QH-C. The structures of two of these components, J1 and J1a, were predicted by PLS-DA whereas the structures of the two others, J2 and J3, were only partly predicted. The structures of J1 and J1a were composed of structural elements found in the 34 known saponins whereas a new acyl substituent, not included in the training set used for calibration of the PLS-DA models, was found in J2 and J3, making these two components outliers. The complete structures of the four components were confirmed by monosaccharide analysis, MS(n) data and (1)H NMR spectroscopy.

Determination of substitution positions in hyaluronic acid hydrogels using NMR and MS based methods

In hydrogels of cross-linked polysaccharides, the total amount of cross-linker and the degree of cross-linking influence the properties of the hydrogel. The substitution position of the cross-linker on the polysaccharide is another parameter that can influence hydrogel properties; hence methods for detailed structural analysis of the substitution pattern are required. NMR and LC-MS methods were developed to determine the positions and amounts of substitution of 1,4-butanediol diglycidyl ether (BDDE) on hyaluronic acid (HA), and for the first time it is shown that BDDE can react with any of the four available hydroxyl groups of the HA disaccharide repeating unit. This was achieved by studying di-, tetra-, and hexasaccharides obtained from degradation of BDDE cross-linked HA hydrogel by chondroitinase. Furthermore, amount of linker substitution at each position was shown to be dependent on the size of the oligosaccharides. For the disaccharide, substitutions were predominantly at ΔGlcA-OH2 and GlcNAc-OH6 while in the tetra- and hexasaccharides, it was mainly at the reducing end GlcNAc-OH4. In the disaccharide there was no substitution at this position. Since chondroitinase is able to completely hydrolyse non-substituted HA into unsaturated disaccharides, these results indicate that the enzyme is prevented to cleave on the non-reducing side of an oligosaccharide substituted at the reducing end GlcNAc-OH4. The procedure can be adopted for the determination of substitution positions in other types of polymers.

Electrospray ionization ion-trap multiple-stage mass spectrometry of Quillaja saponins

Fifteen identified C-18 fatty acyl-containing saponin structures from Quillaja saponaria Molina have been investigated by electrospray ionization ion-trap multiple-stage mass spectrometry (ESI-IT-MS(n)) in positive ion mode. Their MS(1)-MS(3) spectra were analyzed and ions corresponding to useful fragments, important for the structural identification of Quillaja saponins, were recognized. A few key fragments could describe the structural variations in the C-3 and the C-28 oligosaccharides of the Quillaja saponins. A flowchart involving a stepwise procedure based on key fragments from the MS(1)-MS(3) spectra of these saponins, together with key fragments from these saponins and 13 previously investigated saponins, was constructed for the identification of structural elements in Quillaja saponins. Peak intensity ratios in MS(3) spectra were found to be correlated to structural features of the investigated saponins and is therefore of value for the identification of regioisomers.

Multidimensional profiling of components in complex mixtures of natural products for metabolic analysis, proof of concept: application to Quillaja saponins.

A method for separation and detection of major and minor components in complex mixtures has been developed, utilising two-dimensional high-performance liquid chromatography (2D-HPLC) combined with electrospray ionisation ion-trap multiple-stage mass spectrometry (ESI-ITMS(n)). Chromatographic conditions were matched with mass spectrometric detection to maximise the number of components that could be separated. The described procedure has proven useful to discern several hundreds of saponin components when applied to Quillaja saponaria Molina bark extracts. The discrimination of each saponin component relies on the fact that three coordinates (x, y, z) for each component can be derived from the retention time of the two chromatographic steps (x, y) and the m/z-values from the multiple-stage mass spectrometry (z(n), n=1, 2, ...). Thus an improved graphical representation was obtained by combining retention times from the two-stage separation with +MS(1) (z(1)) and the additional structural information from the second mass stage +MS(2) (z(2), z(3)) corresponding to the main fragment ions. By this approach three-dimensional plots can be made that reveal both the chromatographic and structural properties of a specific mixture which can be useful in fingerprinting of complex mixtures.

1D NMR methods for determination of degree of cross-linking and BDDE substitution positions in HA hydrogels.

Hyaluronic acid polymers cross-linked with BDDE are today among the most used hydrogels for biomedical applications. The physical properties of the hydrogels depend, among other parameters, on the degree of cross-linking of HA. Another parameter likely to affect the physical properties is the substitution position of the linker on the HA functional groups. A NMR-based method for the determination of these parameters in hyaluronic acid hydrogels is presented. The method is based on the degradation of HA cross linked hydrogels by chondroitinase ABC followed by one-dimensional 1H and 13C NMR analysis. The necessary structural information to obtain both the degree of cross-linking and the substitution positions can be obtained from the same NMR sample and no chromatographic separation step is required prior to NMR analysis.