Pushing the limits: Quantification of chromophores in real-world paper samples by GC-ECD and EI-GC-MS.

Widening the methodology of chromophore analysis in pulp and paper science, a sensitive gas-chromatographic approach with electron-capture detection is presented and applied to model samples and real-world historic paper material. Trifluoroacetic anhydride was used for derivatization of the chromophore target compounds. The derivative formation was confirmed by NMR and accurate mass analysis. The method successfully detects and quantifies hydroxyquinones which are key chromophores in cellulosic matrices. The analytical figures of merit appeared to be in an acceptable range with an LOD down to approx. 60ng/g for each key chromophore, which allows for their successful detection in historic sample material.

Aging of paper - Ultra-fast quantification of 2,5-dihydroxyacetophenone, as a key chromophore in cellulosics, by reactive paper spray-mass spectrometry.

The detection of individual chromophores that contribute to the overall discoloration of paper or pulp ("yellowing") is a challenge because these substances are only present in extremely small amounts (ppm to ppb range). In this work, paper spray (PS) coupled with mass spectrometry was used to detect a low-concentrated cellulosic key-chromophore, 2,5-dihydroxyacetophenone (DHAP). Sensitivity was enhanced by derivatization with Girard's reagent T (GT). DHAP was successfully detected in historic paper samples and also was applied to model papers in order to investigate different factors that influence its generation: temperature, time, relative humidity, and the presence of iron ions, by means of a full factorial design. The main factors, temperature and relative humidity, have the most impact on the generation of DHAP, but the interactions between the factors are also significant and are therefore important for the degradation process. The historical papers containing DHAP were then compared to the artificially aged samples. The results were confirmed by independent, accurate mass measurements.

Filling the gap: Calibration of the low molar-mass range of cellulose in size exclusion chromatography with cello-oligomers.

Degraded celluloses are becoming increasingly important as part of product streams coming from various biorefinery scenarios. Analysis of the molar mass distribution of such fractions is a challenge, since neither established methods for mono- or disaccharides nor common methods for polysaccharide characterization cover the intermediate oligomer range appropriately. Size exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS), the standard approach for celluloses, suffers from decreased scattering intensities in the lower-molar mass range. The limitation in the low-molecular range can, in principle, be overcome by calibration, but calibration standards for such "short" celluloses are either not readily available or structurally remote and thus questionable. In this paper, we present the calibration of a SEC system- for the first time - with monodisperse cellooligomer standards up to about 3400gmol-1. These cellooligomers are "short-chain celluloses" and can be seen as the "true" standard compounds, by contrast to commonly used standards that are chemically different from cellulose, such as pullulan, dextran, polystyrene, or poly(methyl methacrylate). The calibration is compared against those commercial standards and correction factors are calculated. Calibrations with non-cellulose standards can now be adjusted to yield better fitting results, and data already available can be corrected retrospectively.

A novel method to analyze the degree of acetylation in biopolymers.

A novel approach to measure the degree of acetylation in biopolymers applying a combination of Zemplén-deacetylation by sodium methanolate and GC-MS methodology is introduced. The development focuses on very low limits of detection to cover also samples with extremely low degrees of acetylation which hitherto eluded accurate determination. Free acetic acid or inorganic acetates, often present in biopolymer samples, do not disturb the quantification. Two techniques to measure the Zemplén-released methyl acetate were comparatively assessed, direct injection of the liquid phase and a SPME-based approach, the former being more straightforward, but being inferior to the latter in sensitivity. By applying isotopically labeled methyl acetate released from 4-O-(13C2-acetyl)-vanillin as the internal standard, influences, such as varying moisture contents, are corrected, improving the overall method reliability to a large extent. The combination of Zemplén-release of acetyl groups in biopolymers as methyl acetate, in connection with its accurate quantification by SPME-GC-MS, was found to be the method of choice for routine, yet very accurate analysis of a wide range of acetylation degrees of biopolymers, showing satisfying analytical parameters along with easy handling and widest applicability. Limit of detection for acetylated cellulose samples is 0.09nmol/mg, for hemicellulose samples 0.48nmol/mg.

Reinforcement of bacterial cellulose aerogels with biocompatible polymers.

Bacterial cellulose (BC) aerogels, which are fragile, ultra-lightweight, open-porous and transversally isotropic materials, have been reinforced with the biocompatible polymers polylactic acid (PLA), polycaprolactone (PCL), cellulose acetate (CA), and poly(methyl methacrylate) (PMMA), respectively, at varying BC/polymer ratios. Supercritical carbon dioxide anti-solvent precipitation and simultaneous extraction of the anti-solvent using scCO2 have been used as core techniques for incorporating the secondary polymer into the BC matrix and to convert the formed composite organogels into aerogels. Uniaxial compression tests revealed a considerable enhancement of the mechanical properties as compared to BC aerogels. Nitrogen sorption experiments at 77K and scanning electron micrographs confirmed the preservation (or even enhancement) of the surface-area-to-volume ratio for most of the samples. The formation of an open-porous, interpenetrating network of the second polymer has been demonstrated by treatment of BC/PMMA hybrid aerogels with EMIM acetate, which exclusively extracted cellulose, leaving behind self-supporting organogels.

Ethoximation-silylation approach for mono- and disaccharide analysis and characterization of their identification parameters by GC/MS.

The qualitative and quantitative analysis of complex carbohydrate mixtures is a challenging problem. When tackled by GC/MS, close retention times and largely similar mass spectra with no specific features complicate unambiguous identification, especially of monosaccharides. An optimized pre-capillary ethoximation-silylation GC/MS method for determination of monosaccharides and disaccharides was applied to a wide range of analytes (46 compounds). The two-step derivatization resulted in a pair of syn and anti peaks with specific retention and intensity ratio. The resulting dataset of mass spectra was subjected to a PCA-based pattern recognition. An oxime peak identifier (OPI) of the carbohydrate analytes, based on the combination of an internal standard and the corresponding syn/anti peak ratios, increased the reliability of the identification of reducing carbohydrates. Finally, the introduced EtOx-TMS derivatization method was applied to four different carbohydrate matrices (agave sirup, maple sirup, palm sugar, and honey).

Evaluation of different derivatisation approaches for gas chromatographic-mass spectrometric analysis of carbohydrates in complex matrices of biological and synthetic origin.

Gas chromatographic analysis of complex carbohydrate mixtures requires highly effective and reliable derivatisation strategies for successful separation, identification, and quantitation of all constituents. Different single-step (per-trimethylsilylation, isopropylidenation) and two-step approaches (ethoximation-trimethylsilylation, ethoximation-trifluoroacetylation, benzoximation-trimethylsilylation, benzoximation-trifluoroacetylation) have been comprehensively studied with regard to chromatographic characteristics, informational value of mass spectra, ease of peak assignment, robustness toward matrix effects, and quantitation using a set of reference compounds that comprise eight monosaccharides (C(5)-C(6)), glycolaldehyde, and dihydroxyacetone. It has been shown that isopropylidenation and the two oximation-trifluoroacetylation approaches are least suitable for complex carbohydrate matrices. Whereas the former is limited to compounds that contain vicinal dihydroxy moieties in cis configuration, the latter two methods are sensitive to traces of trifluoroacetic acid which strongly supports decomposition of ketohexoses. It has been demonstrated for two "real" carbohydrate-rich matrices of biological and synthetic origin, respectively, that two-step ethoximation-trimethylsilylation is superior to other approaches due to the low number of peaks obtained per carbohydrate, good peak separation performance, structural information of mass spectra, low limits of detection and quantitation, minor relative standard deviations, and low sensitivity toward matrix effects.

Synthetic chromanol derivatives and their interaction with complex III in mitochondria from bovine, yeast, and Leishmania.

Synthetic chromanol derivatives (TMC4O, 6-hydroxy-2,2,7,8-tetramethyl-chroman-4-one; TMC2O, 6-hydroxy-4,4,7,8-tetramethyl-chroman-2-one; and Twin, 1,3,4,8,9,11-hexamethyl-6,12-methano-12H-dibenzo[d,g][1,3]dioxocin-2,10-diol) share structural elements with the potent inhibitor of the mitochondrial cytochrome (cyt) bc(1) complex stigmatellin. Studies with isolated bovine cyt bc(1) complex demonstrated that these compounds partially inhibit the mammalian enzyme. The aim of this work was to comparatively investigate these toxicological aspects of synthetic vitamin E derivatives in mitochondria of different species. The chromanols and atovaquone as reference compound were evaluated for their inhibition of the cyt bc(1) activity in mitochondrial fractions from bovine hearts, yeast, and Leishmania. In addition, compounds were evaluated in vitro for their inhibitory activity against whole-cell Leishmania and mouse peritoneal macrophages. In these organisms, the chromanols showed a species-selective inhibition of the cyt bc(1) activity different from that of atovaquone. While in atovaquone the side chain mediates species-selectivity, the marked differences for TMC2O and TMC4O in cyt bc(1) inhibition suggests that direct substitution of the chromanol headgroup will control selectivity in these compounds. Low micromolar concentrations of TMC2O (IC(50) = 9.5 ± 0.5 µM) inhibited the growth of Leishmania, and an esterified TMC2CO derivative inhibited the cyt bc(1) activity with an IC(50) of 4.9 ± 0.9 µM. These findings suggest that certain chromanols also exhibit beyond their antioxidative properties antileishmanial activities and that TMC2O derivatives could be useful toward the development of highly active antiprotozoal compounds.

Ubiquinone and tocopherol: dissimilar siblings.

Research on antioxidants and their potential health benefits expanded over the last decades from basic science to the medical and nutritional fields. This included supplementation studies of both vitamin E compounds and the endogenous antioxidant ubiquinone, to prevent or alleviate cardiovascular diseases and their pathophysiological consequences. In many of these studies, only one antioxidant or one group of antioxidants was considered, disregarding the pharmacological and toxicological properties of their metabolites as well as possible cooperative and competitive effects on the overall physiological response. There are many--often indirect--effects, especially in gene regulation, observed on administration of both compound groups in cells, which have been assigned to these molecules without identifying the cellular targets. Therefore, this article focuses on direct chemical and biochemical effects of ubiquinone- and alpha-tocopherol-related compounds, which are evident from direct binding studies or direct interaction leading to chemical modification of the compounds. These groups include para-benzoquinones (ubiquinone and alpha-tocopheryl quinone) and chroma(e)nols (alpha-tocopherol and bicyclic ubiquinone derivatives). Their effects as antioxidants, co-antioxidants, and pro-oxidants as well as direct interactions with proteins are considered, pointing out similarities and dissimilarities of the two compound groups in a wider context. The review of the isolated findings about one or a few of these compounds in the literature, disregarding structurally related compounds, suggests that comprehensive structure/activity relationship studies including related compounds would promote the understanding of biological functions and pharmacological effects of ubiquinone- and alpha-tocopherol-related compounds.

The FDAM method: determination of carboxyl profiles in cellulosic materials by combining group-selective fluorescence labeling with GPC.

A novel method for accurate determination of the carboxyl content in cellulosic materials by fluorescence labeling with 9H-fluoren-2-yl-diazomethane (FDAM) has been developed. The procedure can readily be implemented into a GPC system with RI and MALLS detectors, requiring additional fluorescence detection. The labeling conditions were optimized by means of sugar acid model compounds and were transferred to the cellulose case. Kinetics of the labeling and the influence of reaction parameters were comprehensively studied. For the first time, carboxyl profiles of cellulosics, i.e., the carboxyl content relative to the molecular weight distribution, were obtained.

"Vitamin CE," a novel prodrug form of vitamin E.

Reaction of 5a-bromo-alpha-tocopherol with ascorbic acid produces 5a-tocopheryl ascorbate which is designated "vitamin CE." This novel tocopherol derivative represents an interesting prodrug form of alpha-tocopherol (vitamin E) that is stable under acidic conditions, but regenerates finely dispersed vitamin E in basic media. The reaction mechanism of the base-induced decomposition of vitamin CE involves elimination of ascorbate and production of an ortho-quinone methide intermediate that oxidizes ascorbate, and is reduced to vitamin E. Kinetic experiments showed reaction to proceed in the pH range of 8 to 11 under physiological conditions. Tissue culture measurements demonstrated that vitamin E generated from the novel derivative is absorbed at much higher rates than conventional preparations and can even be absorbed under simulated conditions of malabsorption where there is no uptake of conventional vitamin E medications.