Hydroxyethylmethyl celluloses (MSHE 0.15–0.26, DSMe 1.49–1.82) and hydroxyethyl celluloses (MSHE 1.89–3.03) are analyzed with respect to their substituent distribution in the polymer chains. The cellulose ethers are peralkylated and partially hydrolyzed, and are analyzed by electrospray ionization‐ion trap‐mass spectrometry (ESI‐IT‐MS). The randomness of the partial hydrolysis is proven. Quantitative evaluation of their mass spectra becomes possible after labeling the oligosaccharides at the reducing end. Reductive amination with o‐aminobenzoic acid gives better results than hydrazone formation with cationic Girard's T reagent. Syringe pump infusion gives more‐accurate results compared with liquid chromatography (LC)‐ESI‐MS. Profiles of the substituent distribution in the oligosaccharide fractions of DP 2–DP 7 are compared with the theoretical random distribution of glucosyl units in the chain.magnified image
Three hydroxypropyl methylcellulose samples (HPMC1-3, DS(Me) = 1.45, 1.29, and 1.36; MS(HP) = 0.28, 0.46, and 0.84) were analyzed with respect to their methyl and hydroxypropyl substitution pattern in the polymer chains. Ionization yield of HPMC oligomers in electrospray ionization ion trap mass spectrometry (ESI-IT-MS) is strongly influenced by the hydroxypropyl pattern. Therefore, a sample derivatization procedure, as well as suitable measurement conditions that enable relative quantification were elaborated. Analysis was performed by negative ESI-IT-MS after per(deutero)methylation, partial depolymerization, and reductive amination with m-aminobenzoic acid. Measurement parameters like solvent, trap drive, and voltages of the ion transportation unit were studied with regard to the suitability for quantitative evaluation. Using direct infusion of the samples, strong influence of trap drive and octopole settings was observed. Optimized measurement conditions were used for the determination of the HP pattern of the permethylated samples by direct infusion. The methyl pattern was determined from the perdeuteromethylated samples by high-performance liquid chromatography-electrospray tandem mass spectrometry. For HPMC1, substituents were both found to fit the random distribution model. The other two samples showed pronounced heterogeneity which could be interpreted in more detail by extracting methyl subpatterns depending on the number of HP groups.
Mass spectrometry is widely applied in carbohydrate analysis, but still quantitative evaluation of data is critical due to different ionization efficiencies of the constituents in a mixture. Different size and chemical structure of the analytes cause their uneven distribution in droplets (electrospray ionization, ESI) or matrix spots (matrix-assisted laser desorption/ionization, MALDI). In addition, instrumental parameters affect final ion yields. In order to study and optimize the latter, an equimolar mixture of malto-oligosaccharides (DP1-6) was analyzed using varying target masses for ESI as well as different matrices and laser power for MALDI. The sodium adducts and derivatives for positive ion mode (hydrazones with Girard's T Reagent, GT) and negative ion mode (reductively aminated with o-aminobenzoic acid, oABA) were studied. Negatively charged oABA-labeled malto-oligosaccharides turned out to be unsuitable for quantification of the malto-oligomeric composition. Best agreement was achieved when applying target masses in the range of the highest homolog in the mixture in electrospray ionization ion trap (ESI-IT) (1-2% deviation with GT label or as Na(+) adducts). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) gave best results when the laser power was adjusted significantly over the desorption/ionization threshold (1% deviation with GT label). Both parameters show significant influence on the determined oligomeric composition. Consequently, estimation and even quantitative determination of amounts of oligosaccharides in a mixture can be achieved when the analytes are labeled and the proper instrumental parameters are used.
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