The precise stoichiometry required for the acetylation of surface amines of a poly-(amidoamine) (PAMAM) dendrimer generation 5 (G5) was verified by using potentiometric titration, gel permeation chromatography, and nuclear magnetic resonance spectroscopy. The average number of primary amine groups, absolute molecular weight, and molecular weight distribution of G5 PAMAM were determined by potentiometric titration and GPC. These fundamental parameters were used to design the stoichiometry of an acetylation reaction that yielded acetylation fractions from 0 to 100% of the primary amines on the macromolecule. GPC refractive index detector confirmed that the diameter of the dendrimer related inversely to the degree of acetylation. The acetylated dendrimers do not follow the elution behavior of the conventional polymer molecules most probably because of their spherical shape and polycationic nature. This study clarifies the nature of the acetylation reaction and provides a well-defined acylated macromolecule, which can serve as a scaffold for the development of complex dendrimeric structures.
We discovered that serious issues could arise that may complicate interpretation of metabolomic data when identical samples are analyzed at more than one NMR facility, or using slightly different NMR parameters on the same instrument. This is important because cross-center validation metabolomics studies are essential for the reliable application of metabolomics to clinical biomarker discovery. To test the reproducibility of quantified metabolite data at multiple sites, technical replicates of urine samples were assayed by 1D-1H-NMR at the University of Alberta and the University of Michigan. Urine samples were obtained from healthy controls under a standard operating procedure for collection and processing. Subsequent analysis using standard statistical techniques revealed that quantitative data across sites can be achieved, but also that previously unrecognized NMR parameter differences can dramatically and widely perturb results. We present here a confirmed validation of NMR analysis at two sites, and report the range and magnitude that common NMR parameters involved in solvent suppression can have on quantitated metabolomics data. Specifically, saturation power levels greatly influenced peak height intensities in a frequency-dependent manner for a number of metabolites, which markedly impacted the quantification of metabolites. We also investigated other NMR parameters to determine their effects on further quantitative accuracy and precision. Collectively, these findings highlight the importance of and need for consistent use of NMR parameter settings within and across centers in order to generate reliable, reproducible quantified NMR metabolomics data.
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