The molar substitution (MS) is calculated from the ratio between the signal from the three protons of the methyl group, contained in the hydroxypropyl functional group, and the signal from the proton attached to the carbon C1 (glycosidic proton) of the anhydroglucose units. Use a Fourier-transform nuclear magnetic resonance (NMR) spectrometer having a magnetic field strength of at least 6 Tesla and that is capable of performing quantitative analysis using proton NMR spectroscopy at a temperature of at least 25°.
Sample solution: Mix NLT the equivalent of 10.0 mg of dried Hydroxypropyl Betadex with 0.75 mL of deuterium oxide thoroughly in an NMR tube. Place the tube into an NMR probe. Analysis: Adjust the spectrometer settings so that a high-resolution proton NMR spectrum can be acquired that will provide quantitative data. Acquire a free induction decay (FID) with at least 8 transients using a spectral window from at least 0–6.2 ppm, with the solvent peak located at 4.8 ppm at 25°. Zero fill the spectrum at least three times, and Fourier transform the FID with no Gaussian line broadening and no more than 0.2 Hz of Lorenzian line broadening. Determine the peak areas of the doublet from the methyl protons of the hydroxypropyl functional group at 1.2 ppm (A 1 ) and the peak areas from the glycosidic protons, which are located between 5 and 5.4 ppm (A 2 ). Calculate the MS: Result = A 1 /(3A 2) A 1 = area of the methyl group of hydroxypropyl A 2 = area of the glycosidic proton The degree of substitution is the number of hydroxypropyl groups per molecule of betadex and is obtained by multiplying the MS by 7. Acceptance criteria: 0.40–1.50 and within 10% of the value stated on the label