Effect of Ca2+ on the 1H NMR chemical shift of the methyl signal of oversulphated chondroitin sulphate, a contaminant in heparin.

The chemical shift of the methyl signal of oversulphated chondroitin sulphate (OSCS) is dependent on the type and concentration of the counterion. When OSCS is present as a contaminant in heparin sodium, the reported methyl 1H chemical shift is 2.15 +/- 0.02 ppm. In this report, a value of 2.18 +/- 0.01 ppm is reported for the OSCS in the presence of Ca2+. The chemical shift of the methyl signal of pure OSCS varies linearly from 2.13 ppm to 2.18 ppm with increasing amounts of Ca2+, until reaching the saturation point of four Ca2+ ions per OSCS disaccharide unit, which contains four sulphate groups (a 1:1 ratio between sulphate groups and Ca2+). This Ca2+ effect can be used for OSCS identification as well as to facilitate quantification.

Validation of a quantitative NMR method for suspected counterfeit products exemplified on determination of benzethonium chloride in grapefruit seed extracts.

A 1H-nuclear magnetic resonance (NMR) spectroscopy method for quantitative determination of benzethonium chloride (BTC) as a constituent of grapefruit seed extract was developed. The method was validated, assessing its specificity, linearity, range, and precision, as well as accuracy, limit of quantification and robustness. The method includes quantification using an internal reference standard, 1,3,5-trimethoxybenzene, and regarded as simple, rapid, and easy to implement. A commercial grapefruit seed extract was studied and the experiments were performed on spectrometers operating at two different fields, 300 and 600 MHz for proton frequencies, the former with a broad band (BB) probe and the latter equipped with both a BB probe and a CryoProbe. The concentration average for the product sample was 78.0, 77.8 and 78.4 mg/ml using the 300 BB probe, the 600MHz BB probe and CryoProbe, respectively. The standard deviation and relative standard deviation (R.S.D., in parenthesis) for the average concentrations was 0.2 (0.3%), 0.3 (0.4%) and 0.3mg/ml (0.4%), respectively.