Accurate Determination of Reference Materials and Natural Isolates by Means of Quantitative ¹H NMR Spectroscopy

Abstract

A fast and precise proton nuclear magnetic resonance (qHNMR) method for the quantitative determination of low molecular weight target molecules in reference materials and natural isolates has been validated using ERETIC 2 (Electronic REference To access In vivo Concentrations) based on the PULCON (PULse length based CONcentration determination) methodology and compared to the gravimetric results. Using an Avance III NMR spectrometer (400 MHz) equipped with a broad band observe (BBO) probe, the qHNMR method was validated by determining its linearity, range, precision, and accuracy as well as robustness and limit of quantitation. The linearity of the method was assessed by measuring samples of l-tyrosine, caffeine, or benzoic acid in a concentration range between 0.3 and 16.5 mmol/L (r(2) ≥ 0.99), whereas the interday and intraday precisions were found to be ≤2%. The recovery of a range of reference compounds was ≥98.5%, thus demonstrating the qHNMR method as a precise tool for the rapid quantitation (~15 min) of food-related target compounds in reference materials and natural isolates such as nucleotides, polyphenols, or cyclic peptides.

Accurate quantification of modified cyclic peptides without the need for authentic standards

“…The synthetic peptides subsequently quantified (Table 5 and Figure S11 to S14) showed that compound 13 contained of 21 % of analysed sample, compound 14 contained 34 % of the peptide while 15 and 16 contained 19 % and 33 % of the total solid of the analysed samples respectively. Given the high purity of compounds 13-16 we suggest that the low percentage of compound per total mass of powder can be attributed to significant retention of water during the freeze-drying process, which is supported by a large water peak (~ 3.3 ppm) in the NMR spectra ( Figure S11-14) similar to the finding of Frank et al 72 analytical data presented shows that the actual quantity compounds were under estimated in the preparation of the stock solution. …”

Determination of the purity of pharmaceutical reference materials by 1 H NMR using the standardless PULCON methodology

“…Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for the purpose of quantitative analysis because of the direct proportionality of signal intensity to the number of resonating nuclei and has found many applications in analytical chemistry [1][2][3][4][5]. Usually quantification is provided using a signal from a chemical reference standard added to the sample (internal referencing).…”

Functional Metabolomics – a Useful Tool to Characterize Stress-Induced Metabolome Alterations Opening New Avenues Towards Tailoring Food Crop Quality

“…Therefore, nowadays, determination of the exact concentration of the isotopically labled internal standards can best be achieved by means of quantitative NMR spectroscopy (q NMR) [31].…”