Collaborative Study to Validate Purity Determination by ¹H Quantitative NMR Spectroscopy by Using Internal Calibration Methodology

Abstract

NMR spectroscopy has recently been utilized to determine the absolute amounts of organic molecules with metrological traceability since signal intensity is directly proportional to the number of each nucleus in a molecule. The NMR methodology that uses hydrogen nucleus ( 1 H) to quantify chemicals is called quantitative 1 H-NMR ( 1 H qNMR). The quantitative method using 1 H qNMR for determining the purity or content of chemicals has been adopted into some compendial guidelines and official standards. However, there are still few reports in the literature regarding validation of 1 H qNMR methodology. Here, we coordinated an international collaborative study to validate a 1 H qNMR based on the use of an internal calibration methodology. Thirteen laboratories participated in this study, and the purities of three samples were individually measured using 1 H qNMR method. The three samples were all certified via conventional primary methods of measurement, such as butyl p-hydroxybenzoate Japanese Pharmacopeia (JP) reference standard certified by mass balance; benzoic acid certified reference material (CRM) certified by coulometric titration; fludioxonil CRM certified by a combination of freezing point depression method and 1 H qNMR. For each sample, 1 H qNMR experiments were optimized before quantitative analysis. The results showed that the measured values of each sample were equivalent to the corresponding reference labeled value. Furthermore, assessment of these 1 H qNMR data using the normalized error, E n -value, concluded that statistically 1 H qNMR has the competence to obtain the same quantification performance and accuracy as the conventional primary methods of measurement.

Nontoxic Enantiomeric Reference Materials for Saxitoxins

“…Before investigating the equivalence of analytical properties between ent -STXs ( 4 and 5 ) and STXs ( 2 and 3 ), the concentrations of 4 and 5 were accurately determined by quantitative 1 H NMR spectroscopies using external standards which were based on the PULCON methodology. , The 1 H NMR spectra were acquired, referring to parameters of the validated methods with an internal standard. , The H 2 -13 (4.27 and 4.00 ppm) signals from 4 were used for quantitation. On the other hand, the H-10β (3.54 ppm), H-6 (3.60 ppm), and H-13 signals (3.60 ppm) were used for ent -dcSTX ( 5 ).…”

“…Quantitative 1 H NMR spectroscopy was carried out by the pulse length-based concentration determination method (PULCON) followed by the previous methods. − Aliquots of ent -dcSTX ( 5 ) and ent -STX ( 4 ) were lyophilized and dissolved in 0.6 mL of 4% CD 3 COOD/D 2 O in an NMR tube. DSS- d 6 of 3.828 mM in D 2 O was used as an external standard.…”

The qNMR Summit 5.0: Proceedings and Status of qNMR Technology

“…Since ISO/TC 34 is a technical committee for food products, qNMR will be assessed broadly for the purity determination of measurement standards for food analysis. As supportive evidence, an interlaboratory collaborative study was conducted in 2018, 3 to advice the ongoing ISO standard establishment, where 1 H qNMR with internal calibration methodology has the capability of obtaining the same quantitative performance as the conventional methods including mass balance, coulometric titration, and freezing point depression.…”

The effect of tube quality on externally calibrated quantitative nuclear magnetic resonance analysis: How bad can it be?

“…Substantial efforts have been made in the past few years to optimize laboratory protocols and create internationally recognized standards for the development of internally calibrated qNMR methods. [1][2][3] Conversely, external calibration approaches (i.e., procedures in which the calibrant is kept separate from the test sample) have not gained the same level of acceptance and widespread use despite being available for several decades. [4][5][6][7] This is probably due to the presumed assumption that external calibration will yield less precise and accurate results than internal calibration, even if the former can still meet the acceptance criteria of many quantitative applications.…”