Metrologically traceable quantification of trifluoroacetic acid content in peptide reference materials by¹⁹F solid-state NMR

Abstract

Although solution-state NMR is frequently used in metrologically-traceable quantification studies, this is not the case for solid-state NMR. However, solid-state NMR allows quantification of substances without the need of dissolution, providing a truly non-destructive approach, and extending metrologically-traceable quantitative NMR to sample classes that are difficult to characterize in solution. In this contribution we present a thorough and rigorous protocol for 19 F quantitative solid-state NMR employing a certified reference material as external calibrant to provide metrological traceability to absolutely quantify the content of trifluoroacetic acid (TFA) in a peptide sample, typically the major impurity in synthetic peptides. The protocol includes determining the quantitative volume of the solid-state NMR sample holder (rotor), the ERETIC (electronic reference to access in vivo concentrations) method (Akoka et al 1999 Anal. Chem. 71 2554) to compensate for variations in the sensitivity of the radio frequency resonant circuit when an external calibrant is used, and the EASY (elimination of artefacts in NMR spectroscopy) method (Jaeger and Hemmann 2014 Solid State Nucl. Magn. Reson. 57-58 22) to effectively suppress the 19 F NMR background signal from the probe head. We applied the protocol to quantify the amount of TFA in a candidate NRC certified reference material of the peptide angiotensin II. The results obtained by 19 F quantitative solid-state NMR are in excellent agreement with those obtained by quantitative NMR in solution employing an internal calibrant.

Quantification of amine functional groups on silica nanoparticles: a multi-method approach

“…The probe was tuned to the 19 F Larmor frequency given by 188.7 MHz prior to every measurement. The published protocol for 19 F quantitative solid-state NMR 26 was employed, specically metrological traceability was achieved by using 3,5bis(triuoromethyl)benzoic acid (BTFMBA) as external calibrant. Furthermore, we employed the ERETIC (Electronic REference To access In vivo Concentrations) and EASY (Elimination of Artifacts in NMR Spectroscopy) methods.…”

Establishment of measurement traceability for peptide and protein quantification through rigorous purity assessment—a review

“…The assigned values can be confirmed by microanalysis (% C, H, N content) or by TGA for total non-volatile inorganic impurities. Alternatively, accurate TFA quantification can be achieved by liquid phase quanti tative 19 F-NMR [82,100] or solid state quantitative 19 F-NMR [101].…”