Determination of the purity of a substance traceable to the International System of Units (SI) is important for the production of reference materials affording traceability in quantitative measurements. Post-column isotope dilution using liquid chromatography-chemical oxidation-isotope ratio mass spectrometry (ID-LC-CO-IRMS) has previously been suggested as a means to determine the purity of organic compounds; however, the lack of an uncertainty budget has prevented assessment of the utility this approach until now. In this work, the previously published ID-LC-CO-IRMS methods have not only been improved by direct gravimetric determination of the mass flow of 13C-labelled spike but also a comprehensive uncertainty budget has been established. This enabled direct comparison of the well-characterised ID-LC-CO-IRMS method to quantitative nuclear magnetic resonance spectroscopy (qNMR) for purity determination using valine as the model compound. The ID-LC-CO-IRMS and qNMR methods provided results that were in agreement within the associated measurement uncertainty for the purity of a sample of valine of (97.1 ± 4.7)% and (99.64 ± 0.20)%, respectively (expanded uncertainties, k = 2). The magnitude of the measurement uncertainty for ID-LC-CO-IRMS determination of valine purity precludes the use of this method for determination of purity by direct analysis of the main component in the majority of situations; however, a mass balance approach is expected to result in significantly improved measurement uncertainty.