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Relative Response Factor Determinations by qNMR

Relative response factors (RRFs) are  being included commonly in active pharmaceutical ingredient (API) HPLC method development to allow for accurate API impurity measurement.  A direct area percent measurement of a UV chromatogram at a single wavelength is a quick, useful assessment of purity when comparing one sample to another.  However, it lacks specificity for measuring the actual quantity of any given substance [Figure 1].

Generally, one of two approaches is considered to measure the true level of a specific known API impurity in a given sample:

  1. assay the sample against a well-characterized API impurity reference standard of known purity, or
  2. determine and apply the impurity’s response factor relative to the main component at the chosen detection wavelength.

The second of these choices is more typical, since determination of response factors may be done carefully on a one-time basis, whereas an assay requires a continuous supply of an API impurity reference standard for independent measurements accompanying each testing situation.  Two limitations remain, however:

  • the amount of each API impurity reference standard must be sufficient such that an accurate weight may be measured as part of the overall RRF determination
  • potential sources for error may be introduced if the isolated reference substance proves to be hygroscopic or unstable between the time it is qualified and the time its response factor is measured.

Use of qNMR as an orthogonal means to determine RRFs has significant advantages to address these shortcomings. While NMR has historically been used as a qualitative tool for structure determination, it is also inherently a quantitative technique since the integrated intensity of a proton resonance signal is directly proportional to the number of protons represented by that signal and the sample concentration. Moreover, all protons in a given spectrum are equally sensitive given the appropriate experimental setup. Hence, NMR provides an ideal mechanism by which to quantitate a given substance, assuming the association of a unique reporter proton resonance with the substance and the number of protons the resonance represents can be established.

A single determination of Relative Response Factors (RRFs) using Quantitative NMR (qNMR) allows for simple and accurate quantitation of impurities which eliminates the need for preparation, qualification, and storage of API impurity reference standards.  For more details read our  white paper , published in the European Biopharmaceutical Review, which describes how qNMR-based determination of HPLC relative response factors can provide both a more accurate and cost-effective assessment of impurity levels than UV-VIS response alone.

Contact us to see how we can help you with HPLC relative response factor determinations, and other impurity isolation and identification needs!