We encourage
our authors to consider the use of absolute quantitative 1H NMR spectroscopy to determine the purity of biologically tested
research compounds. The Perspective by Pauli et al. (DOI: 10.1021/jm500734a) in this issue provides an excellent
overview of quantitative 1H NMR spectroscopy methods and
their use for purity determination. The major advantages of these
techniques are that they can be used for very small samples, are nondestructive
to the sample, are fast, and provide high accuracy and precision.
NMR spectroscopy is commonly employed in medicinal chemistry laboratories
for the determination of compound identity, and therefore, adaptation
of absolute quantitative 1H NMR spectroscopy should be
straightforward and can be incorporated readily into the normal workflow
of compound identification by NMR. In the Perspective two methods
are discussed, the 100% percent method and the absolute quantitative 1H NMR method. We consider the latter one as the most suitable
for compounds used in biological assays; thus, we are recommending
this technique to our authors. In collaboration with the Pauli group,
we have developed a detailed procedure that will ensure that absolute
quantitative 1H NMR purity analyses can be performed correctly.
The protocol that details the calibration, the NMR data acquisition,
and the processing of the NMR data with specific examples can be found
in the Guidelines for Authors (http://pubs.acs.org/page/jmcmar/submission/authors.html).The Journal of Medicinal Chemistry requires
purity of >95% for all tested compounds to ensure that the observed
effect is accurate and not related to highly active impurities present
in the test sample. According to the Guidelines for Authors, compound
purity can be established by HPLC, elemental analysis, or any other
“scientifically established method”. We do not accept
other spectral data as evidence of purity. Both of the major methods
that our authors typically employ, HPLC and elemental analysis, have
certain drawbacks. Analysis by HPLC will typically not detect materials
such as solvent residues, water, inorganic impurities, or an impurity
that has the same retention time as the sample under the conditions
employed. While elemental analysis is considered to be superior to
HPLC analysis, fractional amounts of water or solvent are impurities
that are not easy to identify and quantify and therefore require orthogonal
analytical methods to determine their presence. Also, molecules of
similar composition, such as isomers, may not affect the elemental
analysis and may therefore not be detected by this method. As a primary
analytical method, absolute quantitative 1H NMR spectroscopy
offers certain advantages for purity determination. As samples and
the standards are accurately weighed, inorganic impurities and other
nonobservables will be detected and most solvent residues and water
will be identified in the NMR spectrum. Another advantage is that
the method is orthogonal to HPLC, which is often employed for compound
purification. Overlapping NMR peaks from impurities can be detected
by employing 2D NMR techniques.We hope that the Perspective by Pauli and collaborators and our detailed procedure
that gives guidance on the acquisition and purity calculation using
absolute quantitative 1H NMR spectroscopy will promote
the use of this method for purity determination. Absolute quantitative 1H NMR spectroscopy that follows the protocol provided in the
Guidelines for Authors will henceforth be an accepted method for the
establishment of compound purity for papers published in the Journal of Medicinal Chemistry in addition to HPLC and elemental
analysis methods.