Two criteria relevant to Omicron product quality are chemical purity and the
level of isotope enrichment. The former is established by various analytical
methods, including HPLC, GLC, and NMR. Usually one or a combination of these
methods, when used in conjunction with physical tests such as melting point
and optical rotation, proves sufficient to establish the purity of our products.
A related issue is the hydration state of
the product, which bears on the true cost for the consumer (i.e., Are
you paying a high price for waters of hydration?). For crystalline products,
hydration state is determined by X-ray crystallography or from Karl-Fischer
titration in those cases where the information is unavailable from the literature.
For Omicron products sold as syrups, we assume that H2O comprises
20% of the syrup weight, the maximal percentage determined from empirical studies
conducted in our laboratory.
The question of isotope enrichment level
is less easily established with certainty. Two common analytical methods used
for this assay, NMR and mass spectrometry, yield results whose quality depends
highly on the care taken with data collection and analysis, and on the behavior
of the specific compound under investigation. At Omicron, we rely on NMR-based
assays for many, but not all, of these determinations. In the following, we
briefly describe some of the details of the NMR-based assay and the criteria
used to judge its reliability.
The NMR-Based Assay of Enrichment Level
Quantitative NMR requires careful adjustment
of the acquisition parameters and careful preparation of the sample. We illustrate
how isotope enrichment level is determined by 1H NMR using D-[1-13C]glucose
as the test sample.
D-[1-13C]Glucose exists mainly
as α-glucopyranose 1 (38%) and β-glucopyranose 2 (62 %) in aqueous solution at 30°
C (Zhu et al., J. Org. Chem. 2001, 66, 6244-6251).
The anomeric H1 signals of both forms in an unlabeled sample are distinguished
from those of the remaining ring protons by their characteristic chemical shifts
and their multiplicities (the doublets are caused by the presence of 3JH1,H2
whose magnitude depends on the H1-C1-C2-H2 torsion angle).
When 13C is introduced at C1 at significant levels over natural abundance
(1.1 %), four H1 signals appear for each form; the additional large 1JC1,H1
coupling (typically ~160-170 Hz in aldopyranosyl rings) is superimposed
on the smaller 3JH1,H2 coupling, yielding a doublet
of doublets. Thus, in D-[1-13C]glucose, each anomer produces six
H1 signals, two arising from the residual unlabeled molecules and four arising
from the C1-labeled molecules.
This pattern is illustrated in Figure
1, which shows partially-relaxed 1H NMR spectra of D-[1-13C]glucose
(H1 region only) obtained from an inversion-recovery T1 determination
(see below). The inverted signals A-C are attributed to H1 of 1, where
signal B arises from the unlabeled molecules, and signals A and C from the labeled
molecules. An analogous set of signals (D, F and G) is observed for H1 of 2.
Signal E arises from residual HOD in the solvent. It is evident that, in principle,
the level of 13C-enrichment can be determined by appropriate integration
of the H1 signals associated with each anomer (data for each anomer provides
redundant information on the enrichment level at C1). For example, the integration
of signals A+C yields the amount of labeled species and the integration of signal
B yields the amount of unlabeled species.
While this approach appears straightforward,
there are serious pitfalls to be avoided. Signal-to-noise ratio must be excellent,
and signals must be adequately digitized to eliminate undesirable peak-area
truncation. Care must be taken during signal integration to avoid user bias;
the latter can be avoided by using a computerized lineshape analysis commonly
available on many modern spectrometers. Finally, signal area distortions arising
from differential spin-lattice relaxation times must be eliminated. The latter
is addressed through the incorporation of adequate interpulse delay times during
data acquisition to insure complete nuclear relaxation.
Choosing proper interpulse delay times hinges
on knowing the spin-lattice relaxation properties of the molecule. This information
can be obtained through direct measurement of spin-lattice relaxation times,
T1, in the sample under solution conditions similar to those used
for routine determinations of isotope enrichment level. The T1 values
of H1 attached to 12C will differ from those of H1 attached to 13C.
Although the gyromagnetic ratio of 13C is ~1/4 that of 1H,
the short internuclear distance between C1 and H1 (the C-H bond length is ~1.08
Å) renders the 13C at C1 a potent source of dipole-dipole relaxation
for H1. For example, consider the T1 values extracted from the partially-relaxed
(inversion recovery) spectra shown in Figure
1. For H1 of 2, T1 = 1.8 s when attached to 12C
and 0.8 s when attached to 13C. For H1 of 1, T1
= 3.3 s when attached to 12C and 0.9 s when attached to 13C.
The ratio, T1(12C)/T1(13C) is not
constant since this ratio depends highly on molecular geometry. In 1,
H1 is equatorial and thus relaxation mediated by other carbon-bound hydrogens
is less potent (because these protons are too far away from H1; 1/T1
is proportional to rCH-6) than for H1 of 2, which
is axial and thus relatively close to the axial H3 and H5. Consequently, providing
a new relaxation pathway through the 13C nucleus at C1 affects the
T1 of 1 more than that of 2.
Importantly, the data in Figure
1 show that an interpulse delay of 3.3 s x 5 = 16.5 s is required to insure
the attainment of thermal equilibrium between pulses. The value of knowing T1
values is apparent; without this information, selection of the proper interpulse
delay cannot be made. An improper choice can result in inaccurate determinations
of enrichment levels.
Reliability of the NMR-Based Assay for Enrichment Level
In order to validate isotope percent determinations by NMR, a parallel study
of five compounds was undertaken: four samples of D-[1-13C]glucose
containing different levels of 13C-enrichment at C1, and D-[1,6-13C2]glucose.
1H NMR spectra were collected as described above, and the same samples
were analyzed by an independent mass spectrometry facility (Metabolic Solutions,
Inc.). The results are shown in Table 1. The computed
enrichment levels determined from the NMR and MS data are in good agreement,
suggesting that the NMR-based assay yields reliable isotope enrichment levels
to within ± 0.2-0.4% of the actual value (we assume that the MS data yield the
true enrichment level). Based on these results, we rely mainly on NMR-based
methods for routine assays of enrichment level. Clearly, however, this approach
is not applicable to all types of labeled products; for example, assays of uniformly
13C- and 2H-labeled compounds cannot be conducted readily
by NMR. In these cases, mass spectrometry provides the required information.
All Omicron products are accompanied by
one source of data indicating the level of isotope enrichment of the sample.
Additional assays can be requested by the customer; charges for these additional
assays are paid by the customer.
|A Comparison of Nuclear Magnetic Resonance (NMR) and Mass
Spectrometry (MS) Determinations of Percent Isotopic Enrichment in
|aUsing an 8.8 s recycle time.
|bStandard deviation in parentheses, n =
|data collected by Metabolic Solutions Inc.