Hyphenated NMR in Drug Analysis and Drug Metabolism.
The new equipment installed autumn 2002 at the Royal Danish School
of Pharmacy greatly facilitates the use of NMR in drug metabolism
studies
and drug analysis, as the greatly enhanced sensitivity compared with
existing
equipment yields substantially better detection limits. Furthermore,
the 1 mm probe installed and tested on the AV 400 instrument during
2005, extends the detectionlimit of the AV 400 downto the sub-nanomolar
range.
My research will
mainly be centred on the following subheadings.
1. Drug metabolism studies.
When studying drug metabolism identification and quantification of
metabolites
and parent compounds in complex biological fluids is inevitable. A
combination
of separation and spectroscopic techniques is therefore needed to work
efficiently
and obtain reliable results.
Identification of drug metabolites using LC-NMR-MS (Liquid
Chromatography
coupled with Nuclear Magnetic Resonance and Mass Spectroscopy) is very
efficient,
when both NMR and MS data are available for the different metabolites.
Especially
the Solid Phase Extraction (SPE) equipment installed with the new
instrumentation
will make it possible to obtain useful spectra at substantially lower
concentrations.
Especially the fact that one can be fairly sure, that the NMR and MS
spectra
are from the same compound facilitates reliability of analysis. Our
experience
is, that this is a crucial point. The possibility of using smaller
columns
improves detection limits as well as making it affordable to use
deuterated
organic solvents for the Liquid Chromatography which in turn allows
acces
to some important regions of the spectra otherwise obscured by solvent
signals.
Chemometric techniques (subheading 4) will further enhance the spectra
obtained.
The actual compounds analysed are chosen in collaboration with
coworkers
at the Royal Danish School of Pharmacy.
Refs. 3, 8, 9, 13, 16, 19, 22, 23,29, 32, 35, 36, 37,40, 45, 46, 48,
49,
50.
2. Metabonomics
A Ph.D.-project (Michael Lauridsen) was started medio 2004 concerning
metabonomic studies of inflammatory diseases (Morbus Crohn, Colitis
Ulcerosa, and Rheumatoid Arthritis and Osteoarthritis) in collaboration
with the Department of Medicinal Chemistry and the Parker Institue at
HS Frederiksberg.
A collaboration concerning identification of biomarkers for
fruit- and vegetable ingestion is in progress with the Danish
Institute for Food and Veterinary Research.
3. Chiral resolution of NMR spectra of drugs and drug metabolites
using
chiral solvents.
NMR can’t distinguish between enantiomeric compound in itself, but by
utilising
chiral cosolutes (cyclodextrins eg.) or chiral solvents the solutes in
question
form complexes that are diastereomeric, such that enantiomers can be
distinguished
an identified, and enantiomeric excess determined, which is
important
for drugs. Specifically we plan to examine the use of Solid Phase
Extraction
coupled with HPLC and NMR in such a way, that an enantiomer or
enantiomeric
pair is trapped on an SPE-column during HPLC and eluated with a chiral
solvent
into the NMR, where identity is established at the same time as
enantiomeric
excess. Some interesting solvents can be found in the literature,
recently
solvents with DMSO-like properties which is better suited for most
drugs
[51-54].
Refs. 14, 51-54.
4. Drug analysis.
Both analysis of impurities in drugs and analysis of complex drugs or
extracts
obtained from natural sources such as plants are highly amenable to
LC-NMR-MS
analysis. Identity of drug impurities are very important. Knowledge of
the
compounds in complex plant extracts is very important in order to
assure
the quality of the drugs or in order to develop new drugs bases on
material
from natural sources.
As of 2005 an industrial Ph.D. project under DRA in collaboration with
Novo Nordisk, KVL, an dSchool of Pharmacy University College London was
started (Carsten Ravn): Increased
mechanistic
and physico-chemical knowledge of pharmaceutical solid dosage
formulation
design using fast near infrared spectroscopy and chemical imaging
technologies”.(NIR chemical imaging in pharmaceutical formulation
design). The project is implemented with an intention of developning
and evaluating the techniques for Process Analtyical Technology.
Refs. 1, 2, 4, 5, 6, 7, 10, 11, 15, 17, 20, 21, 24, 25, 26, 27, 30, 31,
33,
34, 38, 39, 41, 42, 43, 44, 47.
5 and 6 will largely be performed as an aid to 1, 2, 3, and 4
5. Extraction of NMR spectra and spectral features using chemometric
techniques.
Chemometrics can be defined as the use of mathematical and statistical
techniques
with the object of getting the most out of Your data. The application
of
chemometric techniques to analysis of complex matrices is a rapidly
expanding
area of analytical chemistry, also in the area of complex biological
fluids.
Using chemometric techniques spectra can be extracted from
chromatograms
run in “screening mode”. An NMR-chromatogram can be subjected to factor
analysis
techniques in such a way, that individual spectra of an improved
quality
(compared to extracting slices) can be obtained using the complete
information
in the NMR-chromatogram. A spectrum and its separate chromatogram is
obtained
for each discernible species in the chromatogram.
Spectra of the pure compounds can even be obtained in cases of
overlapping
peaks, which makes it possible to obtain information otherwise obscured
by
solvent peaks, as long as the solvent peak doesn’t move during the
chromatogram
or the solvent signal doesn’t change multiplicity and linewidth.
The improved stability (less drift, better baseline in the spectra) of
the
new NMR-equipment is an important aspect of the future development of
these
methods, as the chemometric techniques can’t separate artifacts created
by
instability of the instrument from “real” changes of features of
interest.
See Reprint of latest poster “Moore or less automatic extraction of
Spectra
from NMR-chromatograms presented at the 23.rd Danish NMR-meeting
24-25/1-2002,
at Hotel Scandic, Roskilde, Denmark.
Lecture at the 22. Danish NMR meeting at Ledernes Kursuscenter, Odense,
Denmark.
“Screening of plant- and other extracts by means of an old NMR
instrument
(keywords: LC NMR, Chemometrics, Plant Extracts)
Ref. 4.
6. Determination of physical parameters of drugs and drug
metabolites
using NMR and other spectroscopi methods.
Physical parameters such as pK-values, solubilities, complexation
reactions
etc. are very important for separations techniques, drug formulations
etc.
Using NMR it is possible in many cases to determine these parameters
even
at low concentrations and for complex mixtures. Interaction of drugs
and excipients with metal-ions, using UV, IR, NIR, Raman, NMR, a.o
Refs 1, 18.
Link to References
Last update: 2/3-2007, cc