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