The Application of High Resolution Proton NMR Spectroscopy to the Detection of Drug Metabolites in Biological Samples
Recent developments in high-resolution 1H NMR allow biological samples to be analyzed for certain low mol. wt. components with little or no sample pre-treatment. The 1H NMR methods require only small sample volumes (0.3 ml), are non-destructive and usually give qualitative results within a few minutes if adequate steps are taken to reduce the intensity of the solvent water signal with an appropriate secondary irradiation field or pulse sequence. Notably, preselection of instrumental conditions to observe different classes of compound is not necessary, e.g. when studying compounds whose metabolic and excretory mechanisms are poorly understood.
The latter is the case for N-methylformamide (NMF), an experimental anti-tumour agent, but not for paracetamol. 1H NMR has been applied to the detection and quantitation of urinary metabolites of these drugs. For paracetamol, the free drug and major metabolites (glucuronide, sulphate, cysteinyl and N-acetylcysteinyl conjugates) were all detected in untreated urine, and there was quantitative agreement with HPLC results. However, two-dimensional (2-D) NMR methods were required to separate the aramatic resonances of the cysteinyl conjugate and the free drug. For NMF, 1H NMR measurements led to detection and identification of previously unknown metabolites including formic acid and a novel N-acetylcysteinyl derivative (N-acetyl-S-[N-methyl-carbamoyl]cysteine) as well as the parent compound and methylamine. Signals for various excreted compounds of endogenous origin are also manifest in 1H NMR spectra of urine, so that information on the toxicological effects of drugs and xenobiotics can be obtained simultaneously with data on their metabolism.
KeywordsDrug Metabolite Indoxyl Sulphate Digital Resolution Methyl Isocyanate Doublet Resonance
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- 1.Bock, J.L. (1982) Clin. Chem.28, 1873–1877.Google Scholar
- 2.Nicholson, J.K., Buckingham, M.J. & Sadler, P.J. (1983) Bioohem. J.211, 605–615.Google Scholar
- 3.Nicholson, J.K., O’Flynn, M., Sadler, P.J., Macleod, A.F. , Juul, S.M. & Sonksen, P.H. (1984) Biochem. J.217, 365–375.Google Scholar
- 4.Bales, J.R., Higham, D.P., Howe, I., Timbrell, J.A. & Sadler, P.J. (1984) Biochem. J.217, 426–432.Google Scholar
- 5.Bales, J.R., Sadler, P.J., Nicholson, J.K. & Timbrell, J.A. (1984) Clin. Chem.30, 1631–1636.Google Scholar
- 6.Nicholson, J.K., Timbrell, J.A. & Sadler, P.J. (1985) Mol. Pharmacol.27, 644–651.Google Scholar
- 7.Bales, J.R., Nicholson, J.K. & Timbrell, J.A. (1985) Clin. Chem.31, 757–762.Google Scholar
- 8.Hore, P. (1983) J. Magn. Resort.55, 283–300.Google Scholar
- 10.Bax, A., Freeman, R. & Morris, G. (1981) J. Magn. Reson.41, 496–501.Google Scholar
- 11.Nagayama, K., Bachmann, P., Wuthrich, K. & Ernst, R.R. (1978) J. Magn. Reson.31, 133–148.Google Scholar
- 13.Tulip, K., Timbrell, J.A. & Nicholson, J.K. (1986) Chemical and Biological Reactive Metabolites (Proc. 3rd Int. Symp.), Plenum, New York, in press.Google Scholar
- 15.Everett, J.R., Jennings, K., Woodnut, G. & Buckingham, M.J. (1984) J. Chem. Soc, Chem. Comm., 894–895.Google Scholar
- 17.Nicholson, J.K., Timbrell, J.A., Bales, J.R. & Sadler, P.J. (1985) Mol. Pharmacol.27, 634–643.Google Scholar