A comparison of quantitative NMR and radiolabelling studies of the metabolism and excretion of Statil™ (3-(4-bromo-2-fluorobenzyl)-4-oxo-3H-phthalazin-1-ylacetic acid) in the rat

The identification and quantitation of the metabolites of Statil(TM) in rat bile and urine were investigated by (1)H- and (19)F-NMR spectroscopy and liquid scintillation counting. Male Wistar rats received a single oral dose of 100 mg/kg of radiolabelled Statil(TM). Statil(TM) is known to produce glucuronide conjugates which are predominantly excreted into the bile in male rats. The complex multiphasic matrix of bile has been shown to make identification of the resonances by (1)H-NMR spectroscopy very difficult as Statil(TM) appeared to be micellar bound giving rise to very broad signals. This not only impaired unambiguous signal characterisation but also quantification. The partial separation by SPEC-(1)H-NMR spectroscopy enabled the disruption of the micellar matrices and hence enabled the identification of Statil(TM) predominantly as aglycone, and to a lesser extent as glucuronide conjugate. In addition, minor acyl migration products of Statil(TM) glucuronide could also be detected as they were separated during the SPEC-process. (19)F-NMR spectroscopic measurements on whole bile confirmed their presence as a number of overlapped signals could be observed. The selectivity, simplicity and signal dispersion characteristic of (19)F-NMR spectroscopy also enabled the calculation of dose related recoveries of Statil(TM) related material in the bile and urine samples without the need for a radiolabel. The aim of this work was to investigate the usefulness and limitations of NMR spectroscopy of intact bile and urine as a means of quantifying levels of drug metabolites. The results obtained from NMR spectroscopy are compared with those obtained using scintillation techniques. Scintillation counting yields unequivocal quantification results, provided the label is preserved in metabolites as has been the case here. In general, quantification by (19)F-NMR results similar to those obtained by scintillation counting (in agreement within about 20%). However, discrepancies have been observed with very small and broad (19)F-NMR signals in bile, Nevertheless, (19)F-NMR spectroscopy of bile is a rapid and facile method for assessing metabolite levels of fluorinated drugs. (C) 2002 Elsevier Science B.V. All rights reserved.