Brian Wright

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 in rat bile and urine were investigated by 1H- and 19F-NMR spectroscopy and liquid scintillation counting. Male Wistar rats received a single oral dose of 100 mg/kg of radiolabelled Statil. Statil 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 1H-NMR spectroscopy very difficult as Statil 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 predominantly as aglycone, and to a lesser extent as glucuronide conjugate. In addition, minor acyl migration products of Statil glucuronide could also be detected as they were separated during the SPEC-process. 19F-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 19F-NMR spectroscopy also enabled the calculation of dose related recoveries of Statil 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 19F-NMR results similar to those obtained by scintillation counting (in agreement within about 20%). However, discrepancies have been observed with very small and broad 19F-NMR signals in bile. Nevertheless, 19F-NMR spectroscopy of bile is a rapid and facile method for assessing metabolite levels of fluorinated drugs.

Size-exclusion chromatography with on-line ultraviolet, proton nuclear magnetic resonance and mass spectrometric detection and on-line collection for off-line Fourier transform infrared spectroscopy

The coupling of HPLC with UV detection and on-line NMR spectroscopy and mass spectrometry combined with a dedicated interface for the collection of the chromatographic eluent for subsequent Fourier transform (FT) IR has been investigated using a number of polymer additives as model compounds. Size-exclusion chromatography was performed using deuterated chloroform as eluent with the separation monitored on-line by UV detection at 254 nm and on-flow 1H-NMR and MS. The effluent from the NMR probe was directed to a dedicated HPLC interface where it was deposited on a germanium plate for subsequent FT-IR. NMR and MS spectra were successfully obtained for 2,6-di-tert.-butyl-4-methylphenol, octadecyl-3-(3,5-di-tert.-butyl-4-hydroxyphenyl) propionate (Irganox 1076) and diisooctyl phthalate on-line and FT-IR spectra for all three compounds were obtained off-line. Practical problems encountered with this multiple hyphenation are described.

Selective deuterium exchange during superheated heavy water chromatography-nuclear magnetic resonance spectroscopy-mass spectrometry of sulfonamides.

Superheated deuterium oxide has been investigated as an eluent for reversed-phase HPLC on a polystyrene-divinylbenzene column with UV, 1H NMR and MS detection using a series of sulfonamides as model compounds. In the course of these studies, a selective, specific and efficient deuteration of the methyl groups on a pyrimidine ring was observed during chromatography of certain of the sulfonamides. The potential of this methodology for producing deuterium-labelled compounds from substances bearing suitable substituents is considered. The utility of HPLC-NMR-MS as a means for studying on-column reactions is discussed.

Superheated deuterium oxide reversed-phase chromatography coupled to proton nuclear magnetic resonance spectroscopy

The on-line coupling of superheated deuterium oxide reversed-phase liquid chromatography and NMR spectroscopy overcomes many of the problems faced by conventional HPLC–NMR systems and provides a new method for the examination of moderately polar organic compounds. Samples of salicylamide and barbiturates were separated on a polystyrene–divinylbenzene (PS–DVB) column and transferred to a NMR spectrometer for on-line or stop-flow detection. This approach enables isothermal and temperature gradient reversed-phase HPLC–NMR spectroscopy to be readily carried out without the use of deuterated organic solvents. Unlike supercritical fluid chromatography–NMR spectroscopy, the method can be carried out with a low pressure HPLC–NMR probe.