Distance measurements in the borderline region of applicability of CW EPR and DEER: a model study on a homologous series of spin-labelled peptides.

Inter-spin distances between 1 nm and 4.5 nm are measured by continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) methods for a series of nitroxide-spin-labelled peptides. The upper distance limit for measuring dipolar coupling by the broadening of the CW spectrum and the lower distance limit for the present optimally-adjusted double electron electron resonance (DEER) set-up are determined and found to be both around 1.6-1.9 nm. The methods for determining distances and corresponding distributions from CW spectral line broadening are reviewed and further developed. Also, the work shows that a correction factor is required for the analysis of inter-spin distances below approximately 2 nm for DEER measurements and this is calculated using the density matrix formalism.

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.

Practical aspects of milk collection in the rat.

It is a requirement as part of the safety evaluation of novel compounds, which are intended to be administered to pregnant women or women with young children, that the potential for the compound or its metabolites to be transferred from a dam to its litter during lactation is measured. The data obtained from the rat milking studies are also used to support the rat peri- and post-natal studies. Therefore it is necessary to collect multiple milk samples from lactating rats. In this paper it was decided to concentrate on three areas: 1. The factors which had to be considered during the development of the rat milking technique; 2. The outline of the basic study design for a rat milking study; 3. The technique for milking rats.