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The urinary excretion of metabolites of 2,3-benzofuran was studied in Sprague-Dawley rats (n = 5) given a single dose of 150 mg/kg i.p. Urine samples were collected at defined intervals up to 7 days postdose and analyzed using (1). H NMR and directly coupled high performance liquid chromatography (HPLC)-NMR, HPLC-(mass spectrometry) MS and HPLC-MS-NMR methods. The principal metabolites were determined to be 2-hydroxyphenylacetic acid and 2-(2-hydroxyethyl)phenyl hydrogen sulfate, representing 24.3 +/- 6.0% and 19.6 +/- 6.4% of the dose, respectively. This indicates that metabolism of benzofuran to the polar species excreted in urine involves cleavage of the furan ring.

The novel application of magic angle spinning 1H NMR spectroscopy, coupled with pattern recognition techniques, has identified biochemical changes in lipid and glutamate metabolism that precede classical nephrotoxicity. These changes occurred in the bank vole (Clethrionomys glareolus) after chronic dosing, at a low level of exposure and at a renal Cd(2+) concentration (8.4 microgram/g dry wt) that was nearly two orders of magnitude below the WHO critical organ concentration (200 microg/g wet wt). These early stage effects of Cd(2+) on the biochemistry of renal tissue may reflect adaptation mechanisms to the toxic insult or the preliminary stages of the toxicological cascade.

In this work, 400 and 600 MHz 1H HPLC-NMR spectroscopic methods were developed and applied to separate and identify the positional glucuronide isomers and anomers of the model nonsteroidal antiinflammatory drug, 6,11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid, in whole human urine. The HPLC methods utilized either an isocratic system, comprising 30% acetonitrile in water at pH 2.5, or a gradient elution system increasing from 30% to 60% acetonitrile, in order to achieve improved separation of the 2-, 3-, and 4-O-acylglucuronide isomers from the faster eluting endogenous urinary metabolites. Directly coupled stop-flow 1H HPLC-NMR spectroscopic measurements were made at the retention times indicated by the UV-monitored chromatographic peaks. The glucuronide isomers were identified from the 1H NMR spectra on the basis of their chemical shifts and spin-spin coupling patterns. The elution order was 4-O-acyl-, 3-O-acyl-, and finally 2-O-acylglucuronide, with tR values of 10.04, 11.68, and 12.64 min, respectively. Although the alpha- and beta-anomers of each of the positional isomers could not be separated in these solvent systems, they could be identified in the individual 1H NMR spectra. This work shows for the first time that directly coupled HPLC-NMR spectroscopy can be used directly to isolate and characterize acyl-migrated isomers of drug glucuronides in whole urine. This approach will be of value in the study of glucuronide acyl migration reactions of nonsteroidal antiinflammatory drugs and other xenobiotic ester glucuronides in whole biofluids.

Ester glucuronides (beta-1-O-acyl-D-glucopyranuronates) of many drugs can undergo a series of acyl migration reactions, resulting in positional isomers and anomers which can react with serum proteins with possible toxicological consequences. We have investigated the acyl migration of the ester glucuronides of the model drug 6,-11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid in pH 7.4 buffer using directly coupled 750 MHz stopped-flow HPLC-NMR spectroscopy. Using a reversed phase isocratic HPLC method with 21% acetonitrile and 79% D2O in the mobile phase, it was possible to separate and hence identify the individual positional isomers of the model drug glucuronide by 750 MHz HPLC-NMR. The order of elution of the isomers from the C18 column was 4alpha-, 4beta-, aglycon, 1beta-, 3beta-, 3alpha-, 2alpha-, 2beta- (alpha- and beta- referring to the anomerization state at C1 on the glucuronide ring and the numbers referring to the carbon number on the glucuronide ring to which the drug moiety has migrated). It is shown that directly coupled ultra-high-field HPLC-NMR spectroscopy offers a unique analytical advantage for obtaining structural information of interconverting compounds in equilibrium mixtures, and this method will be of value in the study of reactive drug glucuronides of toxicological importance.

1. [14C]JV-Ethoxycarbonyl-3-morpholinosydnonimine (molsidomine, Corvaton®) was found to be extensively metabolized following oral dosing to rat and dog and intravenous dosing to rabbit.2. The majority of the radiolabel was rapidly excreted in the urine with the main radiolabelled components being characterized as acidic metabolites resulting from oxidative metabolism of the morpholine ring.3. A new metabolite, (N-cyanomethylenamino-2-aminoethoxy)-aceticacid, was identified and shown to be a major component of the 14C-labelled urinary metabolites in all three species. However, the previously identified metabolite. N-cyancmethylenamino-morpholin-2-one (compound D) was not detected and may therefore have been formed artefactually in the earlier studies.4. The long terminal half-life for plasma radioactivity observed in previous studies was shown to be the result of the production of small amounts of 14C-thiocyanate from the nitrile-containing metabolites of molsidomine.

There is now much interest in the application of NMR techniques to determine the metabolic fate and disposition of foreign compounds [1–4] (& J.K. Nicholson in Vols. 16 & 17, this series — Ed.). For fluorinated compounds 19F NMR can be used with particular advantage due to its large chemical shift range giving good specificity, high sensitivity (83% of 1H) and the absence of endogenous fluorinated compounds in body fluids. In theory, drug recovery studies can be achieved by quantifying the spectra using a known concentration of a standard, thus obviating the need for expensive synthesis of radiolabelled compounds. To illustrate this, we have now investigated the metabolism and urinary excretion of TFMA* and its acetanilide. TLC and GC-MS studies [5] had shown that TFMA is metabolized to its oxanilic acid and a hydroxy metabolite which is probably glucuronidated (Fig. 1).