Development of a liquid chromatographic/tandem mass spectrometric assay for a new endothelin receptor antagonist, and its application to dog plasma samples generated after simultaneous i.v. and p.o. administration of the unlabeled and deuterium-labeled forms of this antagonist.
An assay was developed and applied to determine the bioavailability of a new endothelin receptor antagonist after simultaneous p.o. and i.v. administration of the drug and its stable isotope-labeled analogue. The drug, its main metabolite and the stable isotope-labeled analogues of the drug and the main metabolite were quantified in dog plasma samples using a structural analogue as internal standard. In addition to the calculation of the bioavailability, the formation of the metabolite after p.o. and i.v. administration could be followed independently. The assay covered the concentration range 0.25-1000 ng ml(-1) using sample aliquots of only 50 micro l. Plasma samples were processed after protein precipitation with on-line solid-phase extraction, narrow-bore high-performance liquid chromatography and subsequent tandem mass spectrometric detection. Detection was accomplished with ionspray in the positive ion selected reaction monitoring mode. The inter-assay precision and accuracy of the assay were in the range 4.7-14.2% and 90.3-113.3%, respectively, and the intra-assay precision and accuracy were in the range 1.4-11.5% and 88.4-112.5%, respectively. The fragmentation of the drug was investigated and showed an unexpected shift of a methyl group. Data from MS(n), medium-resolution exact mass tandem mass spectrometry and H-D exchange experiments were employed to clarify the mechanism.
Lausecker
B
B
F. Hoffmann-La Roche, Ltd, Non-Clinical Drug Safety, P.O. Box, CH-4070 Basel, Switzerland. berthold.lausecker@roche.com
Fischer
G
G
D000284:Administration, Oral; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003903:Deuterium; D004285:Dogs; D065128:Endothelin Receptor Antagonists; D004791:Enzyme Inhibitors / Q000008:administration & dosage* / Q000097:blood* / Q000737:chemistry / Q000494:pharmacology; D007275:Injections, Intravenous; D013058:Mass Spectrometry / Q000379:methods*; D015394:Molecular Structure; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D065128:Endothelin Receptor Antagonists; D004791:Enzyme Inhibitors; D003903:Deuterium