title: Narrow-bore liquid chromatography-tandem mass spectrometry with simultaneous radioactivity monitoring for partially characterizing the biliary metabolites of an arginine fluoroalkyl ketone analog of D-MePhe-Pro-Arg, a potent thrombin inhibitor.
abstract: Characterizing components eluting from a HPLC column is enhanced when multiple detectors are incorporated in-line. The performance of a system consisting of a combination of two detectors-electrospray ionization mass spectrometry (and tandem mass spectrometry) and radioactivity monitoring, following gradient separation with a 250 x 2.1 mm I.D. (Vydac Protein and Peptide C18, 5 microns, 300 A) column-is evaluated with respect to chromatographic integrity and detection. The HPLC effluent was split (8:1) and a post-column make-up solvent was added to flow directed towards the radioactivity detector containing a solid glass cell. Trifluoracetic acid (0.1%) was added to the make-up flow solvent to prevent silanol interactions from degrading the profile displayed in the 14C trace. A 14C chromatographic peak representing 550 dpm was detected with signal-to-noise ratio of 3. This system was used for rapidly characterizing the biliary metabolites of an arginine fluoroalkyl ketone analog of D-MePhe-Pro-Arg, a potent thrombin inhibitor currently being evaluated as a drug candidate. These metabolites are shown to comprise of mono- and dihydroxylated drug as well as a reduced ketone form of the drug. Combining the radioactivity monitor in-line with the mass spectrometer ensured that all of the major metabolites (as evident from the 14C profile) were characterized by mass spectrometry.
authors.affiliation: Hoechst Marion Roussel, Kansas City, MO 64134-0627, USA.
authors.forename: T G
authors.initials: TG
authors.lastname: Heath
authors.forename: J P
authors.initials: JP
authors.lastname: Mooney
authors.forename: R
authors.initials: R
authors.lastname: Broersma
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D001646:Bile / Q000737:chemistry* / Q000378:metabolism; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D008297:Male; D013058:Mass Spectrometry; D009842:Oligopeptides / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013917:Thrombin / Q000037:antagonists & inhibitors*
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D002250:Carbon Radioisotopes; C105868:N-methylphenylalanyl-prolyl-arginine 1,1-difluorobutyl ketone; D009842:Oligopeptides; C104714:N-methylphenylalanyl-prolyl-arginine; D013917:Thrombin