IDENTIFICATION OF URINARY AND BILIARY CONJUGATED METABOLITES OF THE NEUROMUSCULAR BLOCKER 51W89 BY LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY

Cisatracurium, (1R, 1'R, 2R, 4-tetrahydro-6,7-dimethoxy-2-methylisoqui nolinium] dibenzenesulphonate (51W89), is an intermediate-acting neuro muscular blocking agent. 51W89 is one of ten isomers contained in Trac rium(R) (atracurium besylate) and represents approximately 15 percent of the atracurium mixture. Clinical studies have indicated that 51W89 is more potent and is significantly weaker as a histamine releaser tha n atracurium. In vitro studies in human plasma have shown that, like a tracurium, 51W89 spontaneously degrades at physiological pH by Hoffman n elimination to form laudanosine and the quaternary monoacrylate. Sub sequent ester hydrolysis of the monoacrylate generates the monoquatern ary alcohol. In rat plasma, 51W89 is also metabolized by non-specific carboxylesterases to the monoquaternary alcohol and the monoquaternary acid, the former being rapidly hydrolysed further to the more stable acid. It has been reported that laudanosine can be further metabolized via N-demethylation to yield tetrahydropapaverine. The rate-limiting step in the degradation of 51W89 in human plasma is Hofmann eliminatio n, whilst in rat plasma, the action of non-specific carboxylesterases is rate limiting. As part of the development of 51W89, the disposition of C-14-51W89 following a single intravenous bolus dose was studied i n various animal species and humans. In the present work, we describe the identification of 51W89 metabolites in urine and bile from these s tudies by high performance liquid chromatography/mass spectrometry usi ng pneumatically-assisted electrospray ionization coupled to an on-lin e radioactivity monitor. This methodology enabled rapid and sensitive screening of biological samples with minimal sample preparation. Struc tural confirmation of metabolites was obtained by tandem mass spectrom etry. In addition to the expected metabolites, a number of urinary and biliary O-glucuronic acid conjugates of monodesmethyl laudanosine and monodesmethyl tetrahydropapaverine were identified, which cochromatog raphed with an early eluting 'unknown' in the radioprofile. A sulphate conjugate of monodesmethyl laudanosine was also identified in fat bil e. The characterization of these metabolites was assisted by the incor poration of on-line radioactivity monitoring during mass spectroscopic analysis, which provided an invaluable means to distinguish drug-rela ted and endogenous material.