J. W. Hubbard

Application of electrospray mass spectrometry in the identification of intact glucuronide and sulphate conjugates of clozapine in rat

1. The phase II metabolites in the bile, urine and faeces of rat dosed with clozapine were investigated by means of electrospray mass spectrometry (ESMS) in both positive and negative ion modes. 2. When operated at a cone voltage of 45 V, this soft ionization technique permitted the detection of quasi molecular ions of both sulphate and glucuronide conjugates of hydroxylated phase I metabolites of clozapine. With the cone voltage set at 90 V, however, the ESMS also contained highly diagnostic ions resulting from the loss of 80 Daltons (sulphur trioxide) or 176 Daltons (the glucuronide moiety) from sulphates and O-glucuronides respectively. 3. A sufficient quantity of one metabolite was isolated from rat bile to permit further analysis by 1H-nmr. This metabolite, which was also found in rat urine, was proved to be 7-O-glucuronyl-7-hydroxyclozapine. The analogous sulphate metabolite was also identified in bile by ESMS. 4. Correspondingly glucuronide and sulphate conjugates of a hydroxylated N-desmethyl clozapine were similarly detected in rat bile. There was insufficient material to permit analysis by 1H-NMR, but it appears likely that conjugation was also at the 7-position of N-desmethylclozapine. 5. Finally, the sulphate conjugate of a hydroxy dechlorinated derivative of clozapine was identified by ESMS in both urine and bile. By analogy with a previous report of a similar metabolite in man, the metabolite was tentatively identified as 8-hydroxy-8-deschloroclozapine.

The identification of two new urinary metabolites of fenfluramine in man

1. Metabolism of the anorectic agent, fenfluramine, was studied in man to detect phenolic and/or alcoholic metabolites.2. Two new metabolites identified as 1-(m-trifluoromethylphenyl)-2-propanol and 1-(m-trifluoromethylphenyl)-1,2-propanediol, were detected in human urine by g.l.c. and g.l.c.-mass spectrometry.

Metabolic O-demethylation of 3,4-dimethoxy-amphetamine in vivo in dog and monkey

1. The disposition of the hallucinogen 3,4-dimethoxyamphetamine in vivo was examined in dogs and monkeys. 2. O-Demethylation is important since 3-O-methyl-alpha-methyldopamine (3-methoxy-alpha-methyltyramine) was found in the urine of both species, and traces of alpha-methyldopamine were found in the urine of dogs. 3. Also found in the urine of dogs were 1-(3,4-dihydroxyphenyl)propan-2-one and 3,4-dihydroxybenzoic acid, which are side-chain modified metabolites of alpha-methyldopamine. 4. 1-(3-Methoxy-4-hydroxyphenyl)propan-2-one, a side-chain modified metabolite of 3-O-methyl-alpha-methyldopamine, was present in the urine of both dogs and monkeys. 5. The 3-O-demethylated isomers 4-O-methyldopamine and 1-(3-hydroxy-4-methoxyphenyl)propan-2-one were not detected.

Identification of phenolic acids in human urine by ion monitoring.

1. Two-different double derivatization techniques and two different g.l.c. systems were used to separate isomeric phenolic carboxylic acids in normal human urine. 2. Carboxylic acids were converted into n-butyl esters and phenolic functions into trifluoroacetic acid esters. 3. n-Butyl trifluoroacetoxybenzoates were separated by g.l.c. and detected by mass spectrometric single-ion monitoring. 4. Trifluoroacetates were hydrolysed under mild conditions, and liberated phenolic groups were subjected to flash methylation. 5. n-Butyl methoxybenzoates were separated by g.l.c. and detected by flame ionization. 6. All derivatives were identified by comparison of retention times and mass spectra with those of authentic reference standards. 7. The urine of a normal vegetarian volunteer was examined. 8. The presence of meta- and para-hydroxybenzoic acids, vanillic acid and isovanillic acid was confirmed by unambiguous techniques.

The metabolism of 3-methoxyamphetamine in dog, monkey and man

1. The metabolism of 3-methoxyamphetamine in vivo was examined in dog, monkey and man. 2. The metabolites identified in all three species were 3-O-methyl-alpha-methyldopamine, 1-(3-methoxy-4-hydroxyphenyl)propan-2-ol, 3-hydroxyamphetamine, 1-(3-hydroxyphenyl)propan-2-one, 1-(3-hydroxyphenyl)propan-2-ol, 1-(3-methoxyphenyl)propan-2-ol and 1-(3-methoxyphenyl)propane-1,2-diol. 3. 1-Hydroxyl-1(3-hydroxyphenyl)propan-2-one was tentatively identified in the urine of all three species. 4. 4-O-Methyl-alpha-methyldopamine was also found in the urine of dog and monkey but not in human urine.

Chapter 13 Radioimmunoassays for Phenalkylamines

Publisher Summary This chapter discusses the radioimmunoassays (RIA) for phenalkylamines. RIA is a highly sensitive assay technique which was developed in the 1950s. Following the injection of porcine insulin into humans or guinea-pigs, they observed the production of a globulin (antibody) with an extraordinary specific affinity for insulin. Their RIA depends upon competition between natural insulin and I-labeled insulin for binding sites on a limited amount of the antibody. After separation of the antibody-bound insulins from the free, unbound insulins, the amount of radioactivity in each fraction is determined. The ratio of radioactivity in the free fraction to radioactivity in the bound fraction is related to the concentration of natural insulin in the sample. RIA procedures have been developed subsequently for other macromolecules such as growth hormone, parathyroid hormone and adrenocorticotrophic hormone and also for a wide variety of low molecular weight compounds.