Identification of cis-3'-hydroxycotinine as a urinary nicotine metabolite

1. cis-3'-Hydroxycotinine was detected as an S(-)-nicotine metabolite in the urine of smokers as well as in the urine of rats and hamsters dosed with nicotine.

2. The excreted amount of cis-3'-hydroxycotinine is lower than that of the trans-isomer.     

Synthesis of (3'R,5'S)-trans-3'-hydroxycotinine, a major metabolite of nicotine. Metabolic formation of 3'-hydroxycotinine in humans is highly stereoselective

A method for the synthesis of (3'R,5'S-trans-3'-hydroxycotinine, a major metabolite of nicotine in humans, is described. The method involves deprotonation of (S)-cotinine with lithium diisopropylamide (LDA) followed by oxidation with the transition metal peroxide oxodiperoxymolybdenum(pyridine)(hexamethylphosphoric triamide) (MoOPH) to give an 80:20 mixture of trans-/cis-3'-hydroxycotinine. The pure (greater than 98%) trans isomer is obtained by conversion to the solid hexanoate ester, recrystallization, and cleavage of the ester by heating with n-butylamine. GC-MS analysis of urine extracts from several smokers indicated that in humans metabolic 3'-hydroxycotinine is 95-98% trans.     

A convenient synthesis of trans-3'-hydroxycotinine, a major nicotine metabolite found in the urine of tobacco users

trans-3'-Hydroxycotinine is a major urinary metabolite of nicotine in smokers, but no straightforward method is available for its synthesis. A simple method was developed for preparation of trans-3'-hydroxycotinine from cotinine in two steps by using NaN[(CH3)3Si]2 and dibenzyl peroxydicarbonate, followed by base-catalyzed hydrolysis.     

Identification of a urinary metabolite of perazine as a piperazine-2,5-dione derivative

A non-basic perazine metabolite has been isolated from the urine of schizophrenic patients ingesting perazine. Identification of this compound as 10-[3‘-(2“,5”-dioxo-4“-methyl-piperazinyl)-propyl]-phenothiazine sulphoxide was achieved using ultraviolet, infrared, nuclear magnetic resonance and mass spectroscopy.     

Evidence for urinary excretion of glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine in smokers.

Urinary nicotine metabolic output was profiled for 11 smokers who smoked their regular cigarette brands ad libitum. Thermospray liquid chromatography/mass spectrometry was used to monitor nicotine and eight metabolites, including glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine that were determined indirectly using enzyme hydrolysis. These results were used to estimate an average, steady-state concentration in a 24-hr urine sample during ad libitum smoking and to assess interindividual variability in the excretion of these metabolites. The variability in absolute amount among the nine analytes ranged from 35 to 70% for these smokers. The glucuronide conjugates constituted an average of 29% of all urinary metabolites monitored in this study. trans-3'-Hydroxycotinine in the free form constitutes the largest single metabolite in smokers' urine, with an average of 35% of the total. The sums of nicotine metabolites determined here are very close to the Federal Trade Commission yields of nicotine for the total number of cigarettes smoked by these subjects during the urine collection interval. These results indicate that a large proportion of the nicotine absorbed while smoking can be accounted for as urinary metabolites of nicotine, including glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine.     

Usefulness of salivary trans-3'-hydroxycotinine concentration and trans-3'-hydroxycotinine/cotinine ratio as biomarkers of cigarette smoke in pregnant women

Nicotine is rapidly and extensively metabolized in humans and negatively impacts the developing fetus. The concentrations of nicotine, cotinine, trans-3'-hydroxycotinine (hydroxycotinine), and norcotinine in pregnant smokers' oral fluid were evaluated to determine usefulness as biomarkers of cigarette smoking. Sixteen participants were divided into two groups: eight light smokers (LS) who smoked < or =10 cigarettes/day and eight heavy smokers (HS) who smoked > or =20 cigarettes/day. Oral fluid specimens (n=415) were collected throughout pregnancy and analyzed with solid-phase extraction followed by gas chromatography-mass spectrometry-electron impact selected ion monitoring. Median concentrations of nicotine, cotinine, and hydroxycotinine in oral fluid of LS ranged from 241.1 to 622.0, 80.6 to 387.5, and 14.4 to 117.7 ng/mL and for HS 146.5-1372.2, 66.0-245.8, and 38.3-184.4 ng/mL, respectively. Salivary cotinine and hydroxycotinine concentrations were significantly correlated in LS (r = 0.55, p < 0.01) and HS (r = 0.74, p < 0.01). Ratios of hydroxycotinine/cotinine in oral fluid from pregnant women averaged 0.30 +/- 0.18 (range, 0.07-1.05) for LS and 0.68 +/- 0.25 (range, 0.29-1.83) for HS. Based on these preliminary data, the best ratio to differentiate light from heavy pregnant smokers was 0.41. Salivary hydroxycotinine and cotinine concentrations are both good biomarkers of cigarette smoking. Determining the hydroxycotinine/cotinine ratio may differentiate light from heavy tobacco use and help predict increased fetal tobacco exposure.     

Synthesis of a hapten to be used in development of immunoassays for trans-3'-hydroxycotinine, a major metabolite of cotinine.

4-Carboxyl-substituted analogues of trans-3'-hydroxycotinine were synthesized to be covalently linked to macromolecules for antibody production. 3-Pyridyl-N-methylnitrone was condensed with dimethyl fumarate to give two isomeric isoxazolidines. Hydrogenolysis of the major product [2RS-(2 alpha,3 alpha,3 beta)]-3-carbomethoxy-3- [[(benzyloxy)carbonyl]oxy]-1-methyl-5-oxo-2-(3-pyridinyl)pyrrolidine with Pd/C followed by hydrolysis gave [2RS-(2 alpha,3 beta,4 beta)]-4-hydroxy-1-methyl-5-oxo-2-(3-pyridinyl)-3- pyrrolidinecarboxylic acid. The same compound was also prepared in two steps in high yield starting with dibenzyl fumarate and 3-pyridyl-N-methylnitrone.     

Identification of N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine as a urinary metabolite of benzene, phenol, and hydroquinone

The metabolite 2-(S-glutathionyl)hydroquinone is formed when a microsomal incubation mixture containing either benzene or phenol is supplemented with glutathione. This metabolite is derived from the conjugation of benzoquinone, an oxidation product of hydroquinone. However, neither the glutathione conjugate or its mercapturate, N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine, have been identified as metabolites resulting from in vivo metabolism of benzene, phenol, or hydroquinone. To determine if a hydroxylated mercapturate is produced in vivo, we treated male Sprague-Dawley rats with either benzene (600 mg/kg), phenol (75 mg/kg), or hydroquinone (75 mg/kg) and collected the urine for 24 hr. HPLC coupled with electrochemical detection confirmed the presence of a metabolite that was chromatographically and electrochemically identical to N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine. The metabolite was isolated from the urine samples and treated with diazomethane to form the N-acetyl-S-(2,5-dimethoxyphenyl)-L-cysteine methyl ester derivative. The mass spectra obtained from these samples were identical to that of an authentic sample of the derivative. The results of these experiments indicate that benzene, phenol, and hydroquinone are metabolized in vivo to benzoquinone and excreted as the mercapturate, N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine.     

Identification of detomidine carboxylic acid as the major urinary metabolite of detomidine in the horse.

Horse urine was investigated for metabolites by chromatography and mass spectrometry following the oral administration of the large animal analgesic sedative detomidine to two stallions and intravenous administration of [3H]-detomidine to a mare. Detomidine carboxylic acid and hydroxydetomidine glucuronic acid conjugate were identified in the urine after the oral doses. In addition, traces of free hydroxydetomidine were observed. About half of the radioactivity of [3H]-detomidine was excreted in the urine in 12 h after the i.v. dose (80 micrograms/kg). Most of the excretion occurred between 5 and 12 h in contrast to urine output which was highest 2-5 h after the dosing. The major radioactive metabolite in the urine was detomidine carboxylic acid. It comprised more than two thirds of the total metabolites in all the urine fractions collected. Its excretion profile was similar to that of total radioactivity. Hydroxydetomidine glucuronide was also excreted. It contributed 10-20% of the total metabolites in the urine. The free aglycone was only seen in the samples collected during the peak urine flow. A minor metabolite was tentatively characterized as the glucuronide of N-hydroxydetomidine.     

3-O-methyl-alpha-methyldopamine, a urinary metabolite of p-methoxyamphetamine in dog and monkey

Metabolites of p-methoxyamphetamine in the urine of dogs and monkeys were separated by gas-liquid chromatography as their trifluoroacetyl derivatives, and identified by comparison of the chromatographic and mass spectrometric behavior of these derivatives with those of authentic synthetic compounds. The three new metabolites identified in both dogs and monkeys were 3-O-methyl-alpha-methyldopamine, 4-hydroxy-3-methoxybenzyl methyl ketone, and 4-hydroxybenzyl methyl ketone. A fourth new metabolite was tentatively identified in the absence of the appropriate reference standard.     

Identification of 2-benzimidazolinone as a metabolite of droperidol in man

2(3H)-Benzimidazolinone has been identified in human urine as a metabolite of the neuroleptic droperidol. Comparative GC-MS examination of both purified urine extracts and synthesised references has shown that no more than 5% of the administered droperidol dose was excreted as 2-benzimidazolinone.     

Hopantenic acid beta-glucoside as a new urinary metabolite of calcium hopantenate in dogs

The metabolism of calcium hopantenate (HOPA) was studied in beagle dogs. After oral administration of 14C-labeled HOPA, 25.5% of the administered radioactivity was excreted in the urine within 24 hr, mostly in the form of unchanged drug. The only metabolite, accounting for 4.2% of the radioactivity in the urine, was isolated by HPLC. The metabolite was hydrolyzed by the treatment of beta-glucuronidase (Helix pomatia), acid phosphatase, or beta-glucosidase. These enzyme activities were not inhibited by treatment with D-glucaric acid 1,4-lactone or PO4(3-), but with D-gluconic acid 1,5-lactone, demonstrating that the metabolite is a glucose conjugate. The compound was identified as HOPA-glucoside, 4'-O-(beta-D-glucopyranosyl)-D-hopantenic acid, by GC/MS analyses after derivatization of the metabolite and the synthetic compound. This is the first reported instance of glucose conjugation to a non-acidic hydroxyl group in the metabolism of xenobiotics in mammals.