Quantitation of phenobarbital and its main metabolites in human urine

  • N. Kållberg
  • S. Agurell
  • Ö. Ericsson
  • E. Bucht
  • B. Jalling
  • L. O. Boréus


A method for the quantitative determination of phenobarbital and free and conjugatedp-hydroxyphenobarbital in urine samples is described. The method includes initial extraction, purification on a small chromatographic column and finally determination by gas chromatography. The barbituric acids are methylated by trimethylanilinium hydroxide which serves as a “flash heater” methylating agent. The conjugate ofp-hydroxyphenobarbital, which appears to be a glucuronide, is hydrolysed with hydrochloric acid.

Key words

Conjugate-cleavage drug metabolism gas chromatography phenobarbital urinary excretion 


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  1. 1.
    Algeri, E.J., McBay, A.J.: Metabolite of phenobarbital in human urine. Science123, 183–184 (1956)Google Scholar
  2. 2.
    Atkinson, A.J., MacGee, J., Strong, J., Garteiz, D., Gaffney, T.E.: Identification of 5-meta-hydroxyphenyl-5-phenylhydantoin as a metabolite of diphenylhydantoin. Biochem. Pharmacol.19, 2483–2491 (1970)Google Scholar
  3. 3.
    Brochmann-Hanssen, E., Oke, T.O.: Gas chromatography of barbiturates, phenolic alkaloids, and xanthine bases: flash-heater methylation by means of trimethylanilinium hydroxide. J. pharm. Sci.58, 370–371 (1969)Google Scholar
  4. 4.
    Butler, T.C.: The metabolic hydroxylation of phenobarbital. J. Pharmacol. exp. Ther.116, 326–336 (1956)Google Scholar
  5. 5.
    Butler, T.C.: The metabolic conversion of 5,5-diphenylhydantoin to 5-(p-hydroxyphenyl)-5-phenylhydantoin. J. Pharmacol. exp. Ther.119, 1–11 (1957)Google Scholar
  6. 6.
    Chang, T., Glazko, A.J.: Quantitative assay of 5,5-diphenylhydantoin (Dilantin®) and 5-(p-hydroxyphenyl)-5-phenylhydantoin by gas-liquid chromatography. J. Lab. clin. Med.75, 145–155 (1970)Google Scholar
  7. 7.
    Chang, T., Savory, A., Glazko, A.J.: A new metabolite of 5,5-diphenylhydantoin (Dilantin®). Biochem. biophys. Res. Commun.38, 444–449 (1970)Google Scholar
  8. 8.
    Glasson, B., Benakis, A.: Etude du phénobarbital C-14 dans l'organisme du rat. Helv. Physiol. Pharmacol. Acta19, 323–334 (1961)Google Scholar
  9. 9.
    Grover, P.L., Hewer, A., Sims, P.: Epoxides as microsomal metabolites of polycyclic hydrocarbons. FEBS Letters18, 76–80 (1971)Google Scholar
  10. 10.
    Harvey, D.J., Glazener, L., Stratton, C., Nowlin, J., Hill, R.M., Horning, M.G.: Detection of a 5-(3,4-dihydroxy-1,5-cyclohexadien-1-yl)-metabolite of phenobarbital and mephobarbital in rat, guinea pig and human. Res. Commun. chem. Pathol. Pharmacol.3, 557–565 (1972)Google Scholar
  11. 11.
    Horning, E.C., Horning, M.G.: Metabolic Prophiles: Gas-phase methods for analysis of metabolites. Clin. Chem.17, 802–809 (1971)Google Scholar
  12. 12.
    Horning, M.G., Butler, C., Harvey, D.J., Hill, R.M., Zion, T.E.: Metabolism of N,2-dimethyl-2-phenylsuccinimide (methsuximide) by the epoxide-diol pathway in rat, guinea pig and human. Res. Commun. chem. Pathol. Pharmacol.6, 565–578 (1973)Google Scholar
  13. 13.
    Jalling, B., Boréus, L.O., Kållberg, N., Agurell, S.: Disappearance from the newborn of circulating prenatally administered phenobarbital. Europ. J. clin. Pharmacol.6, 234–238 (1973)Google Scholar
  14. 14.
    Jalling, B.: Plasma and cerebrospinal fluid concentration of phenobarbital in infants given single doses. Develop. Med. Child Neurol.16, 781–793 (1974)Google Scholar
  15. 15.
    Kållberg, N., Agurell, S., Boréus, L.O., Jalling, B.: Rapid gas chromatographic determination of phenobarbital in small plasma samples. Europ. J. clin. Pharmacol.3, 185–188 (1971)Google Scholar
  16. 16.
    Modin, R., Tilly, A.: Quantitative determinations by ion pair extraction. Ion pairs between quaternary ammonium ions and carboxylic acids and phenol. Acta pharm. suec.5, 311–322 (1968)Google Scholar
  17. 17.
    Nicholson, J.D.: The urinary excretion of phenobarbitone and pentobarbitone in the horse. Biochem. Pharmacol.17, 1–8 (1968)Google Scholar
  18. 18.
    Oesch, F., Morris, N., Daly, J.W., Gielen, J.E., Nebert, D.W.: Genetic expression of the induction of epoxide hydrase and aryl hydrocarbon hydroxylase activities in the mouse by phenobarbital or 3-methylcholanthrene. Mol. Pharmacol.9, 692–696 (1973)Google Scholar
  19. 19.
    Pierce, A.E., Rising, M.M.: Chemical studies of the mechanism of the narcosis induced by hypnotics. The synthesis of colored derivatives of phenobarbital. J. Amer. chem. Soc.58, 1361–1363 (1936)Google Scholar
  20. 20.
    Svendsen, A.B., Brochmann-Hanssen, E.: Gas chromatography of barbiturates II. Application to the study of their metabolism and excretion in humans. J. pharm. Sci.51, 494–495 (1962)Google Scholar

Copyright information

© Springer-Verlag 1975

Authors and Affiliations

  • N. Kållberg
    • 1
  • S. Agurell
    • 1
  • Ö. Ericsson
    • 2
  • E. Bucht
    • 3
  • B. Jalling
    • 3
  • L. O. Boréus
    • 3
  1. 1.The Karolinska PharmacyStockholmSweden
  2. 2.Dept. of Organic Chemistry, Faculty of PharmacyUniversity of UppsalaStockholmSweden
  3. 3.Dept. of Clinical PharmacologyKarolinska HospitalStockholmSweden

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