Analytical and Bioanalytical Chemistry

, Volume 388, Issue 8, pp 1643–1651 | Cite as

Autism and urinary exogenous neuropeptides: development of an on-line SPE–HPLC–tandem mass spectrometry method to test the opioid excess theory

  • K. DettmerEmail author
  • D. Hanna
  • P. Whetstone
  • R. Hansen
  • B. D. Hammock
Original Paper


Autism is a complex neurodevelopmental disorder with unknown etiology. One hypothesis regarding etiology in autism is the “opioid peptide excess” theory that postulates that excessive amounts of exogenous opioid-like peptides derived from dietary proteins are detectable in urine and that these compounds may be pathophysiologically important in autism. A selective LC–MS/MS method was developed to analyze gliadinomorphin, β-casomorphin, deltorphin 1, and deltorphin 2 in urine. The method is based on on-line SPE extraction of the neuropeptides from urine, column switching, and subsequent HPLC analysis. A limit of detection of 0.25 ng/mL was achieved for all analytes. Analyte recovery rates from urine ranged between 78% and 94%, with relative standard deviations of 0.2–6.8%. The method was used to screen 69 urine samples from children with and without autism spectrum disorders for the occurrence of neuropeptides. The target neuropeptides were not detected above the detection limit in either sample set.


Autism Neuropeptides β-Casomorphin Gliadinomorphin Opioid peptide excess theory On-line SPE–HPLC–MS/MS 



autistic spectrum disorder














benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate


solid-phase extraction







This work was supported by grants from NIEHS Grant R37 ES02710, NIEHS Superfund Basic Research Program Grant P42 ES04699, National Institute of Environmental Health Sciences Center, R01 ES013933, P30 ES05707, and NIEHS Center for Children’s Environmental Health & Disease Prevention Grant P01 ES11269, the UC Davis M.I.N.D. Institute, and in part by the German Research Foundation and Baygene.


  1. 1.
    Wiggins LD, Baio J, Rice C (2006) J Dev Behav Pediatr 27:S79–S87CrossRefGoogle Scholar
  2. 2.
    Association AP (2000) Diagnostic and statistical manual of mental disorders IV—Test Revision. American Psychiatric Association, Washington, DCGoogle Scholar
  3. 3.
    Fombonne E (2003) JAMA 289:87–89CrossRefGoogle Scholar
  4. 4.
    Muhle R, Trentacoste SV, Rapin I (2004) Pediatrics 113:e472–e486CrossRefGoogle Scholar
  5. 5.
    Bailey A, Le Couteur A, Gottesman I, Bolton P, Simonoff E, Yuzda E, Rutter M (1995) Psychol Med 25:63–77CrossRefGoogle Scholar
  6. 6.
    Bailey A, Palferman S, Heavey L, Le Couteur A (1998) J Autism Dev Disord 28:369–392CrossRefGoogle Scholar
  7. 7.
    Panksepp J (1979) Trends Neuroscience 2:174–177Google Scholar
  8. 8.
    Knivsberg AM, Reichelt KL, Hoien T, Nodland M (2002) Nutr Neurosci 5:251–261CrossRefGoogle Scholar
  9. 9.
    Kidd PM (2002) Altern Med Rev 7:472–499Google Scholar
  10. 10.
    Kidd PM (2002) Altern Med Rev 7:292–316Google Scholar
  11. 11.
    Reichelt KL, Knivsberg AM, Nodland M, Lind G (1994) Dev Brain Dysfunct 7:71–85Google Scholar
  12. 12.
    Reichelt KL, Knivsberg AM (2003) Nutr Neurosci 6:19–28CrossRefGoogle Scholar
  13. 13.
    Horvath K, Perman JA (2002) Curr Gastroenterol Rep 4:251–258CrossRefGoogle Scholar
  14. 14.
    Wakefield AJ, Puleston JM, Montgomery SM, Anthony A, O’Leary JJ, Murch SH (2002) Aliment Pharmacol Ther 16:663–674CrossRefGoogle Scholar
  15. 15.
    D’Eufemia P, Celli M, Finocchiaro R, Pacifico L, iozzi L, Zaccagnini M, Cardi E, Giardini O (1996) Acta Paediatr 85:1076–1079Google Scholar
  16. 16.
    Ashwood P, Anthony A, Pellicer AA, Torrente F, Walker-Smith JA, Wakefield AJ (2003) J Clin Immunol 23:504–517CrossRefGoogle Scholar
  17. 17.
    Jyonouchi H, Sun S, Itokazu N (2002) Neuropsychobiology 46:76–84CrossRefGoogle Scholar
  18. 18.
    Horvath K, Papadimitriou JC, Rabsztyn A, Drachenberg C, Tildon JT (1999) J Pediatr 135:559–563CrossRefGoogle Scholar
  19. 19.
    Pavone L, Fiumara A, Bottaro G, Mazzone D, Coleman M (1997) Biol Psychiatry 42:72–75CrossRefGoogle Scholar
  20. 20.
    Arnold GL, Hyman SL, Mooney RA, Kirby RS (2003) J Autism Dev Disord 33:449–454CrossRefGoogle Scholar
  21. 21.
    Bowers L (2002) J Hum Nutr Diet 15:141–144CrossRefGoogle Scholar
  22. 22.
    Hunter LC, O’Hare A, Herron WJ, Fisher LA, Jones GE (2003) Dev Med Child Neurol 45:121–128CrossRefGoogle Scholar
  23. 23.
    Kaiser E, Colescott RL, Bossinger CD, Cook PI (1970) Anal Biochem 34:595–598CrossRefGoogle Scholar
  24. 24.
    Einhorn J, Einhorn C, Luche JL (1991) Synlett 1:37–38Google Scholar
  25. 25.
    Rutter M, Couteur AL, Lord C (2003) Autism diagnostic interview—revised, WPS edition. Western Psychological Services, Los Angeles, CAGoogle Scholar
  26. 26.
    Lord C, Rutter M, DiLavore PC, Risi S (1999) Autism diagnostic observation schedules, WPS edition. Western Psychological Services, Los Angeles, CAGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • K. Dettmer
    • 1
    • 2
    Email author
  • D. Hanna
    • 2
  • P. Whetstone
    • 2
  • R. Hansen
    • 3
  • B. D. Hammock
    • 2
  1. 1.Institute of Functional GenomicsUniversity of RegensburgRegensburgGermany
  2. 2.Department of EntomologyUniversity of California at DavisDavisUSA
  3. 3.Department of Pediatrics, M.I.N.D. InstituteUniversity of California Davis School of MedicineSacramentoUSA

Personalised recommendations