High-performance liquid-chromatographic separation and fluorescence measurement of biogenic amines in plasma, urine, and tissue

T. P. Davis, C. W. Gehrke, C. W. Gehrke, T. D. Cunningham, K. C. Kuo, K. O. Gerhardt, H. D. Johnson, C. H. Williams

Research output: Contribution to journalArticle

110 Scopus citations

Abstract

The authors describe a high-performance liquid-chromatographic method for measuring histamine, norepinephrine, octopamine, normetanephrine, dopamine, serotonin, and tyramine in plasma (2 ml), brain (0.2 g), or urine. These amines are modified by pre-column derivatization with o-phthalaldehyde, which stabilizes the molecules, facilitates extraction, and improves detection of nanogram amounts. Before separation, samples were neutralized with KOH and immediately derivatized and extracted into ethyl acetate, in which derivatives were stable for longer than 24 h. Interfering amino acids were removed from ethyl acetate by partitioning twice with Na2HPO4 buffer (pH 10.0). Separation was complete in about 90 min on a 'μ Bondapak/phenyl' column, with which a stepwise gradient of methanol/phosphate buffer (pH 5.1) was used. A variable-wavelength fluorometer was used (exciting wavelength, 340 nm; emission wavelength, 480 nm). Amount and response were linearly related from 1 to 200 pmol. Precision (CV) for retention times was 1%, for derivatization and injection 2.5%. Analytical recoveries of the seven amines from 2 ml of plasma fortified with 200 pmol averaged 65% (CV ~ 8%). Data on rat-brain tissue samples are compared with results by the trihydroxyindole method. Application of the method to urine from normal persons and a patient with a brain tumor is demonstrated.

Original languageEnglish (US)
Pages (from-to)1317-1324
Number of pages8
JournalUnknown Journal
Volume24
Issue number8
DOIs
StatePublished - 1978
Externally publishedYes

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

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    Davis, T. P., Gehrke, C. W., Gehrke, C. W., Cunningham, T. D., Kuo, K. C., Gerhardt, K. O., Johnson, H. D., & Williams, C. H. (1978). High-performance liquid-chromatographic separation and fluorescence measurement of biogenic amines in plasma, urine, and tissue. Unknown Journal, 24(8), 1317-1324. https://doi.org/10.1093/clinchem/24.8.1317