Lipoic acid analogs and L-tryptophan do not interfere with the assay of biotin by HPLC/avidin-binding assay

J. Zempleni, P. M. Mock, S. L. Stratton, D. B. McCormick

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


There are structural similarities in the side chain and heterocyclic ring among biotin, lipoic acid analogs (LAA), and L-tryptophan (TRP). The large association constant for the avidin - lipoic acid complex and the cleavage of lipoyllysine by biotinidase further indicate a close relationship of LAA and biotin. We assessed the potential interference of LAA and TRP with the measurement of biotin by the HPLC/avidin-binding assay. In this assay, compounds are separated by HPLC, followed by assay of each HPLC fraction based on binding to avidin-horseradish peroxidase. Compounds that interfere can be separated from biotin and its metabolites by chromatography. Using physiologic concentrations, LAA (d-lipoate, 1-lipoale, d.l-lipoamide, bisnor-, β-hydroxybisnor-, and tetranorlipoates) did not compete for avidin-binding in our assay, whereas TRP showed minor avidin binding (0.0003% compared to biotin, 100%). Spectrophotometric determination of HPLC retention times showed that none of the LAA co-eluted with any of 16 available naturally occurring biotin metabolites, whereas TRP co-eluted (22 min) with bisnorbiotin methyl ketone. Because avidin affinity of TRP was 5 orders of magnitude smaller than that of bisnorbiotin methyl ketone, we conclude that neither LAA nor TRP interfere with the measurement of biotin or its metabolites by the HPLC/avidin-binding assay.

Original languageEnglish (US)
Pages (from-to)A802
JournalFASEB Journal
Issue number3
StatePublished - 1996
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics


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