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Clinical Chemistry 19: 459-462, 1973;
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Clinical Chemistry, Vol 19, 459-462, Copyright © 1973 by the American Association for Clinical Chemistry

Urinary Homogentisic Acid: Determination by Thin-Layer Chromatography

Jerome M. Feldman 1 and June Bowman 1

1 From the Division of Endocrinology, Department of Medicine, Duke University Medical Center; and Durham Veterans Administration Hospital, Durham, N. C. 27710.

A new method is described for qualitative and quantitative determination of urinary homogentisic acid. The method involves extraction of urine with ethyl acetate, thin-layer chromatography of the extract on Silica Gel G, elution of the homogentisic acid into water, and color development with Folin’s phenol reagent. Absorbance is maximum at 750 nm and linear to a concentration of at least 5 mg of homogentisic acid per milliliter of urine. The method is highly specific for homogentisic acid; added gentisic acid, 3,4-dihydroxyphenylacetic acid, ascorbic acid, or L-3,4 dihydroxyphenylalanine do not interfere. The coefficient of variation "in-run" is 5.3%, "between-run" 6.8%. Using this method we have demonstrated a marked variation in day-to-day homogentisic acid excretion in a patient with alcaptonuria. This method, which offers some advantages over existing techniques, should be suitable for use in a clinical laboratory.


Key Words: alcaptonuria • inherited metabolic disorders • day-to-day variation

Submitted on December 27, 1972
Accepted on February 9, 1973






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Copyright © 1973 by the American Association for Clinical Chemistry.