For several decades, the major method for the measurement of body fluid amino acids has been ion-exchange chromatographic separation with postcolumn ninhydrin derivatization and dual-wavelength spectrophotometric detection. Ninhydrin reacts with primary amines, including most amino acids, and some secondary amines. Identification is based on peak retention time and relative dual spectrophotometric signals. A number of drugs, including some commonly used antibiotics, have long been known to also react with ninhydrin and to interfere with amino acid measurement, particularly in urine (1).
This report highlights a case of a very large interference in the chromatographic region around tryptophan in a patient's urine that might have had inappropriate diagnostic implications had the investigation into the true nature of the ninhydrin-positive peak not been undertaken. Vigabatrin is one of the latest generation of anticonvulsant medications that interferes with the endogenous metabolism of γ-aminobutyric acid aminotransferase. It is also a primary amine that is excreted unchanged in the urine. In the ion-exchange chromatography used in this report, the elution of urinary vigabatrin was sufficiently close to that of tryptophan to appear as a tryptophan peak and to initially indicate hypertryptophanuria. Such a finding was not consistent with the clinical phenotype of the patient, who was under investigation for a probable mitochondrial energy disorder that may have been associated with a generalized aminoaciduria. The subsequent differential diagnosis for tryptophanuria would have included Hartnup condition, a disorder caused by a mutation in the SLC6A19 transporter, in which urinary neutral amino acids are increased because of a failure of tubular reabsorption (2). Tryptophanuria associated with dwarfism is seen in the exceedingly rare defect initially described by Tada et al. and postulated to be a defect in the kynurenine pathway (3).
The true identity of the interfering compound was made via Ultra Performance Liquid Chromatography (UPLC) separation and peak identification by tandem mass spectrometry, which also demonstrated a nonpathologic urinary tryptophan concentration. The important point that this case report makes is the advantage of methods with positive identification that use mass spectrometry over methods that rely on retention time alone. This point can be applied to all separation procedures with non–mass spectrometric detection, including all HPLC, UPLC, capillary electrophoresis, and gas chromatography methods that rely on other detector types.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: M.J. Bennett, Clinical Chemistry, AACC.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: None declared.
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
- Received for publication December 7, 2010.
- Accepted for publication December 14, 2010.
- © 2011 The American Association for Clinical Chemistry