Identification of thyroxine in human breast milk by gas chromatography-mass spectrometry

In several previous publications it has been reported that human breast milk contains significant amounts of T4. Great differences in concentrations were found by different authors. In all these publications T4 was assayed by RIA, and it seems probable that the varying results obtained are due to varying specificity of the antibodies used in the assay. Definite demonstration of the presence of T4 in breast milk requires a more specific method than RIA. In the present work we have used combined gas chromatography-mass spectrometry. T4 was isolated from human breast milk by combined anion- and cation-exchange chromatography. The purified material was treated with HCl in methanol and trifluoroacetic acid anhydride and was analyzed by single-ion monitoring of the ions at m/e 983, m/e 870, and m/e 799, major ions in the mass spectrum of the N,O-bis(trifluoroacetyl) methyl ester of T4. Significant peaks confirmed the presence of T4 in human milk. The estimated concentration was less than 10 ng/ml. Attempts were made to quantitate the T4 by selected ion monitoring of the ions at m/e 870 and m/e 822 after addition of monobromotriiodothyronine as internal standard. According to this latter method, the concentration of T4 in four samples of centrifuged breast milk was less than 4 ng/ml. The possible physiological significance of the finding is discussed.