in the Assessment of Testosterone in a New Reference Method Procedure To the Editor:

We read with interest the recent publication by Botelho and col-leagues describing a candidate ref-erence method procedure for measuring serum testosterone to support the CDC’sHormone Stan-dardization (HoSt) Program (1). The aims of this program are to standardize serum testosterone measurements across laboratories and platforms and to support the generation of age- and sex-specific reference intervals for testosterone. After careful consideration, we have identified a potential error in the assay that could affect the suc-cess of the program. Specifically, the final testosterone concentra-tion is determined by multiplying the response ratio of the analyte peak to the internal-standard peak by the concentration of the inter-nal standard. By design, the re-sponse ratio is near 1.0, which has previously been demonstrated to reduce the imprecision of mea-surement (2). The error in this approach lies in the use of a testosterone internal standard labeled with the 13C3 sta-ble isotope, whichwill lead to 2 im-portant problems for the HoSt program. First, the reference inter-vals for testosterone will be inaccu-rate, and second,HoSt participants who use gravimetric preparation techniques for their calibration materials will observe that the pro-gram produces a bias from the ex-pected results. Mass spectrometry typically measures the dominant monoiso-topic species of the analyte to be measured. Testosterone (C19H28O2) has a monoisotopic mass of 288.209 with an expected protonatedmolec-ular ion [MH] ofm/z 289.2. Be-cause 1.1 % of the carbon is 13C, a mass spectrometer measuring the dominantmonoisotopicmassof tes-tosterone would measure approxi-mately 77 % of the molecule (atm/z 289.2), with approximately 21 % oc-curring as the 1 isotope (m/z 290.2) and approximately 2 % as the 2 isotope (m/z 291.2) (3). By enriching the internal stan-dard with 3 13C atoms, the internal standard has an altered proportion of the dominant monoisotopi