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Objectives Thyroid dysfunction is a common medical condition affecting an estimated 30 million people in the US alone. Employing gold standard Liquid chromatography–tandem mass spectrometry (LC–MSMS) methods we have examined the extent of inaccuracy of immunoassay (IA) measurement for total T3 (TT3) at low, normal and high concentrations. Design and Methods 268 TT3 Roche Cobas 6000 immunoassay TT3 values (covering the low, normal, and high ranges) were compared with LC–MSMS results. Results At TT3 concentrations between 50 and 113 ng/dL (conversion factor for TT3 to SI Units is ng/dL × 0.0154 = nmol/L), n = 122, LC–MSMS values were lower than immunoassay with 72% found to be below the 2.5th percentile by LC–MSMS compared to 27% for immunoassay. Strikingly 45% of the patients classified as normal TT3 by immunoassay were defined as lower than the 2.5th percentile by LC–MSMS. Only 38 of the 122 patients with low T3's were not receiving T4. In this latter group all of whom had TSH's > 3.7 mIU/L, 74% of results by LC–MSMS were below the 2.5th percentile while only 21% were below the 2.5th percentile by IA. The clinical consequences of these inaccuracies may affect whether dosing with T4 or combination of T4 with T3 is selected for treatment. Finally the correlation of TT3 with TSH was far superior when TT3 was measured by LC–MSMS. A typical case which demonstrates our message is included. Conclusion T3 being the active hormone needs to be reliably measured and if the patient has low TT3 and hypothyroid symptoms persist; treatment with T3 should be considered. A typical case report is included to illustrate the problems of inaccurate immunoassay results for TT3. Measurement of TT3 by immunoassay at low concentrations is less than optimal and often provides the clinician with a normal result when the LC–MSMS method and the patient's clinical condition suggests that supplementation with T3 (as in combination therapy) may be required to optimize patient care.

Our goals were to (1) develop an improved micro-method usable for neonates for steroid profile measurements and a method to measure androsterone, a key steroid in the recently described androgen backdoor pathway together, with dehydroepiandrosterone and (2) to assess if dehydroepiandrosterone diurnal concentration fluctuations exist potentially necessitating strict adherence to time of blood sample draw and requirement of separate time-dependent reference intervals. Liquid chromatography-tandem mass spectrometry was performed with an atmospheric pressure photoionization source [1]. For each sample 50μL (100μL for the backdoor pathway) of serum was deproteinized by adding 75μL (150μL for the backdoor pathway) of acetonitrile containing the internal standards. After centrifugation, 75μL (150μL for the backdoor pathway) of supernatant was diluted with 250μL of water and injected onto a Poroshell 120 EC-C8 column (SB-C8 column for the backdoor pathway). Within-run coefficients of variation ranged from 2.4 to 10.4% and between-day coefficients of variation from 2.9 to 11.2%. Comparison studies yielded correlation coefficient between 0.97 and 1.00 with recoveries of 90% or greater. Our methods analyze a 9 steroid profile and an additional 2 steroid profile (backdoor pathway) with minimal sample volume (usable in neonates optimizing early diagnosis of endocrinopathies and genetic diseases). Low limits of quantitation make these methods ideal for steroid measurement in women and prepubertal children. As diurnal variations of dehydroepiandrosterone and other steroids [2] concentrations are clinically significant we recommend that separate reference intervals be developed for 8 am, 8 pm, and midnight sample draws. The use of this approach in improving the diagnosis of patients with adrenal insufficiency and congenital adrenal hyperplasia is discussed.