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Development of Reference Methods and Reference Materials for the Determination of Hormones in Human Serum


Hormones are chemical messengers that regulate many life functions, and deviations from normal levels can indicate serious health problems. Measurement of levels of steroid and/or thyroid hormones in serum can be beneficial in diagnosing and monitoring a number of diseases. Routine clinical tests for hormones are generally immunoassay-based, and intercomparison studies have revealed problems in precision, accuracy, and specificity for some of these assays, particularly at low hormone concentrations. NIST has developed reference measurement procedures and a new reference material, SRM 971 Hormones in Human Serum, to address the need for improved accuracy of routine clinical assays for hormones and to ensure that timely and effective treatment decisions are made.


Steroid hormones (cortisol, progesterone, estradiol-17β, testosterone) and thyroid hormones (thyroxine, triiodothyronine) serve an essential function by regulating gene transcription. Cortisol is a steroid hormone that is produced by the adrenal gland. It is often called the "stress hormone" because it is secreted at higher levels in response to stress. Prolonged elevation of cortisol levels in the bloodstream can have negative effects on thyroid function, metabolism, and immune function. Measurement of cortisol in serum can be used to diagnose problems in the adrenal or pituitary gland, as well as stress-related disorders. Previous work has indicated that routine clinical measurements may significantly overestimate the cortisol levels present in serum, making accurate diagnosis of cortisol-related disorders difficult. Similar limitations have been observed in routine clinical assays for other hormones as well. Improving the accuracy of clinical assays for hormones will improve diagnoses and result in earlier treatments. The new reference methods and SRM 971 Hormones in Human Serum will help achieve this goal and will also provide higher-order reference systems for traceability.

Major Accomplishments

  • Reference measurement procedures have been developed for cortisol, estradiol-17β, progesterone, testosterone, thyroxine (T4), and triiodothyronine (T3)
  • A Standard Reference Material for hormones in human serum has been developed (SRM 971).



  1. Julianne Cook Botelho, Christopher Shacklady, Hans C Cooper, Susan S-C Tai, Katleen Van Uytfanghe, Linda M Thienpont and Hubert W Vesper. Isotope Dilution Liquid Chromatography Tandem Mass Spectrometry Candidate Reference Method for total Testosterone in Human Serum. Clin Chem 59, 372-380 (2013).
  2. Tai, S.S-C., Xu B., Welch M.J., and Phinney K.W., "Development and evaluation of a candidatereference measurement procedure for the determination of testosterone in human serum using isotope dilution liquid chromatography/tandem mass spectrometry," Anal. Bioanal. Chem., 388:1087–94 (2007).
  3. Tai, S.S.-C., Xu, B., and Welch, M.J., "Development and Evaluation of a Candidate Reference Measurement Procedure for the Determination of Progesterone in Human Serum Using ID-LC/MS/MS," Anal. Chem., 78:6628-6633 (2006).
  4. Tai, S.S-C. and Welch, M.J., "Development and Evaluation of a Reference Measurement Procedure for the Determination of Estradiol-17β in Human Serum Using ID-LC/MS/MS," Anal. Chem., 77:6359-6363 (2005).
  5. Tai, S.S-C., Bunk, D.M., White V, E., and Welch, M.J., "Development and Evaluation of a Reference Measurement Procedure for the Determination of Total 3,3',5-Triiodothyronine in Human Serum Using ID-LC/MS/MS," Anal. Chem., 76:5092-5096 (2004).
  6. Tai, S.S-C. and Welch, M.J., "Development and Evaluation of a Candidate Reference Method for the Determination of Total Cortisol in Human Serum Using ID-LC/MS and LC/MS/MS," Anal. Chem., 76:1008-1014 (2004).
  7. Tai, S.S-C., Sniegoski, L.T., and Welch, M.J., "Determination of Thyroxine in Human Serum by ID-LC/MS with Electrospray Ionization," Clin. Chem., 48:637-642 (2002).

SRM 971 Hormones in Human Serum consists of two serum pools, one from normal adult males, and one from normal, premenopausal adult females. Both pools are unfortified. In order to value assign the analytes of interest in SRM 971, a suite of isotope dilution liquid chromatography-mass spectrometry (LC/MS and LC-MS/MS) methods were developed. These methods are recognized by the Joint Committee for Traceability in Laboratory Medicine (JCTLM) as reference measurement procedures (RMPs) of a higher-order. Certified values have been assigned for cortisol, progesterone, and testosterone in SRM 971, and additional certified values will be added for estradiol-17β, thyroxine, and triiodothyronine.

Created March 29, 2009, Updated December 11, 2019