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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 reference materials to address the need for improved accuracy of routine clinical assays for hormones, and to ensure that timely and effective treatment decisions are made.


]:  Photograph of SRM 971a consisting of a labeled box and labeled vials of  containing male serum and female serum.

SRM 971a Hormones in Frozen Human Serum

Credit: Lane Sander

Steroid hormones and thyroid hormones serve an essential function by regulating gene transcription. Improving the accuracy of clinical assays for hormones will improve diagnoses and result in earlier treatments. Therefore, reference methods for the measurement of steroid and thyroid hormones have been developed at NIST and Standard Reference Materials 971a, Hormones in Frozen Human Serum, and 1949, Frozen Human Prenatal Serum, will help 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 male and female human serum has been developed (SRM 971, now in second generation, SRM 971a)
  • A Standard Reference Material for thyroid hormones and related proteins in the serum of reproductive age women and women in each trimester of pregnancy has been developed (SRM 1949)

  • A collaboration with the Centers for Disease Control and Prevention (CDC) has been formed to continually add new steroids and hormone related analytes to these materials

Associated Product(s)


  1. Boggs, A. S. P., Kilpatrick, L. E., Burdette, C. Q., Tevis, D. S., Fultz, Z. A., Nelson, M. A., Jarrett, J. M., Kemp, J. V., Singh, R. J., Grebe, S. K. G., Wise, S. A., Kassim, B. L., and Long, S. E., "Development of a pregnancy-specific reference material for thyroid biomarkers, vitamin D, and nutritional trace elements in serum," Clinical Chemistry and Laboratory Medicine, 59, 671-679 (2021). 

  1. Boggs, A. S. P., Bowden, J. A., Galligan, T. M., Guillette, L. J., and Kucklick, J. R., "Development of a multi-class steroid hormone screening method using Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS)," Analytical and Bioanalytical Chemistry, 408, 4179-4190 (2016). 

  1. Phinney, K. W., Ballihaut, G., Bedner, M., Benford, B. S., Camara, J. E., Christopher, S. J., Davis, W. C., Dodder, N. G., Eppe, G., Lang, B. E., Long, S. E., Lowenthal, M. S., Mcgaw, E. A., Murphy, K. E., Nelson, B. C., Prendergast, J. L., Reiner, J. L., Rimmer, C. A., Sander, L. C., Schantz, M. M., Sharpless, K. E., Sniegoski, L. T., Tai, S. S. C., Thomas, J. B., Vetter, T. W., Welch, M. J., Wise, S. A., Wood, L. J., Guthrie, W. F., Hagwood, C. R., Leigh, S. D., Yen, J. H., Zhang, N. F., Chaudhary-Webb, M., Chen, H. P., Fazili, Z., Lavoie, D. J., Mccoy, L. F., Momin, S. S., Paladugula, N., Pendergrast, E. C., Pfeiffer, C. M., Powers, C. D., Rabinowitz, D., Rybak, M. E., Schleicher, R. L., Toombs, B. M. H., Xu, M., Zhang, M., and Castle, A. L., "Development of a Standard Reference Material for Metabolomics Research," Analytical Chemistry, 85, 11732-11738 (2013). 

  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). 

  1. 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). 

  1. 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). 

  1. 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). 

  1. 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). 

  1. 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). 

  1. 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). 

Created March 29, 2009, Updated November 3, 2023