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SRM 971a Page 1 of 9
Date of Issue:
27 March 2023
Standard Reference Material? 971a
Hormones in Frozen Human Serum
CERTIFICATE OF ANALYSIS
Purpose: The certified values delivered by this Standard Reference Material (SRM) are intended for validating
methods for determining testosterone and progesterone in human serum and similar materials.
Description: A unit of SRM 971a consists of two vials each of two materials: one from a pool of healthy,
premenopausal adult females and one from a pool of healthy adult males. Both materials are unfortified. Each vial
contains approximately 2 mL of human serum.
Certified Values: A NIST certified value is a value for which NIST has the highest confidence in its accuracy in that
all known or suspected sources of bias have been investigated or taken into account [1]. Metrological traceability is
to the International System of Units (SI) derived unit of mass fraction and converted to be expressed as nanomoles
per liter and nanograms per deciliter. The certified values apply only to serum thawed to room temperature (20 °C to
25 °C; see “Storage” and “Use”). Table 1 certified values are reported on an as-received basis [2].
Table 1. Certified Concentration Values for SRM 971a for Female Level and Male Level(a)
Analyte Level
Molar
Concentration(b)
(nmol/L)
Lower 95 %
Confidence
Interval
(nmol/L)
Upper 95 %
Confidence
Interval
(nmol/L)
Mass
Concentration(c)
(ng/dL)
Lower 95 %
Confidence
Interval
(ng/dL)
Upper 95 %
Confidence
Interval
(ng/dL)
Testosterone Female 1.12 ? 0.01 1.10 1.14 32.31 ?? 0.50 31.81 32.81
Progesterone Female 8.36 ? 0.60 7.17 9.54 263 ? 19 231 296
Testosterone Male 20.14 ? 0.16 19.83 20.44 580.8 ? 9.0 571.8 589.8
Progesterone Male 0.134 ? 0.011 0.112 0.156 4.21 ? 0.35 3.61 4.80
(a) Each certified concentration value is the consensus value of results from an isotope dilution liquid chromatography tandem mass
spectrometry (ID-LC-MS/MS) reference measurement procedure at NIST and ID-LC-MS/MS reference measurement procedure
at the Centers for Disease Control and Prevention (CDC). The uncertainty provided with each value is an expanded uncertainty
about the consensus to cover the measurand with approximately 95 % confidence, consistent with the ISO/JCGM Guide [3].
The expanded uncertainty is calculated as U = kuc, where uc is the combined uncertainty that incorporates within-method
uncertainty and Type B uncertainty components related to the analysis, and k is the coverage factor corresponding to
approximately 95 % confidence for each analyte.
(b) Amount concentration values, nmol/L, are calculated from the mass concentration results, nanogram per deciliter, via
multiplication by 10/M, where M is the molar mass, grams per mol, of the analyte. The molar masses used are:
testosterone = 288.424 g/mol and progesterone = 314.461 g/mol. These molar masses have associated standard
uncertainty u(M) = 0.011 g/mol for testosterone and 0.012 g/mol for progesterone.
(c) Mass concentrations were calculated from the mass fractions for each compound using the measured serum density
of 1.0238 ? 0.0012 g/mL at 22.3 ºC for the female level and at 1.0252 ? 0.0012 g/mL at 21.6 ºC for the male level.
Non-Certified Values: Non-certified values are provided in Appendix A.
Period of Validity: The certified values delivered by SRM 971a are valid within the measurement uncertainty
specified until 01 May 2029. The certified values are nullified if the material is stored or used improperly, damaged,
contaminated, or otherwise modified.
Maintenance of Certified Values: NIST will monitor this SRM over the period of its validity. If substantive
technical changes occur that affect the certification, NIST will issue an amended certificate through the NIST SRM
website (https://www.nist.gov/srm) and notify registered users. SRM users can register online from a link available
on the NIST SRM website or fill out the user registration form that is supplied with the SRM. Registration will
facilitate notification. Before making use of any of the values delivered by this material, users should verify they have
the most recent version of this documentation, available through the NIST SRM website (https://www.nist.gov/srm).
Carlos A. Gonzalez, Chief Steven J. Choquette, Director
Chemical Sciences Division Office of Reference Materials
Certificate Revision History on Page 4
SRM 971a Page 2 of 9
Safety: SRM 971a IS INTENDED FOR RESEARCH USE. This is a human-source material. Handle product as a
biohazardous material potentially capable of transmitting infectious disease. The supplier has reported that each donor
unit of serum used in the preparation of this product was tested by FDA-licensed tests and found to be negative for
human immunodeficiency virus (HIV), HIV-1 antigen, hepatitis B surface antigen, and hepatitis C. However, no
known test method can offer complete assurance that hepatitis B virus, hepatitis C virus, HIV, or other infectious
agents are absent from this material. Accordingly, this human blood-based product should be handled at
Biosafety Level 2 as recommended by the Centers for Disease Control and Prevention/National Institutes of Health’s
Biosafety in Microbiological and Biomedical Laboratories [4] for human-derived blood products where the presence
of infectious agent(s) may be unknown.
This SRM was developed after an appropriate human subjects research determination by NIST.
Storage: The serum is shipped frozen (on dry ice) and, upon receipt, should be stored frozen until ready for use. The
material is kept at –80 °C for long-term storage at NIST. Under these conditions, analytes are expected to be stable.
The SRM should not be exposed to sunlight or ultraviolet radiation. Storage of thawed material at room or refrigerator
temperatures may result in changes in analyte concentrations.
Use: SRM 971a is provided as frozen serum that should be allowed to thaw at room temperature for at least 30 min
under subdued light. After the material is thawed, it should be used immediately. The contents of the vial should be
mixed thoroughly by inverting and/or rolling (DO NOT CENTRIFUGE OR VORTEX MIX) before aliquots are
withdrawn. Precautions should be taken to avoid exposure to strong UV light and direct sunlight.
Source and Preparation: SRM 971a was prepared from serum collected by Bioreclamation IVT, now BioIVT
(Westbury, NY). The serum materials are comprised from 50 male and 50 female donors. The male samples were
required to originate from non-smokers between the ages of 21 and 40. The female samples were required to originate
from premenopausal non-smokers between the ages of 21 and 40. Donors were excluded if they were taking over the
counter or prescription medications that would alter hormone concentrations such as steroidal pain relievers, birth
control, or thyroid medications. All serum was collected according to the CLSI C37-A guideline "Preparation and
Validation of Commutable Frozen Human Serum Pools as Secondary Reference Materials for Cholesterol
Measurement Procedures” [5]. Briefly, serum was collected off clot with no additives in an ice bath and bottled.
Whole blood was centrifuged within 5 minutes of collection, allowed to clot overnight at 4 °C, centrifuged, then the
clear serum was aseptically transferred to a sterile container. Serum was tested for clotting factors and the process
repeated if tested positive. The materials were shipped frozen on dry ice to Solomon Park (Burian, WA) for pooling
and bottling. The frozen serum was thawed at 4 °C to 8 °C on a shaker system. Serum was then passed through sterile
cheese cloth to remove any larger particles, mixed overnight, and then filtered through 0.22 μm spiral filters.
Aliquoting was conducted using a peristaltic pump dispenser into vials which were on cooled pads. Nitrogen was
introduced into the vials as they were being stoppered with the Teflon stoppers. Once the entire pool was aliquoted,
boxes were frozen at –70 °C.
Analytical Approach for Determination of Testosterone: The NIST reference measurement procedure for
testosterone [6] involves spiking the serum with testosterone-d3, acidifying the sample, isolating testosterone from the
serum matrix using a solid-phase extraction cartridge (C18), further purifying testosterone by a liquid-liquid
extraction, drying the sample, and reconstituting in methanol containing 0.5 mL/L acetic acid. Samples were analyzed
by isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS). Selected reaction monitoring
was used with following transitions: m/z 289.0 → m/z 97.0 (quantification) and m/z 289.0 → m/z 109.0
(confirmation) for testosterone, and m/z 292.0 → m/z 97.0 (quantification) and m/z 292.0 → m/z 109.0 (confirmation)
for testosterone-d3. Chemical purity characterizations of calibrants with metrological traceability to SI units were
assessed using a quantitative 1H nuclear magnetic resonance spectroscopy (1H-qNMR) procedure.
The CDC measurement method for testosterone [7] involves spiking the serum with testosterone-13C3, acidifying the
sample, isolating testosterone with two liquid-liquid extractions, drying the sample, and reconstituting in a
water:acetonitrile mixture (90:10, volume fraction) with 0.1 % formic acid (volume fraction). Selected reaction
monitoring was used with following transitions: m/z 289.3 → m/z 97.0 (quantification) and m/z 289.3 → m/z 109.0
(confirmation) for testosterone, and m/z 292.3 → m/z 100.0 (quantification) and m/z 292.3 → m/z 112.0
(confirmation) for testosterone-13C3.
Analytical Approach for Determination of Progesterone: The NIST reference measurement procedure for
progesterone [8] involves spiking the serum with progesterone-13C2, equilibrating at room temperature under reduced
laboratory lighting, then amending the pH to 9.8 ? 0.2 with 0.1 g/mL carbonate buffer (pH 9.8). Two rounds of hexane
extraction were performed before drying the extract and reconstituting in methanol with 0.5 mL/L acetic acid.
Samples were analyzed by ID-LC-MS/MS. Selected reaction monitoring was used with the following
transitions: m/z 315.2 → m/z 97.2 (quantification) and m/z 315.2 → m/z 109.2 (confirmation) for progesterone, and
m/z 317.2 → m/z 97.2 (quantification) for progesterone-13C2. Chemical purity characterizations of calibrants with
metrological traceability to SI units were assessed using a quantitative 1H nuclear magnetic resonance
spectroscopy (1H-qNMR) procedure.
CDC measurements were conducted using a CDC Steroid Panel Method in serum by ID-LC-MS/MS with a
progesterone-13C3 internal standard [9,10]. Progesterone was dissociated from carrier proteins by the addition of
acetate buffer at pH 5.5 followed by a liquid-liquid extraction using ethyl acetate. Selected reaction monitoring was
used with the following transitions: m/z 315 → m/z 97 (quantification) and m/z 315 → m/z 109 (confirmation).
REFERENCES
[1] Beauchamp, C.R.; Camara, J.E.; Carney, J.; Choquette, S.J.; Cole, K.D.; DeRose, P.C.; Duewer, D.L.;
Epstein, M.S.; Kline, M.C.; Lippa, K.A.; Lucon, E.; Molloy, J.; Nelson, M.A.; Phinney, K.W.; Polakoski, M.;
Possolo, A.; Sander, L.C.; Schiel, J.E.; Sharpless, K.E.; Toman, B.; Winchester, M.R.; Windover, D.;
Metrological Tools for the Reference Materials and Reference Instruments of the NIST Material Measurement
Laboratory; NIST Special Publication (NIST SP) 260-136, 2021 edition; U.S. Government Printing Office:
Washington, DC (2021); available at https://nvlpubs.nist.gov/nistpubs/SpecialPublications/NIST.SP.260-136-
2021.pdf (accessed Mar 2023).
[2] Hahm, G.; Nelson, M.; Camara, J.; Toman, B.; Certification of Standard Reference Material® s 2969 and 2970:
Vitamin D Metabolites in Frozen Human Serum (Total 25-Hydroxyvitamin D Low Level) and
(25-Hydroxyvitamin D2 High Level); NIST Special Publication 260-210 (2021); available at
https://nvlpubs.nist.gov/nistpubs/SpecialPublications/NIST.SP.260-210.pdf (accessed Mar 2023).
[3] JCGM 100:2008; Evaluation of Measurement Data ― Guide to the Expression of Uncertainty in Measurement
(GUM 1995 with Minor Corrections); Joint Committee for Guides in Metrology (JCGM) (2008); available at
https://www.bipm.org/en/committees/jc/jcgm/publications (accessed Mar 2023); see also Taylor, B.N.;
Kuyatt, C.E.; Guidelines for Evaluating and Expressing the Uncertainty of NIST Measurement Results; NIST
Technical Note 1297, U.S. Government Printing Office: Washington, DC (1994); available at
https://www.nist.gov/pml/nist-technical-note-1297 (accessed Mar 2023).
[4] CDC/NIH: Biosafety in Microbiological and Biomedical Laboratories, 6th ed.; HHS Publication
No. (CDC) 300859; Meechan, P.J.; Potts, J.; Eds.; U.S. Department of Health and Human Services, Public
Health Service, Centers for Disease Control and Prevention, National Institutes of Health;
US Government Printing Office: Washington, DC (2020); available at https://www.cdc.gov/labs/BMBL.html
(accessed Mar 2023).
[5] CLSI Publication C37-A; Preparation and Validation of Commutable Frozen Human Serum Pools as Secondary
Reference Materials for Cholesterol Measurement Procedures; Clinical Laboratory Standards Institute:
Wayne, PA (1999).
[6] Tai, S.S.C.; Xu, B.; Welch, M.J.; Phinney, K.W.; Development and Evaluation of a Candidate Reference
Measurement Procedure for the Determination of Testosterone in Human Serum Using Isotope Dilution Liquid
Chromatography/Tandem Mass Spectrometry; Anal. Bioanal. Chem., Vol. 388, pp. 1087–1094 (2007).
[7] Botelho, J.C.; Shacklady, C.; Cooper, H.C.; Tai, S.S.C.; Van Uytfanghe, K.; Thienpont, L.M.; Vesper, H.W.;
Isotope-Dilution Liquid Chromatography—Tandem Mass Spectrometry Candidate Reference Method for Total
Testosterone in Human Serum; Clin. Chem., Vol. 59, pp. 372?380 (2013).
[8] Tai, S.S.C.; Xu, B.; Welch, M.J.; Development and Evaluation of a Candidate Reference Measurement
Procedure for the Determination of Progesterone in Human Serum Using Isotope-Dilution Liquid
Chromatography/Tandem Mass Spectrometry; Anal. Chem., Vol. 78, pp. 6628–6633 (2006).
[9] Ortega, M.T.; McGrath, J.A.; Carlson, L.; Poccia, V.F.; Larson, G.; Douglas, C.; Sun, B.Z.; Zhao, S.; Beery, B.;
Vesper, H.W.; Duke, L.; Botelho, J.C.; Filie, A.C.; Shaw, N.D.; Longitudinal Investigation of
Pubertal Milestones and Hormones as a Function of Body Fat in Girls; J. Clin. Endocrinol. Metab., Vol. 106,
pp. 1668–1683 (2021).
[10] Duke, L.; Kim, P.H.; Botelho, J.C.; Ziegler, R.G.; Vesper, H.; A new Sensitive, Accurate Liquid
Chromatography–Tandem Mass Spectrometry (LC-MS-MS) Method to Measure Circulating Estrogens,
Androgens, and Progesterone in Postmenopausal and Premenopausal Women; American Association for
Cancer Research Annual Meeting, Abstract 2206 (2018).
[11] Netzel, B.C.; Grant, R.P.; Hoofnagle, A.N.; Rockwood, A.L.; Shuford, C.M.; Grebe, S.K.G.; First Steps
Toward Harmonization of LC-MS/MS Thyroglobulin Assays; Clin. Chem., Vol. 62, pp. 297–299 (2016).
[12] Netzel, B.C.; Grebe, S.K.G.; Carranza Leon, B.G.; Castro, M.R.; Clark, P.M.; Hoofnagle, A.N.; Spencer, C.A.;
Turcu, A.F.; Algeciras-Schimnich, A.; Thyroglobulin (Tg) Testing Revisited: Tg Assays, TgAb Assays, and
Correlation of Results With Clinical Outcomes; J. Clin. Endocrinol. Metab., Vol. 100, pp. E1074–E1083 (2015).
 
SRM 971a Page 3 of 9
Certificate Revision History: 27 March 2023 (Updated storage conditions to remove short term storage temperature and add NIST storage
temperature; editorial changes); 02 February 2022 (Addition of certified values for progesterone; addition of molar concentration and
uncertainty values for testosterone; addition of non-certified values for 17-hydroxyprogesterone, androstenedione, dehydroepiandrosterone
sulfate, 17β-estradiol, estrone, estrone sulfate, anti-Müllerian hormone, follicle stimulating hormone, luteinizing hormone, sex hormone binding
globulin, thyroglobulin, and thyroglobulin antibodies; updated format; editorial changes); 16 August 2019 (Original certificate date).
Certain commercial equipment, instruments, or materials may be identified in this Certificate of Analysis to
adequately specify the experimental procedure. Such identification does not imply recommendation or endorsement
by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified
are necessarily the best available for the purpose.
Users of this SRM should ensure that the Certificate of Analysis in their possession is current. This can be accomplished
by contacting the Office of Reference Materials 100 Bureau Drive, Stop 2300, Gaithersburg, MD 20899-2300;
telephone (301) 975-2200; e-mail srminfo@nist.gov; or the Internet at https://www.nist.gov/srm.
* * * * * * * * * * * * End of Certificate of Analysis * * * * * * * * * * * *
SRM 971a Page 4 of 9
SRM 971a Page 5 of 9
APPENDIX A
Non-Certified Values: Non-certified values are suitable for use in method development, method harmonization, and
process control but do not provide metrological traceability to the International System of Units (SI) or other
higher-order reference system. Non-certified values are provided below.
Table A1. Non-certified Molar Concentration Values of Steroids for SRM 971a Female Level(a)
Analyte
Molar
Concentration(b)
Lower 95 %
Confidence
Interval
Upper 95 %
Confidence
Interval
Molar
Concentration
Units
17-Hydroxyprogesterone 2.640 ? 0.049 2.545 2.736 nmol/L
Androstenedione 2.829 ? 0.016 2.798 2.860 nmol/L
Dehydroepiandrosterone Sulfate 3.310 ? 0.076 3.161 3.459 µmol/L
17β-Estradiol 1073 ? 11 1051 1094 pmol/L
Estrone 680.2 ? 7.0 666.4 693.9 pmol/L
Estrone Sulfate 4139 ? 45 4050 4227 pmol/L
(a) Each concentration value is the mean value of results from an ID-LC-MS/MS reference measurement procedure at CDC. The
uncertainty provided with each value is an expanded uncertainty about the mean to cover the measurand with approximately
95 % confidence, consistent with the ISO/JCGM Guide [3]. The expanded uncertainty is calculated as U = kuc, where uc is the
combined uncertainty that incorporates within-method uncertainty and Type B uncertainty components related to the analysis,
and k is the coverage factor corresponding to approximately 95 % confidence for each analyte.
(b) Amount concentration values of nanomol per liter are calculated from the mass concentration results, nanogram per deciliter, via
multiplication by 10/M, where M is the molar mass, gram per mol, of the analyte. Amount concentration values
of picomol per liter are calculated from the mass concentration results, picogram per milliliter, via multiplication by 1000/M.
Amount concentration values of micromol per liter are calculated from the mass concentration results, microgram per deciliter,
via multiplication by 10/M. The molar masses used are: 17α-hydroxyprogesterone = 330.460 g/mol,
androstenedione = 286.408 g/mol, dehydroepiandrosterone sulfate = 368.489 g/mol, 17β-estradiol = 272.381 g/mol,
estrone = 270.365 g/mol, and estrone sulfate = 350.431 g/mol. These molar masses have associated standard
uncertainty u(M) = 0.011 g/mol for androstenedione, estrone, and 17β-estradiol and u(M) = 0.012 for 17-hydroxyprogesterone,
dehydroepiandrosterone sulfate, and estrone sulfate.
Table A2. Non-certified Mass Concentration Values of Steroids for SRM 971a Female Level(a)
Analyte
Mass
Concentration(b)
Lower 95 %
Confidence
Interval
Upper 95 %
Confidence
Interval
Mass
Concentration
Units
17-Hydroxyprogesterone 87.25 ? 1.61 84.08 90.40 ng/dL
Androstenedione 81.03 ? 0.45 80.16 81.93 ng/dL
Dehydroepiandrosterone Sulfate 122.0 ? 2.8 116.3 127.6 µg/dL
17β-Estradiol 292.3 ? 3.0 286.3 298.5 pg/mL
Estrone 183.9 ? 1.9 180.2 187.5 pg/mL
Estrone Sulfate 1450 ? 16 1419 1483 pg/mL
(a) Each concentration value is the mean value of results from an ID-LC-MS/MS reference measurement procedure at CDC. The
uncertainty pr