WADA - WADA Technical Document – ISL TD2027BSM

WADA Technical Document – ISL TD2027BSM Document number: ISL TD2027BSM Version number: 1.0

Written by: Reviewed by:

WADA Science/EAAS Working Group WADA Steroidal ABP WG/ Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 17 March 2026 Effective date: 1 January 2027

ISL TD2027BSM - Version 1.0 – 01 January 2027 Page 1 / 7 Analytical and Reporting Requirements for the Blood Markers of the Steroidal Module of the Athlete Biological Passport 1.0 Introduction The purpose of this Technical Document (TD), which constitutes an integral part of the International Standard for Laboratories (ISL) [1], is to harmonize the analysis and reporting of the blood (serum) Markers of the Steroidal Module of the Athlete Biological Passport (ABP) to uncover the Use of synthetic forms of Endogenous Anabolic Androgenic Steroids (EAAS), in particular testosterone and its precursors. 1.1 Procedure for Analysis of the Blood Steroid Markers The Analytical Testing Procedure (ATP) involves the measurement of the serum concentrations of two (2) naturally occurring EAAS, namely Testosterone (T) and Androstenedione (Androst -4ene-3,17-dione, A4), and the automatic calculation of the T/A4 ratio in ADAMS. a) The ATP for the blood steroid Markers is not a mandatory ATP (see ISL TD ATP [2]) and is not applied to all serum Samples. Therefore, the blood steroid Markers shall be measured in serum Sample(s) (see Article 2.0) by Laboratories with appropriate analytical capacity and upon request by the Testing Authority (TA) or WADA, and results shall be reported in ADAMS. b) The analysis of the blood steroid Markers follows a two (2)-step procedure:

1. An Initial Testing Procedure (ITP) based on the quantification of the concentrations of the

upon request by the Testing Authority (TA) or WADA, and results shall be reported in ADAMS. b) The analysis of the blood steroid Markers follows a two (2)-step procedure:

1. An Initial Testing Procedure (ITP) based on the quantification of the concentrations of the blood steroid Markers by Liquid Chromatography combined with Mass Spectrometry (LCMSn) (see Article 3.2), and ii. A subsequent Confirmation Procedure (CP) may be performed, which consists of the LCMSn quantification and identification (as per ISL TD IDCR [3]) of the blood steroid Markers

(see Article 4.0). A CP shall be performed when at least one primary blood steroid Marker (T or T/A4) in the Sample constitutes an outlier in the corresponding Passport for elevated values, as determined by the Adaptive Model, triggering an Atypical Passport Finding – Confirmation Procedure Request (ATPF-CPR) in ADAMS. A CP may also be perf ormed upon request to the Laboratory (see Article 4.2).WADA Technical Document – ISL TD2027BSM Document number: ISL TD2027BSM Version number: 1.0

Written by: Reviewed by:

WADA Science/EAAS Working Group WADA Steroidal ABP WG/ Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 17 March 2026 Effective date: 1 January 2027

ISL TD2027BSM - Version 1.0 – 01 January 2027 Page 2 / 7 2.0 Assay Pre-analytical Procedure a) The Laboratory should (usually) receive refrigerated (not frozen1) “A” and “B” blood Samples, which have been collected in blood “serum tubes” containing an inert polymeric serum separator gel and a clotting activation factor (for example: BD Vacutainer ® SSTTM-II Plus tubes, EU ref 367955; BD

have been collected in blood “serum tubes” containing an inert polymeric serum separator gel and a clotting activation factor (for example: BD Vacutainer ® SSTTM-II Plus tubes, EU ref 367955; BD Vacutainer® SSTTM-II Plus Advance tubes, EU ref 367954; BD Vacutainer® SSTTM tubes, US ref 367986) in accordance with the International Standard for Testing (IST) [4]. The use of alternative collection devices shall be validated by the relevant Laboratory(ies) and approved by WADA prior to use for Sample collection. b) Alternatively, if the clotting and centrifugation of the blood Sample is performed prior to reception at the Laboratory (for example, at the site of Sample collection) or when a blood Sample is shipped from another Laboratory for subcontracted analyses , Samples may be received at the Laboratory as frozen/refrigerated Samples either in the same Sample collection tubes or as separated serum in new tubes. c) The Laboratory shall check the status of the Sample(s) and the integrity of the collection tubes (e.g., evidence of breakage of the separating gel). The Laboratory shall note any unusual condition of the Sample and record such condition(s) in the Lab Results in ADAMS. d) Any Samples delivered to the Laboratory in tubes containing an anti-coagulant (for example, whole blood Samples collected in EDTA tubes), or as separated plasma, shall not be analyzed for the blood steroid Markers. e) The Laboratory shall notify and seek advice from the Testing Authority (TA) regarding rejection or Analytical Testing of Samples for which irregularities are noted (see ISL [1]).

1 Unless the blood matrix components have been separated before shipment to the Laboratory.WADA Technical Document – ISL TD2027BSM Document number: ISL TD2027BSM Version number: 1.0

Written by: Reviewed by:

WADA Science/EAAS Working Group WADA Steroidal ABP WG/ Laboratory Expert Advisory Group

Approved by: WADA Executive Committee

Document number: ISL TD2027BSM Version number: 1.0

Written by: Reviewed by:

WADA Science/EAAS Working Group WADA Steroidal ABP WG/ Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 17 March 2026 Effective date: 1 January 2027

ISL TD2027BSM - Version 1.0 – 01 January 2027 Page 3 / 7 2.1. Blood Samples Received as Non-separated in “Serum Tubes” Sample Processing upon Reception − Both “A” and “B” Samples shall be centrifuged for 10-15 min at 1300-1500 g as soon as possible after reception at the Laboratory. − “A” Sample If the “A” Sample is not opened to be analyzed within five (5) days from Sample collection, then the Laboratory may: • Keep the centrifuged “A” Sample in the Sample collection tube and step-freeze it (at approx. -15°C or less and according to the tube manufacturer’s instructions ) until thawing and aliquoting for analysis, or • Aliquot the separated serum fraction into new vials (ensuring that appropriate Laboratory Chain of Custody (see ISL TD LCOC) [5] is maintained), which shall be stored frozen (at approx. -15°C or less) until thawing for analysis. − “B” Sample The centrifuged “B” Sample shall be step-frozen and stored (at approx. -15°C or less and according to the tube manufacturer’s instructions ) until use, if needed (see below). [Comment: If the Laboratory transfers the Aliquot into new vials for frozen storage, the vials should ensure proper sealing for optimal storage (cryovials with an “O -ring”). Thawing of Sample(s) for analysis should be done stepwise; Samples shall not be thawed under hot water or any other similar process that risks raising the temperature of the Sample above room

should ensure proper sealing for optimal storage (cryovials with an “O -ring”). Thawing of Sample(s) for analysis should be done stepwise; Samples shall not be thawed under hot water or any other similar process that risks raising the temperature of the Sample above room temperature. Thawing overnight under refrigeration (2-8 °C) is recommended.] Sample Processing for Analysis a) ITP An Aliquot of the “A” Sample serum fraction shall be taken for the ITP of the blood steroid Markers, and shall be processed as follows: • It may be analyzed immediately after aliquoting; or • It may be stored refrigerated (2-8 °C) if analyzed within a maximum of five (5) days from Sample collection; or • It shall be stored frozen (at approx. -15 °C or less) if the analysis will be conducted more than five (5) days from Sample collection. The remaining “A” serum fraction may be kept in the Sample collection tube or aliquoted into new vial(s) and shall be stored frozen (at approx. -15 °C or less) if the analysis will be conducted more than five (5) days from Sample collection2. b) CP The CP shall be performed on a new Aliquot of the remaining “A” Sample serum fraction and shall be conducted immediately after aliquoting.

2 It is recommended that the Laboratory stores the serum Samples frozen (at approx. -70 °C or less) if the TA (or WADA) has requested the Laboratory to place them into long-term storage (> 3 months) for Further Analysis purposes (see also ISL Article 5.3.7.2).WADA Technical Document – ISL TD2027BSM Document number: ISL TD2027BSM Version number: 1.0

Written by: Reviewed by:

WADA Science/EAAS Working Group WADA Steroidal ABP WG/ Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 17 March 2026 Effective date: 1 January 2027

Written by: Reviewed by:

WADA Science/EAAS Working Group WADA Steroidal ABP WG/ Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 17 March 2026 Effective date: 1 January 2027

ISL TD2027BSM - Version 1.0 – 01 January 2027 Page 4 / 7 2.2 Blood Samples Received as Centrifuged and Frozen/Refrigerated Sample Processing upon Reception a) If Samples are received frozen, they shall remain frozen until thawing and aliquoting for analysis. b) If Samples are received refrigerated, the “A” Sample should be processed to obtain an Aliquot for analysis as soon as possible (as per Article 2.1.), while the “B” Sample shall be stored frozen (at approx. -15 °C or less ) until aliquoting for analysis. Aliquot, Storage and Analysis a) ITP

1. Once a serum Aliquot of the “A” Sample is taken for the ITP of the blood steroid Markers, it should: • Be analyzed immediately or may be stored refrigerated (2-8 °C) if analyzed within a maximum of five (5) days from Sample collection; or • Stored frozen (at approx. -15 °C or less) if the analysis is to be conducted after five (5) days from Sample collection 3. ii. The remaining “A” serum fraction shall be stored as per Article 2.1 above. b) CP The CP shall be performed on a new Aliquot of the remaining “A” Sample serum fraction and shall be conducted immediately after aliquoting.

[Comment to Articles 2.1 and 2.2: When analyses specific to the ABP are requested, only the “A” Sample shall be considered for the ITP and CP. In cases where the “A” Sample is not suitable for the performance of the ABP Markers analysis (e.g.,

[Comment to Articles 2.1 and 2.2: When analyses specific to the ABP are requested, only the “A” Sample shall be considered for the ITP and CP. In cases where the “A” Sample is not suitable for the performance of the ABP Markers analysis (e.g., there is insufficient Sample volume; the Sample container has not been properly sealed or has been broken; the Sample’s integrity has been compromised in any way; the “A” Sample is missing), a splitting procedure of the “B” Sample could be performed, as detailed in the ISL [1].] 3.0 Analytical Testing Procedure Validation and Analysis Requirements For the implementation of ATP for the analysis of the blood steroid Markers in routine Doping Control analysis, the Laboratory shall fulfil the following requisites: a) Validate the Quantitative Procedures (for ITP and CP) for measuring the Marker concentrations, as well as the Qualitative Procedure (for CP) for Marker identification, as per the ISL TD VAL [6] requirements. b) The validated ATPs shall meet the acceptance values for the parameters of assay performance applicable to the separate quantification of T and A4 concentrations as specified in Article 3.1. c) The Laboratory shall apply the validated ATPs in accordance with the ATP Analysis Requirements specified in Article 3.2.WADA Technical Document – ISL TD2027BSM Document number: ISL TD2027BSM Version number: 1.0

Written by: Reviewed by:

WADA Science/EAAS Working Group WADA Steroidal ABP WG/ Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 17 March 2026 Effective date: 1 January 2027

ISL TD2027BSM - Version 1.0 – 01 January 2027 Page 5 / 7 3.1 ATP Validation Requirements (see also ISL TD VAL [6]) Steroid Marker Testosterone (T) Androstenedione (Androst-4-ene-3,17-dione, A4), Identification of Markers

3.1 ATP Validation Requirements (see also ISL TD VAL [6]) Steroid Marker Testosterone (T) Androstenedione (Androst-4-ene-3,17-dione, A4), Identification of Markers The Laboratory shall validate the Qualitative Procedure for confirmation of the identity of the Markers in accordance with the requirements of the ISL TD IDCR [3] and ISL TD VAL [6]. Limit of Quantification (LOQ) ≤ 0.1 ng/mL Working Range 0.1 – 10 ng/mL Relative Standard Combined Measurement Uncertainty, uc (%) ≤ 30% at LOQ ≤ 20% at > 0.3 ng/mL 3.2 ATP Analysis Requirements Steroid Marker Testosterone (T) Androstenedione (Androst-4-ene-3,17-dione, A4), Target Steroid Markers Total (free and protein bound) unconjugated fraction Total (free and protein bound) unconjugated fraction Test Method and Instrumentation Quantitative Procedure (ITP and CP): LC-MSn Qualitative Procedure (CP): LC-MSn (in compliance with the ISL TD IDCR [3]) Aliquot The measurement of the T and A4 concentrations shall be conducted (ITP and CP) in singlicate (1x) on one (1) serum Aliquot not greater than (≤) 100 μL. Internal Standards Adequate isotopically labelled internal standards shall be used for both blood steroid Markers, e.g., T-d3 (16,16,17-d3) and A4-d3 (19-d3). Calibration Calibration standard(s) shall be included in each sequence of analysis. Quality Controls The QCs shall be prepared either from authentic serum or by spiking standard steroid solution(s), independent from that used for the calibrator(s), into serum. At least one (1) serum QC sample representative of the low part of the working

Quality Controls The QCs shall be prepared either from authentic serum or by spiking standard steroid solution(s), independent from that used for the calibrator(s), into serum. At least one (1) serum QC sample representative of the low part of the working range (e.g., within the first quartile of the working range) and one (1) serum QC sample representative of the high part of the working range ( e.g., within the fourth quartile of the working range) shall be used. For the CP, at least one QC sample, depending on the ITP quantification results for the Markers, shall be included in each confirmatory analytical batch.WADA Technical Document – ISL TD2027BSM Document number: ISL TD2027BSM Version number: 1.0

Written by: Reviewed by:

WADA Science/EAAS Working Group WADA Steroidal ABP WG/ Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 17 March 2026 Effective date: 1 January 2027

ISL TD2027BSM - Version 1.0 – 01 January 2027 Page 6 / 7 4.0 Confirmation Procedure for the Blood Steroid Markers 4.1 Confirmation Procedure Requests for Blood Steroid Markers Triggered through ADAMS a) Once the ITP data of the blood steroid Markers is entered and matched with the corresponding Doping Control Form (DCF) in ADAMS, the Adaptive Model automatically updates the steroidal Passport. If an outlier is identified based on an abnormally high T and/or T/A4 value, an ATPFCPR is triggered and sent automatically to the Laboratory through ADAMS. The Laboratory shall ensure the reception and management of CPR notifications using a dedicated ADAMS account(s). b) The TA3 shall inform the Laboratory whether to proceed or not with the CP of the blood steroid Markers, within fourteen (14) days of the receipt of the ATPF-CPR notification.

1. Upon receipt of confirmation to proceed with the CP, the Laboratory shall proceed with the

account(s). b) The TA3 shall inform the Laboratory whether to proceed or not with the CP of the blood steroid Markers, within fourteen (14) days of the receipt of the ATPF-CPR notification.

1. Upon receipt of confirmation to proceed with the CP, the Laboratory shall proceed with the CP of the blood steroid Markers as soon as possible. ii. Any justification from the TA or the Passport Custodian (PC)3 to not proceed with the CP shall be provided in writing according to Article 8.6 of the ISL TD APMU [7]. In such cases, the Laboratory shall update the Lab Results in ADAMS for the Sample with a comment stating that the TA or the PC3, as applicable, requested to not perform the CP, and the reasons given. iii. In the absence of communication from the TA or the PC3 within fourteen (14) days from the ATPF-CPR notification, the Laboratory shall proceed with the CP of the blood steroid Markers. c) When the Laboratory receives an ATPF-CPR for a Sample for which Adverse Analytical Finding(s) (AAF) have been reported for other Prohibited Substance(s) or Prohibited Method(s), the Laboratory shall consult the TA about the need to conduct the CP for the blood steroid Markers 4.2 Confirmation Procedure Requests from the Testing Authority, the Passport Custodian, the

Athlete Passport Management Unit, or WADA. The Adaptive Model will also flag abnormally low or variable steroid Markers. However, in such cases the Laboratory will not receive an automatic notification through ADAMS. Instead, the APMU will advise the PC (who will advise the TA, if different) on whether the Sample, or other Samples from the corresponding Passport, shall be subjected to a CP for the blood steroid Markers. Therefore, in these cases the Laboratory shall receive a written request from the TA3, or WADA, before proceeding with the CP.

3 The APMU or PC, where the PC is not the TA, may contact, in writing, the Laboratory regarding performance of a CP of the blood

these cases the Laboratory shall receive a written request from the TA3, or WADA, before proceeding with the CP.

3 The APMU or PC, where the PC is not the TA, may contact, in writing, the Laboratory regarding performance of a CP of the blood steroid Markers on behalf of the TA. In such cases, the APMU (which may have been bestowed such authority by the PC) or the PC shall copy the relevant TA.WADA Technical Document – ISL TD2027BSM Document number: ISL TD2027BSM Version number: 1.0

Written by: Reviewed by:

WADA Science/EAAS Working Group WADA Steroidal ABP WG/ Laboratory Expert Advisory Group Approved by: WADA Executive Committee Date: 17 March 2026 Effective date: 1 January 2027

ISL TD2027BSM - Version 1.0 – 01 January 2027 Page 7 / 7 4.3 Confirmation of Blood Steroid Marker Values The CP for the blood steroid Markers following receipt of an ATPF-CPR, or upon request, consists of the application of the same ATP (see Article 3) for the quantification of the concentrations of T and A4 in the ITP as well as their identification (in compliance with the ISL TD IDCR [3]). 5.0 Reporting Initial Testing Procedure and Confirmation Procedure Results for the Blood Steroid Markers a) The T and A4 concentrations shall be reported in ADAMS in nanograms per milliliter (ng/mL). [Comment to Article 5.0 a): For the purposes of the Steroidal Module of the ABP, the T/A4 ratio does not need to be calculated or reported by the Laboratory; it will be automatically calculated in ADAMS]. b) If the measured concentration(s) of T and/or A4 is below the LOQ of the assay, the Laboratory shall report a value of “-1” for the affected concentration in ADAMS.

or reported by the Laboratory; it will be automatically calculated in ADAMS]. b) If the measured concentration(s) of T and/or A4 is below the LOQ of the assay, the Laboratory shall report a value of “-1” for the affected concentration in ADAMS. c) If the concentration(s) of T and/or A4 cannot be determined or the Marker(s) cannot be identified when performing the CP, the affected Marker(s) shall be reported as “-1” and the Laboratory shall make a corresponding comment in the Lab Results in ADAMS (e.g., matrix interferences). 6.0 References [1] The World Anti-Doping Code International Standard for Laboratories. [2] WADA Technical Document ISL TD ATP: Analytical Testing Procedures [3] WADA Technical Document ISL TD IDCR: Minimum Criteria for Chromatographic -Mass Spectrometric Confirmation of the Identity of Analytes for Doping Control Purposes. [4] The World Anti-Doping Code International Standard for Testing. [5] WADA Technical Document ISL TD LCOC: Laboratory Chain of Custody [6] WADA Technical Document ISL TD VAL: Minimum Requirements for Validation of Analytical Testing Procedures for Doping Control. [7] WADA Technical Document ISL TD APMU: Athlete Passport Management Unit Requirements and Procedures

[Comment to Article 6.0: Current versions of WADA International Standards and Technical Documents may be found at https://www.wada-ama.org/en/what-we-do/international-standards]