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The remaining “A” serum fraction may be kept in the Sample collection tube or aliquoted into new vial (s) with label(s) ensuring Laboratory Internal Chain of Custody is maintained .
For the confirmatory quantification, a new Aliquot of the “A” Sample shall be analyzed immediately after aliquoting.
ABP Operating Guidelines – Version 9.0 – July 2023 Page 44/91 5.0 Analytical Testing Procedure Requirements 5.1 Analytical Testing Procedure Validation Requirements Prior to the implementation of the Analytical Testing Procedure for the quantification of blood endogenous steroids in routine Doping Control analysis, the Laboratory shall fulfil the following requisites: − Validate the Analytical Testing Procedure , including the determination of the assays’ Limit of Quantification (LOQ ), Repeatability (sr), Intermediate Precision (sw), Bias and Measurement Uncertainty (uc); − The Analytical Testing Procedure shall meet the acceptance values for the parameters of assay performance applicable to the separate determination of T and A4 concentrations as specified in Table 1 below.
5.2 Analytical Testing Procedure Accreditation Requirements − Demonstrate readiness for assay implementation through method validation data and successful participation in at least one WADA -approved educational External Quality Assessment Scheme (EQAS ) round or inter -Laboratory collaborative study.
In cases of identified deficiencies, proper corrective action(s) shall be documented and implemented; − Obtain ISO/IEC 17025 accreditation for the Analytical Testing Procedure for quantification of endogenous steroids in blood from an A ccreditation Body that is a full member of the International Laboratory Accreditation Cooperation (ILAC) and a signatory to the ILAC Mutual Recognition Agreement (ILAC MRA) .
6.0 Analytical Testing Procedure and Reporting of Test Results 6.1 Initial Quantification of the Markers − One (1) Aliquot taken from the original “A” Sample shall be analyzed once (x1) to quantify T and A4; − QC Sample(s), at low - and high- levels of the Marker s (see Table 1), shall be included in each initial quantification analytical batch; − The T and A4 Marker concentrations shall be reported in ADAMS in nanograms per milliliter (ng/mL) ; [Comment: for the purposes of the Steroidal Module of the ABP, the T/A4 ratio does not need to be c alculated or reported by the Laboratory ; it will be automatically calculated in ADAMS].
− If the measured Marker concentration is below the LOQ of the assay , the Laboratory shall report a value of “ -1” for its concentration in ADAMS and the Laboratory shall make a comment in the Test Report on why the Marker could not be quantified (e.g., the measurement of the Marker is not possible due to unusual matrix interferences) ; − An observation of hemolysis of the Sample should be recorded in the comment s section of the Laboratory Test Report in ADAMS.
ABP Operating Guidelines – Version 9.0 – July 2023 Page 45/91 6.2 Confirmatory Quantification of the Markers If requested by the Testing Authority (TA), Results Management Authority (RMA ) or WADA , the Laboratory shall proceed with the confirmatory quantification of the Markers of the blood Steroidal Module.
[Comment: An APMU or Passport Custodian (PC), where the PC is not the TA, may request a confirmatory quantification on behalf of the TA or RMA .
In such case s, the APMU or PC shall copy the relevant TA or RMA , as applicable, on all written requests to the Laboratory for confirmatory quantification.]
When a confirmatory quantification analysis is requested: − One (1) new Aliquot taken from the original “A” Sample shall be analyzed once (x1) to identify (as per the TD IDCR 8) and to quantify T and A4.
− At least one QC Sample (see Table 1), depending on initial quantification results, shall be included in each confirmatory quant ification analytical batch; − The T and A4 Marker concentrations shall be reported in ADAMS in nanograms per milliliter (ng/mL).
− If the measured Marker concentration is below the LOQ of the assay, the Laboratory shall report a value of “ -1” for its concentration in ADAMS and the Laboratory shall make a comment in the Test Report on why the Marker could not be quantified (e.g., the measurement of the Marker is not possible due to unusual matrix interferences) ; − An observation of hemolysis of the Sample should be recorded in the comments section of the Laboratory Test Report in ADAMS.
ABP Operating Guidelines – Version 9.0 – July 2023 Page 46/91 Table 1 : Analytical Testing Procedure Validation and Performance Requirements for the initial and confirmatory quantification of blood (serum) endogenous steroid Markers .
Markers Testosterone (T), total unconjugated fraction Androstenedione (Androst -4-ene-3,17- dione, A4), total unconjugated fraction Method and Instrumentation Liquid Chromatography combined with tandem Mass Spectrometry based on triple quadrupole or HRMS analyzer (LC -MSn; n ≥ 1).
Range of the Method Shall cover the ranges of Marker concentrations normally found in males and females and demonstrate linearity between 0.1 – 10 ng/mL (~ 0.35 – 35 nmol/L), at least.
Limits of Quantification (LOQ ) The LOQ shall be determined during method validation and is defined as the lowest concentration with an associated uc (%) not greater than (≤) 30% and shall be not greater than (≤) 0.1 ng/mL (~ 0.35 nmol/L) .
Relative Standard Combined Measurement Uncertainty , uc (%) The estimated uc (%) shall be no greater than (≤) 30% at the LOQ ; and not greater than (≤) 20% when the Marker concentration is greater than (>) 0.3 ng/mL.
Sample Marker quantification shall be conducted on one serum Aliquot of no greater than (≤) 100 μL .
Internal Standards Adequate isotopic -labelled internal standards shall be used for both Markers (e.g., Testosterone- d3 (16,16,17- d3)ii and Androstenedione- d3 (19- d3)iii).
Calibration Calibration standard(s) shall be included in each sequence of analysis.
The “Multilevel Serum Calibrator Set ” from Chromsystemiv is recommended.
Other calibrators may be used as long as the method performance criteria are met.
Quality Control At least two (2) quality control (QC) samples in serum containing representative low ( e.g., 0.5 ng/mL) and high ( e.g., 5 ng/mL) concentrations of the Markers shall be included in each analytical batch .
The QCs should be prepared from authentic samples, or by spiking with a standard solution independent from that used for the calibrator(s).
Applicable links: ii https://www.lipomed- usa.com/en/testosterone- d3, for example.
iii https://www.lgcstandards.com/US/en/Androstenedione- d3/p/TRC -A637552- 1MG , for examp le.
iv https://chromsystems.com/en/6plus1r -multilevel -serum -calibrator -set-masschromr -steroid- panel -2-72039.html ABP Operating Guidelines – Version 9.0 – July 2023 Page 47/91 7.0 Bibliography 1.
World Anti -Doping Agency.
International Standard for Laboratories - Version 11.0 .
; 2021.
Accessed June 8, 2023. https://www.wada -ama.org/en/resources/world- anti-doping-program/international -standard- laboratories -isl#resource- download 2.
Elings Knutsson J, Andersson A, Baekken LV, Pohanka A, Ekström L, Hirschberg AL.
Disposition of Urinary and Serum Steroid Metabolit es in Response to Testosterone Administration in Healthy Women.
J Clin Endocrinol Metab.
2021;106(3):697- 707. doi:10.1210/clinem/dgaa904 3.
Salamin O, Nicoli R, Langer T, et al.
Longitudinal evaluation of multiple biomarkers for the detection of testosterone gel administration in women with normal menstrual cycle.
Drug Test Anal.
Published online April 5, 2021. doi:10.1002/dta.3040 4.
Ponzetto F, Mehl F, Boccard J, et al.
Longitudinal monitoring of endogenous steroids in human serum by UHPLC -MS/MS as a tool to detect testosterone abuse in sports.
Anal Bioanal Chem .
2016;408(3):705 -719. doi:10.1007/s00216- 015-9185- 1 5.
Nair VS, Sharpe K, Husk J, et al.
Evaluation of blood parameters by linear discriminant models for the detection of testosterone administration.
Drug Test Anal .
2021;13(7):1270 -1281. doi:10.1002/dta.3017 6.
Handelsman DJ, Bermon S. Detection of testosterone doping in female athletes.
Drug Test Anal .
Published online September 3, 2019:dta.2689.
doi:10.1002/dta.2689 7.
World Anti -Doping Agency.
International Standard for Testing and Investigations.
Published 2023.
Accessed June 6, 2023. https://www.wada- ama.org/en/resources/world- anti-doping-program/international -standard- testing- and-investigations -isti 8.
World Anti -Doping Agency.
Technical Document - Minimum Criteria for Chromatographic -Mass Spectrometric Confirmation of the Identity of Analytes for Doping Control Purposes.
; 2023.
Accessed January 24, 2023. https://www.wada- ama.org/en/resources/lab-documents/td2023idcr#resource- download ABP Operating Guidelines – Version 9.0 – July 2023 Page 48/91 3.6.
Measurement and Reporting of Endogenous Anabolic Androgenic Steroid (EAAS) Markers of the Urinary Steroid Profile WADA Technical Document – TD2021EAAS Document Number: TD2021 EAAS Version Number: 2.0 Written by: Reviewed by: WADA Science/EAAS Working Group WADA Laboratory Expert Group Approved by: WADA Executive Committee Date: 20 May 2021 Effective Date: 1 June 2021 1.0 Introduction The purpose of this Technical Document (TD) is to harmonize the measurement and reporting of the “steroid profile” of urine Samples in support of the steroidal module of the Athlete Biological Passport (ABP) (the steroidal Passport ).
1.1 The Steroid Profile The measurement of steroidal Markers [concentrations and ratios of defined Endogenous Anabolic Androgenic Steroids (EAAS)] in a urine Sample form the steroid profile for that Sample (see Table 1).
The steroid profiles of a series of urine Samples collected from an Athlete over a period of time constitute the steroidal Passport of that Athlete .
The administration of synthetic forms of EAAS can alter one or more of the Markers of the urinary steroid profile, resulting in increased or decreased concentrations and/or ratios of specific pairs of steroid Markers [1-3].
This effect forms the basis for the use of the steroidal Passport as a tool for the detection of doping with EAAS, in particular testosterone (T), its precursors (for example, 4 -androstenediol, andr ostenedione and prasterone), its active Metabolite [dihydrotestosterone (DHT)], or its epimer epitestosterone (E).
The steroidal module of the ABP utilizes the Adaptive Model in ADAMS to trigger Atypical Passport Findings (ATPFs ), which can lead to the performance of Confirmation Procedures (CP), Target Testing of an Athlete , or to establish Use of a Prohibited Substance and/or Prohibited Method as per Code Article 2.2 (see International Standard for Results Management , Annex C [4]).
1.2 Procedure for Determination of the Steroid Profile Each urine Sample shall be analyzed to determine its steroid profile.
The determination and reporting of a Sample’s steroid profile follows a two -step procedure: i.
An Initial Testing Procedure (ITP) is conducted to estimate the steroid profile of the Sample , and ABP Operating Guidelines – Version 9.0 – July 2023 Page 49/91 ii.
A subsequent CP is performed when the reported steroid profile constitutes an ATPF , as determined by the Adaptive Model , or upon request from the Athlete Passport Management Unit (APMU ), the Testing Authority or WADA .
Table 1.
Markers of the Urinary Steroid Profile.
Type of Marker Steroid Profile Markers Determination Concentrations of Steroids - Androsterone (A); - Etiocholanolone (Etio); - 5α-Androstane- 3α,17β-diol (5αAdiol); - 5β-Androstane-3 α,17β-diol (5βAdiol); - Testosterone (T); and - Epitestosterone (E).
Determined by the Laboratory by GC -MSn from the combination of the free steroid fraction and the conjugated fraction released after hydrolysis with β-glucuronidase from E. coli .
Ratios of Steroids - T/E As reported by the Laboratory in ADAMS.
- A/T; - A/Etio; - 5αAdiol/5β Adiol; and - 5αAdiol/E Automatically computed in ADAMS from respective steroid concentrations after the reporting of the steroid profile by the Laboratory .
1.3 Factors Impacting the Steroid Profile In addition to the effects mediated by the administration of EAAS, alteration of the urinary steroid profile can occur for a number of other reasons including, but not limited to, the following factors [1-3]: • Intake of alcohol (ethanol); • The administration of other anabolic androgenic steroids ( e.g.
stanozolol); • The administration of human chorionic gonadotrophin (hCG) in males; • The administration of aromatase inhibitors and anti -estrogenic substances; • The administration of inhibitors of 5α -reductase ( e.g.
finasteride, dutasteride); • The administration of ketoconazole or other similar compounds ( e.g.
fluconazole, miconazole); • The use of masking agents ( e.g.
probenecid) and diuretics; • Microbial activity; • Sample manipulation.
2.0 Initial Testing Procedure (ITP) 2.1 ITP Method Requirements The quantification of the Markers of the steroid profile shall be based on gas chromatography combined with mass spectrometry (GC -MSn; n ≥ 1).
ABP Operating Guidelines – Version 9.0 – July 2023 Page 50/91 Table 2.
Requirements of the ITP for Quantification of the Markers of the Steroid Profile.
2.1.1 ITP Validation Requirements Range of the Method Shall cover the ranges of Marker concentrations normally found in males and females.
Enzymatic Hydrolysis Assess the efficiency of the enzymatic hydrolysis using β-glucuronidase from E. coli Derivatization Assess the efficiency of the trimethylsilyl (TMS) derivatization Limits of Quantification (LOQ ) The LOQ shall be determined during method validation as the lowest concentration that can be measured with an uc (%) not greater than (≤) 30% and shall meet the following criteria: • T, E ≤ 1 ng/mL; • 5αAdiol, 5β Adiol ≤ 10 ng/mL; • A, Etio ≤ 500 ng/mL Measurement Uncertainty , uc (%) Level A Etio T E Adiols (5α-, 5β-) T/E The estimated uc (%) shall be not greater than (≤) the uc_Max (%) value given below at LOQ ≤ 30% at 5 x LOQ ≤ 20% ≤ 25% (T, E) > 5 ng/mL ≤ 15% (T, E) ≤ 5 ng/mL ≤ 30% 2.1.2 ITP Analysis Requirements Sample The ITP for the quantification of the Markers of the steroid profile shall be conducted on a single Aliquot .
When needed, the volume of the Aliquot may be adjusted as a function of its specific gravity (SG) and of the sex of the Athlete .
Calibration Calibration standard(s) or a calibration curve shall be included in each sequence of analysis.
Quality Control At least two (2) quality control (QC) urine samples containing representative low and high concentrations of the Markers of the steroid profile shall be included in each sequence of analysis.
Enzymatic Hydrolysis Purified β-glucuronidase from E. coli shall be used for the hydrolysis of the glucuroconjugated urinary steroids, and the completeness of hydrolysis shall be monitored in each Aliquot with isotopically labeled A-glucuronide (or an equivalent scientifically recogniz ed alternative).
H. pomatia mixtures shall not be used.
Derivatization The Markers of steroid profile shall be analyzed as TMS derivatives (TMS enol ethers and/or TMS ethers).
ABP Operating Guidelines – Version 9.0 – July 2023 Page 51/91 Completeness of the derivatization shall be controlled in each Aliquot through the monitoring of mono- O-TMS vs. di -O-TMS derivative of A. T/E Ratio The T/E ratios shall be determined from the ratios of chromatographic peak areas or peak heights after correction against a calibrator or a calibration curve.
Factors Impacting the Steroid Profile The Laboratory shall: • Monitor for signs of microbial activity [ e.g.
presence of indicators of 3α-hydroxysteroid dehydrogenase (HSD) activity]; [Comment: The direct enzymatic hydrolysis of urine Samples may increase the effects of microbial contamination.]
Test for the presence of conjugated Metabolite(s) of ethanol [ e.g.
ethanol glucuronide (EtG)], 5 α-reductase inhibitors ( e.g.
finasteride, dutasteride) and ketoconazole (and similar substances).
2.2 Reporting the Sample’s Steroid Profile from the ITP Following the performance of the ITP, the Laboratory shall report in ADAMS the steroid profile for each Sample analyzed.
The Laboratory shall report in ADAMS: i.