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Sample preparation) and identification requirements (e.g. |
number of diagnostic ions or precursor -product ion transitions) applica ble to Initial Testing Procedures and Confirmation Procedures , their detection capabilities may differ. |
Therefore, it may occur that a Sample is reported as an Adverse Analytical Finding for a Non-Threshold Substance at concentrations lower than the estimated LOD of the Initial Testing Procedure . |
Furthermore, since LOD values are estimations based on method validation with a limited number of representative samples, a Laboratory may be able to effectively confirm the presence of a target Non-Threshold Substance (or its representative Metabolite or characteristic Marker) in a given Sample at levels below the validated LOD (e.g. |
in a Sample with low background or less matrix interferences). |
A Confirmation Procedure for a Non-Threshold Substance shall allow the unequivocal identification of the Non-Threshold Substance (or its ISL – January 2021 Page 83 of 160 representative Metabolite (s) or characteristic Marker (s)) in compliance with the TD IDCR. |
If successfully identified, a Non-Threshold Substance can be reported at a concentration below the estimated LOD of the Initial Testing Procedure or the LOI of the Confirmation Procedure .] |
- Robustness: The Confirmation Procedure shall be demonstrated to produce similar results with respect to minor variations in analytical conditions, which may affect the results of the analysis. |
Those conditions that are critical to ensuring reproducible results shall be considered ; - Carryover: T he conditions required to eliminate carryover of the substance of interest from Sample to Sample during processing or instrumental analysis ; [Comment: Elimination of ‘injection memory’ effect is demonstrated by injecting a blank control sample for the Analyte in question, prepared in the Sample matrix , immediately prior to the Sample of interest.] |
5.3.5.2 Validation of Analytical Testing Procedures for Threshold Substances As part of the validation process for chromatography -mass spectrometric Analytical Methods applied to the analysis of Threshold Substances , the Laboratory shall develop acceptable standard solutions for identification of Threshold Substances using Referenc e Materials . |
For Confirmation Procedures , Certified Reference Materials should be used for quantification, if available. |
For the application of affinity -binding assays to the analysis of Threshold Substances , the Laboratory shall follow the applicable Tech nical Document (e.g. |
Technical Document on human Growth Hormone, TD GH) or Laboratory Guidelines . |
a) Validation of Initial Testing Procedures for Threshold Substances The Laboratory shall validate Initial Testing Procedures that are Fit-for-Purpose , in accordance with relevant Technical Document (s), Technical Letter (s) or Laboratory Guidelines . |
For chromatograph ic-mass spectrometr ic Initial Testing Procedures , the Laboratory shall validate the Selectivity , LOD and dynamic range from the analysis of an adequate number of representative samples prepared in the appropriate matrix of analysis 12. |
The Laboratory shall validate and document the concentration levels which will require quantitative Confirmation Procedu re(s)12. |
12 Unless otherwise specified in a Technical Document , Technical Letter or Laboratory Guidelines . |
ISL – January 2021 Page 84 of 160 [Comment: In order to account for a possible underestimation of concentrations of Threshold Substances during non -quantitative Initial Testing Procedures , the Laboratory shall establish, and document in the Test M ethod ’s SOP, criteria (e.g. |
concentration levels ), determined during the Initial Testing Procedure method validation, to evaluate initial results as Presumptive Adverse Analytical Finding s and ensure that all potentially positive Samples are subjected to quanti tative Confirmation Procedures . |
Unless otherwise specified in a Technical Document, Technical Letter or Laboratory Guidelines , the Laboratory may also choose to forward all Samples containing an exogenous Threshold Substance to confirmation analysis, in or der to ensure that all potential Presumptive Adverse Analytical Findings are subjected to Confirmation Procedure (s).] |
The estimation of Measurement Uncertainty (MU) is not required during the validation of Initial Testing Procedures 12. b) Validation of Confirmation Procedures for Threshold Substances Factors to be investigated during the method validation to demonstrate that a quantitative Confirmation Procedure for a Threshold Substance is Fit-for-Purpose include but are not limited to: - Selectivity , LOI, Robustness, Carryover (see Article 5.3.5.1); - Limit of Quantification (LOQ): The Laboratory shall demonstrate that a quantitative Confirmation Procedure has an established LOQ of no more than 50% of the Threshold value or in accordance with the LOQ values required in relevant Technical Document (s) or Laboratory Guidelines ; - Dynamic Range: The range of the quantitative Confirmation Procedure shall be documented from at least 50% to 200% of the Threshold value ; - Repeatability (sr): The quantitative Confirmation Procedure shall allow for the reliable repetition of the results over a short time, using a single operator, item of equipment, etc. |
Repeatability at levels close to the Threshold shall be determined ; - Intermediate Precision (sw): The quantitative Confirmation Procedure shall allow for the reliable repetition of the results at different times and with different operators and instruments, if applicable, performing the assay. |
Intermediate Precision at levels cl ose to the Threshold shall be determined ; - Bias (b): The Bias of the measurement procedure shall be evaluated either using Certified Reference Materials or traceable Reference Materials , if available, or from comparison with a reference method or with the c onsensus values obtained from an inter -Laboratory comparison study or EQAS participation. |
Bias at the levels close to the ISL – January 2021 Page 85 of 160 Threshold shall be determined ; - Measurement Uncertainty (MU): The MU associated with the results obtained with the quantitative Confirmation Procedure shall be estimated in accordance with the Technical Document on Decision Limits (TD DL) or other applicable Technical Document (e.g. |
TD GH) , Technical Letter or Laboratory G uidelines . |
At least, MU at levels close to the Threshold shall be addressed during the validation of the quantitative Confirmation Procedure . |
Confirmation Procedure method validation data (including the estimation of MU) is evaluated during the assessment process for inclusion of the quantitative Confirmation Procedure within the Laboratory ’s Scope of ISO/IEC 17025 Accreditation. |
Therefore, for those Confirmation Procedures that are included within the Laboratory ’s Scope of ISO/IEC 17025 Accreditation, the Laboratory is not required to produce method validation data or other evidence of method validation in any legal proceeding. |
5.3.6 Sample Analysis Laboratories shall analyze Samples collected by Anti-Doping Organizations using In-Competition or Out-of-Competition Analytical Testing menus to detect the presence of Prohibited Substances or Prohibited Methods only (as defined in the Prohibited List ). |
In addition, Laboratories may analyze Samples for the following , in which case the results of the analysis shall not be reported as an Atypical Finding or an Adverse Analytical Finding : - Non-prohibited substances or methods that are included in the WADA Monitoring Program (see Code Article 4.5 ); - Non-prohibited substances for results interpretation purposes ( e.g. |
confounding factors of the “steroid profile”, non -prohibited substances that share Metabolite (s) or degradation products with Prohibited Substances ), if applicable ; - Non-prohibited substances or methods requested as part of a Results Management process by the Results Management Authority , a hearing body or WADA ; - Non-prohibited substances or methods requested by the Testing Authority as part of its safety code , code of conduct or other regulations (see comments to Code Articles 5.1 and 23.2.2) ; or - Additional analyses for quality assurance/quality improvement/ method development or research purposes, in accordance with the requirements indicated in Article 5.3.12 . |
[Comment: An Anti -Doping Organization has the discretion to apply anti -doping rules to an Athlete who is neither an International -Level Athlete nor a National -Level Athle te and may elect ISL – January 2021 Page 86 of 160 to request that Samples collected from these Athletes are analyzed for less than the full menu of Prohibited Substances and Prohibited Methods . |
The Anti -Doping Organization is responsible for providing the Laboratory with the appropriate written justification for a reduced Testing menu.] |
At minimum, all Laboratories are required to implement all mandatory Analytical Testing Procedures , as determined by WADA in specific Technical Document (s), Technical Letter (s) or Laboratory Guidelines . |
Laboratories may implement additional methods for the analysis of particular Prohibited Substances or Prohibited Methods . |
[Comment: Mandatory Analytical Testing Procedures are those Analytical Methods for which all Laboratori es shall have available analytical capacity, in compliance with relevant Technical Document(s), Technical Letter (s) or Laboratory Guidelines , and therefore should have the Analytical Method included in their Scope of ISO/IEC 17025 Accreditation. |
However, b ased on an In -Competition or Out -of-Competition Analytical Testing menu, a mandatory Analytical Testing Procedure is not necessarily applied to all Samples. |
For some Samples, Testing Authorities may decide to request Analytical Testing for specific Prohibi ted Substances or Prohibited Methods only. |
These requests shall be detailed in the Sample chain of custody. |
On occasion, however, certain Analytical Testing Procedures (e.g. |
gene doping) or the analysis of certain Prohibited Substances (e.g. |
some large pep tides) or Prohibited Methods (e.g. |
homologous blood transfusion) with a given Analytical Testing Procedure may not be mandatory for all Laboratories . |
WADA will maintain the list of mandatory Analytical Methods for reference by the Anti -Doping Organizations.] |
Analytical Testing Procedure (s) included in the Laboratory ’s Scope of ISO/IEC 17025 Accreditation shall be considered as Fit-for-Purpose and therefore the Laboratory shall not be required to provide method validation documentation or EQAS performance data in support of an Adverse Analytical Finding . |
However, if the Analytical Testing Procedure has not been included yet in the Laboratory ’s Scope of ISO/IEC 17025 Accreditation, the Laboratory shall validate the procedure in compliance with the ISL and the applicable Technical Document (s), Technical Letter (s) or Laboratory Guidelines prior to its application to the analysis of Samples . |
In s uch cases, the Laboratory may be required to provide method validation documentation or EQAS performance data in support of an Adverse Analytical Finding (see Article 4.4.2.2) . |
Laboratories may, on their own initiative and prior to reporting a test result , apply additional Analytical Testing Procedures to analyze Samples for Prohibited Substances or Prohibited Methods not included in the standard Analytical Testing menu or in the Technical Document for sport-specific analysis (TD SSA), provide d that the additional work is conducted at the Laboratory ’s expense and does not significantly affect the possibility to submit the Sample , as identified by the Testing Authority or WADA , to Further Analysis . |
Results from any such analysis shall be reported in ADAMS and have the same validity and Consequences as any other analytical result . |
ISL – January 2021 Page 87 of 160 5.3.6.1 Application of Initial Testing Procedures The objective of the Initial Testing Procedure is to obtain information about the potential presence of Prohibited Substance (s) or Metabolite (s) of Prohibited Substance (s), or Marker (s) of the Use of a Prohibited Substance or Prohibited Method. |
Results from Initial Testing Procedure (s) can be included as part of longitudinal studies ( e.g. |
endogenous steroid or hematological profiles), provided that the method is Fit-for-Purpose . |
The Initial Testing Procedure (s) shall fulfil the following requirements: - The Initial Testing Procedure shall be Fit-for-Purpose ; - The Initial Testing Procedure shall be performed on Aliquot (s) taken from the container identified as the “A” Sample ; [Comment: In cases when the “A” Sample cannot be used for the Initial Testing Procedure (s), the Initial Testing Procedure may be performed on an Aliquot of the first bottle of the split “B” Sample, which is to be used as the “A” Sample (see Article 5.3.3.2).] |
- The Initial Testing Procedure shall b e recorded, as part of the Sample (or Sample batch) record, each time it is conducted ; - All batches undergoing an Initial Testing Procedure shall include appropriate negative and positive quality controls prepared in the matrix of analysis 13; - The Initial Testing Procedures for Non-Threshold Substanc es shall include appropriate controls of representative substance(s) at or below the MRPL ; - The Initial Testing Procedures for Threshold Substances shall include appropriate controls close to the Threshold 14; - Results from Initial Testing Procedures are not required to consider the associated MU 14; - The Laboratory shall establish criteria, based on its method validation and in accordance with its SOP , to evaluate results from an Initial Testing Procedure as a Presumptive Adverse Analytical Finding , which would trigger confirmation analyses. |
13 Unless otherwise specified in a Technical Document , Technical Letter or Laboratory Guidelines . |
ISL – January 2021 Page 88 of 160 5.3.6.2 Application of Confirmation Procedures The objective of the Confirmation Procedure is to obtain a result, which supports or does not support the reporting of an Adverse Analytical Finding or Atypical Finding . |
A Confirmation Procedure for a Non-Threshold Substance with a Minimum Reporting Level may also be performed if the result estima ted from the Initial Testing Procedure is lower than the applicable Minimum Reporting Level , as determined by the Laboratory in accordance with the method’s validation results. |
A result obtained in the Initial Testing Procedure for a Threshold Substance higher than the Threshold requires a Confirmation Procedure , even if this result is below the relevant Decision Limit 14. |
A Confirmation Procedure may also be performed if the result obtained in the Initial Testing Procedure is lower than the Threshold , as determined by the Laboratory or as specifically required by the Testing Authority (or Results Management Authority , if different) or WADA . |
Irregularities in the Initial Testing Procedure (s) shall not invalidate an Adverse Analytical Finding , which is adequately established by a Confirmation Procedure . |
The Confirmation Procedure (s) shall fulfil the following requirements: - The Confirmation Procedure (s) shall be Fit-for-Purpose , including the estimation of the MU associated with a quantitative Confirmation Procedure ; - The Confirmation Procedure (s) shall be recorded, as part of the Sample (or Sample batch) record , each time it is conducted ; - The Confirmation Procedur e shall have equal or greater Selectivity than the Initial Testing Procedure and shall provide accurate quantification results (applicable to Threshold Substances ). |
The Confirmation Procedure should incorporate, when possible and adequate, a different Sample extraction protocol and/or a different analytical methodology 14; - All batches undergoing a Confirmation Procedure shall include appropriate negative and positive quality controls prepared in the matrix of analysis. |
5.3.6.2.1 Confirmation Procedure Methods Mass spectrometry (MS) coupled to chromatographic separation ( e.g. |
gas or liquid chromatography) is the analytical technique of choice for confirmation of most Prohibited Substances , Metabolite (s) of a 14 Unless otherwise specified in a Technical Document , Technical Letter or Laboratory Guidelines ISL – January 2021 Page 89 of 160 Prohibited Substance , or Marker (s) of the Use of a Prohibited Substance or Prohibited Method . |
These are acceptable methods for both the Initial Testing Procedure and the Confirmation Procedure . |
Affinity -binding assays ( e.g. |
Immunoassays), electrophoretic methods and other Analytical Methods are also routinely used for detection of macromolecules in Samples . |
[Affinity -binding assays applied for the Initial Testing Procedure (s) and Confirmation Procedure( s) shall use affinity reagents ( e.g. |
antibodies) recognizing different epitopes of the macromolecule analyzed, unless a purification ( e.g. |
immunopurification) or separation method ( e.g. |
electrophoresis, chromatography) is used prior to the application of the affinity -binding assay to elimina te the potential of cross -reactivity. |
The Laboratory shall document, as part of the method validation, that any such purification or separation method is Fit-for-Purpose . |
In affinity -binding assays which include multiple affinity reagents (such as sandwich immunoassays), at least one (1) of the affinity reagents (either applied for capture or detection of the target Analyte ) used in the affinity -binding assays applied for the Initial Testing Procedure( s) and Confirmation Procedure( s) must differ. |
The other affinity reagent may be used in both affinity -binding assays . |
For Analytes that are too small to have two (2) independent antigenic epitopes, two (2) different purification methods or two (2) different Analytical Methods shall be applied. |
Multiplexed affin ity-binding assays, protein chips, and similar simultaneous multi -Analyte testing approaches may be used . |
Antibodies may also be used for specific labelling of cell components and other cellular characteristics. |
When the purpose of the test is to identify populations of blood constituents, the detection of multiple Markers on the cells as the criteria for an Adverse Analytical Finding replaces the requirement for two (2) antibodies recognizing different antigenic epitopes. |
An example is the detection of surface Markers on red blood cells (RBCs) using flow cytometry. |
The flow cytometer is set up to selectively recognize RBCs. |
The presence on the RBCs of more than one surface Marker (as determined by antibody labelling) as a cri terion for an Adverse Analytical Finding may be used as an alternative to multiple antibodies to the same Marker .] |
ISL – January 2021 Page 90 of 160 5.3.6.2.2 “A” Confirmation Procedure : - Aliquots The “A” Confirmation Procedure shall be performed using new Aliquot (s) taken from the container identified as the “A” Sample . |
At this point, the link between the Sample external code as shown in the Sample container and the Laboratory internal Sample code shall be verified. |
[Comment: In cases when the “A” Sample cannot be used, the “A” Confirmation Procedure may be performed on an Aliquot of the split “B” Sample (see Article 5.3.3.2).] |
- Target Analyte (s) If the presence of more than one (1) Prohibited Substance , Metabolite (s) of a Prohibited Substance , or Marker (s) of the Use of a Prohibited Substance or Prohibited Method is detected by the Initial Testing Procedure (s), the Laboratory shall confirm as many of the Presumptive Adverse Analytical Findings as reasonably possible (such decision should take into account the volume s available in the “A” and “B” Sample s). |
The confirmation(s) shall prioritize the identification and/or quantification of the Prohibited Substance (s) or Prohibited Method (s) that carry the longest potential period of Ineligibility . |
The decision shall be made in consultation with the Testing Authority (or Results Management Authority , if different) and documented . |
- Existence of approved Therapeutic Use Exemption (TUE) When there is a Presumptive Adverse Analytical Finding for hCG, hGH (Biomarkers Test), Beta -2 Agonists, Diuretics, Amfetamine, Methylphenidate, Glucocorticoids or Beta -blockers , the Laboratory may contact the Testing Authority (or Results Management Authority , if different) to enquire whether an approved Therapeut ic Use Exemption (TUE) exists for the Prohibited Substance(s) detected. |
[Comment: Unless there is a prior agreement between the Testing Authority (or Results Management Authority , if different) and the Laboratory , contacting the Testing Authority (or Results Management Authority , if different) in such cases is not a requirement for the Laboratory . |
The Laboratory may proceed, at its discretion, to confirm the Presumptive Adverse Analytical Finding for hCG, hGH (Biomarkers Test), Beta -2 Agonists, Diureti cs, Amfetamine, Methylphenidate, Glucocorticoids or Beta -blockers and report an Adverse Analytical Finding in ADAMS according to the confirmation results obtained.] |
ISL – January 2021 Page 91 of 160 [Comment: In principle, the enquiry by Laboratories regarding the existence of an approved TUE for a Beta -2 Agonist may be applied not only to those Beta -2 Agonists which are prohibited under any condition, but also to those which are permitted up to a maximum dose by inhalation only , as specified in the Prohibited List . |
In such cases, the Labor atory may enquire about the existence of an approved TUE for the Use of a prohibited route of administration or a supra -therapeutic inhalation dose.] |
When possible, the Laboratory should provide an estimated concentration of the Analyte (s) from the Initial Testing Procedure . |
Any such contact with the Testing Authority (or Results Management Authority , if different) shall be confirmed in writing (for further guidance, refer to the Laboratory Guidelines on TUE enquiries). |
The instruction by the Testin g Authority (or Results Management Authority , if different) on whether the Laboratory shall proceed or not with the confirmation based on an approved TUE shall be provided to the Laboratory in writing. |
If not proceeding with the confirmation, then the Testing Authority (or Results Management Authority , if different) shall provide WADA with a copy of the approved TUE or the associated TUE number if the TUE has been submitted into ADAMS . |
- Repetition of the “A” Confirmation Procedure The Laboratory may repeat the Confirmation Procedure for an “A” Sample , if appropriate , (e.g. |
quality control failure, chromatographic peak interferences, inconclusive “A” confirmation results). |
In that case, the previous test result shall be nullified. |
Each repeat confirmation shall be performed using (a) new Aliquot (s) taken from the “A” Sample container and shall be recorded. |
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