The importance of reference materials in doping-control analysis
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- Mackay, L.G. & Kazlauskas, R. Anal Bioanal Chem (2011) 401: 483. doi:10.1007/s00216-011-5049-5
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Currently a large range of pure substance reference materials are available for calibration of doping-control methods. These materials enable traceability to the International System of Units (SI) for the results generated by World Anti-Doping Agency (WADA)-accredited laboratories. Only a small number of prohibited substances have threshold limits for which quantification is highly important. For these analytes only the highest quality reference materials that are available should be used. Many prohibited substances have no threshold limits and reference materials provide essential identity confirmation. For these reference materials the correct identity is critical and the methods used to assess identity in these cases should be critically evaluated. There is still a lack of certified matrix reference materials to support many aspects of doping analysis. However, in key areas a range of urine matrix materials have been produced for substances with threshold limits, for example 19-norandrosterone and testosterone/epitestosterone (T/E) ratio. These matrix-certified reference materials (CRMs) are an excellent independent means of checking method recovery and bias and will typically be used in method validation and then regularly as quality-control checks. They can be particularly important in the analysis of samples close to threshold limits, in which measurement accuracy becomes critical. Some reference materials for isotope ratio mass spectrometry (IRMS) analysis are available and a matrix material certified for steroid delta values is currently under production. In other new areas, for example the Athlete Biological Passport, peptide hormone testing, designer steroids, and gene doping, reference material needs still need to be thoroughly assessed and prioritised.
KeywordsCertified reference materialsReference materialsDoping
Matrix reference materials have a different use [3, 5]. They are most commonly used in the validation of test methods to enable completely independent assessment of method bias, because the matrix material can be taken through the entire measurement procedure. This assessment may then be used for estimation of measurement uncertainty for the test method with the observed bias and the CRM uncertainty being incorporated into the measurement uncertainty budget for the method [4, 6]. Matrix CRMs should also be used in ongoing quality-control (QC) checks during routine analysis. This is particularly important for analyses of samples close to threshold limits. They can also be used to calibrate in-house-prepared QC materials which can then be used with every analysis batch .
Matrix CRMs may or may not form part of the traceability chain for the measurement result [3, 5]. If a correction factor is applied to the result to correct for the difference between the certified and observed values then they are being used in the calibration of the result and they do form part of the traceability chain. If they are simply used as a QC check, and no correction is actually carried out, then they provide a valuable recovery check but do not provide traceability. Estimation of measurement uncertainty for these two cases will be subtly different .
The main problem laboratories face is lack of availability of certified reference materials prepared to the appropriate ISO standard (vide infra). The World Anti-Doping Agency (WADA) has funded the production of a range of matrix materials in support of the key threshold substances of 19-norandrosterone, testosterone, epitestosterone, and testosterone/epitestosterone (T/E) ratio . However a literature review on doping reference materials shows only a limited number of groups working in this area [8–26]. In a measurement area in which the results may be subjected to substantial legal scrutiny CRM production and effective use of the resulting CRMs should be priorities.
Certification requirements for CRMs
There are many misconceptions among users about what constitutes a certified reference material. This is perhaps not helped by the fact that there are two international definitions for a reference material, one from the International Vocabulary of Metrology—basic and general concepts and associated terms (VIM3 or ISO Guide 99 as it is also known ) and one from ISO’s Reference Material Committee, ISO REMCO .
Reference material: material, sufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit for its intended use in a measurement process.
Certified reference material: reference material characterized by a metrologically valid procedure for one or more specified properties, accompanied by a certificate that provides the value of the specified property, its associated uncertainty, and a statement of metrological traceability.
Both the VIM and ISO REMCO definitions are basically in agreement and the key issues are that a CRM must be certified using valid procedures, and have a certified measurement uncertainty and a defined traceability. These are not trivial issues and the establishment of procedures to provide property values with completely defined uncertainty budgets and that can provide sufficient evidence to ensure appropriate traceability to the International System of Units (SI) is a substantial task. In some cases traceability will also be required to be to other reference systems, for example International Units (IU) as used in the clinical community to measure biological activity.
ISO Guide 34 (ISO REMCO’s General requirements for the competence of reference material producers) was reissued in 2009  and provides guidance on how certifiers should produce, characterize, and test the homogeneity and stability of materials. It also includes an annex dedicated to the metrological traceability of certified property values of reference materials. In addition, ISO Guide 35 provides further statistical advice on these aspects . Guide 35 provides examples of homogeneity and stability-testing regimes for reference materials and defines how the data should be treated mathematically. It defines how the stability and homogeneity components of the uncertainty for the certified value of a CRM should be calculated and incorporated with other factors.
Homogeneity testing involves ensuring that a representative selection of units across the batch have been tested to ensure that the certified property values are applicable to the entire batch. For example a typical batch of a urine matrix material may be 1,000 bottles and 30 would be tested and analysed via ANOVA to determine the within-bottle and between-bottle variation [18, 28, 29].
Stability testing involves testing the long-term stability under the prescribed storage conditions. These data will be used to assess if the material is fit for its intended purpose and a long-term stability uncertainty is assessed. Also, when applicable, transport stability under the likely conditions involved in distribution of the units should also be tested and a separate estimate of uncertainty determined [18, 28, 29].
The long-term stability of reference materials is an important issue and reference material producers should have strict procedures in place for ongoing stability testing. In several areas of doping analysis different groups have studied the stability of a range of analytes, for example amphetamines , ephedrine , salbutamol , diuretics , testosterone glucuronide , and 19-norandrosterone glucuronide . These have covered urine in both its liquid and freeze-dried forms and have examined factors such as the effects of freeze–thaw cycles .
Reference materials should be characterized using appropriate technically valid methods that provide property values which have a fit for purpose measurement uncertainty. For certified materials the approach must provide appropriate traceability [18, 28].
Guide 34 covers many of the other requirements of reference material production and Guide 31 covers aspects such as information that must be included in a reference material certificate .
If reference material producers are adhering to the requirements in these guides then the resulting materials should be of an acceptable standard. However, many producers would not be meeting these requirements but would still be claiming they are producing reference materials and even certified reference materials. Unfortunately it then becomes the job of the user to make an appropriate assessment as to the fitness of the material for purpose.
The international recognition of CRMs
Institutes can also submit data for inclusion in the International Committee of Weights and Measures database of measurement services and CRMs. This database contains one steroid in urine CRM from the National Measurement Institute, Australia, (NMIA) and a range of pure substance doping CRMs (refer to http://kcdb.bipm.org/appendixC/search.asp?reset=1&met=QM to search for materials). These materials have all been reviewed by a team of international experts from other NMIs before acceptance into the database. This database is thus one way of checking the quality of materials.
The role of CRMs for non-threshold substances
By far the majority of substances in the WADA Prohibited List  do not have a threshold value and thus are reported as simply being present with no quantification required. WADA Technical Document TD2010IDCR Identification Criteria for Qualitative Assays Incorporating Column Chromatography and Mass Spectrometry  outlines the use of reference materials to confirm the identity of detected substances, for example in respect of retention time, etc. Thus it is the identity of the substance which is crucial and a reference material which has appropriately confirmed identity is essential.
A large number of pure substance reference materials are available, many from NMIs [38, 39]. There are also many commercial suppliers of such compounds in their pure or solution form. The techniques used for confirmation of the identity of such materials must be critically evaluated. In the certification of pure materials a simple comparison with a melting point or IR spectrum can be highly dangerous for substances where many structurally similar analogues with subtly different regiochemistry and stereochemistry can exist. This is highly relevant for steroid materials, for example. A more rigorous approach involving mass spectral analysis, IR, and NMR is more commonly accepted as best practice for identity confirmation . Users of materials should check the techniques which have been used in the certification of RMs to ensure that they would withstand appropriate legal scrutiny.
There is a continual need for new materials as new substances are added to the WADA Prohibited List and new designer drugs are detected and their metabolites identified [40–45]. Several groups have been working on the synthesis of new standards to meet these needs [23, 38]. Novel techniques such as enzyme-assisted production of materials have been reported  to assist in the formation of appropriate metabolites.
The role of CRMs in the quantification of threshold substances
The WADA Prohibited List  prescribes nine substances which have threshold limits (19-norandrosterone, carboxy-THC, epitestosterone, salbutamol, morphine, cathine, ephedrine, methylephedrine and pseudoephedrine). Human growth hormone is also classed as a threshold substance on the basis of the ratio between its different isoforms. For these substances their detection at levels greater than the WADA decision limit (DL) warrants an “adverse analytical finding” (AAF) which must be reported . The WADA decision limit is determined from the threshold limit (T) via the WADA-defined maximum combined standard uncertainty (ucMax). WADA’s laboratory committee have calculated expected maximum combined standard uncertainties for all threshold substances based on the performance of WADA-accredited laboratories in relevant rounds of the WADA External Quality Assessment Scheme (EQAS) . The decision limit thus becomes the threshold limit value plus a guard band representing a 95% confidence interval. Clients must be careful not to confuse the intended use of DL and the threshold, and to ensure they do not use the DL as a new threshold to which uncertainty is applied a second time.
For these substances quantification of the analyte concentration must occur. This will typically take place during a separate analysis after initial detection in the screening method. For all of these substances the calibration material is an important RM which will be either the pure substance or a solution of the material. A matrix reference material provides information on overall method bias for these threshold substances.
Pure substance and solution RMs for threshold substances
For analytes for which quantification will be performed, the identity and purity value of the RM are equally important and thus the methods used to assess both of these should be considered. International best practice in pure substance reference material purity evaluation is being examined by the International Bureau of Weights and Measures (BIPM) in a series of international comparisons (CCQM-K55) . These comparisons have shown the need to use a full range of techniques to assess all potential impurities. The most common reason for poor agreement in purity assessment is underestimation of impurities because an insufficient range of techniques is being used . A typical list of purity-assessment techniques would be gas chromatography with flame ionization detector (GC–FID), high-performance liquid chromatography (HPLC), thermogravimetric analysis (TGA), Karl–Fischer titration (KF), head space–gas chromatography–mass spectrometry (HS–GS–MS), NMR, and microanalysis [15–17]. These techniques together enable cross-checking of purity value assignment data. Many suppliers of materials are still using only one or two techniques to assign purity and this can easily lead to overestimation of purity, because some impurities can be difficult to detect or can be missed entirely. For example TGA will often underestimate water content and should be accompanied by a KF direct check for water content, and the use of either UV or mass spectral detection needs to account for varying response factors especially if the identity of an impurity is not known. Many analytes, for example steroid glucuronides, can absorb water over time and thus rigorous stability testing of the pure materials is required by the RM producer, and users should be aware of these problems.
Additionally these RMs need to have purity values with appropriately estimated measurement uncertainties. The use of a range of techniques can ensure that an appropriate realistic measurement uncertainty is assigned to the purity value. The information which should be provided with the material will enable the user to assess whether the material really meets the criteria of a CRM, RM, or neither of these. The measurement uncertainty should be critically reviewed for each substance as some producers sell a range of materials with exactly the same measurement uncertainty which probably indicates limited individual analysis of such materials.
During the use of pure substance reference materials many laboratories underestimate factors such as static effects when weighing small amounts of materials in the preparation of standard solutions. Evaporation of solution standards over time is also important. For these reasons not only are pure reference materials of appropriate quality needed initially but the maintenance and monitoring of the working solutions is crucial, especially when the measurements are only performed on adverse findings which only occur rarely. One common reason for analytical bias in quality-assurance schemes is problems with the working calibration solutions.
Solution reference materials are also available [18, 38] and may improve the situation. These materials remove the need to weigh small quantities of pure material; information on their stability must be available, however. The certification methods and ongoing stability testing of these materials should be factors which users critically assess.
For many analytes, laboratories require isotopically-labelled analogues as internal standard materials. A large range of these materials is available [38, 48] with although there is still a growing need as testing requirements change and laboratories move to more liquid chromatography–mass spectrometry-based technologies.
Matrix reference materials for threshold substances
Matrix RMs will provide crucial evidence that the laboratory’s method is under control and that the laboratory measurement uncertainty is below that outlined by WADA as the maximum combined standard uncertainty for that analyte in WADA TD2010DL . The laboratory may correct their initial result using the value obtained for a matrix CRM (in comparison with its certified value) in which case the CRM then becomes an ideal matrix-matched calibrant for the method.
Matrix CRMs in urine have been prepared for several analytes . These are freeze-dried urine materials because of the greater stability of this form and the excellent ability of the matrix to be reconstituted. The urine is stabilized with sodium azide after collection and typically fortified with the relevant metabolite to levels close to the threshold values . When prepared this way these materials have excellent ongoing stability [10, 18]. Some specific materials are described in more detail below.
An example of the use of CRMs for 19-norandrosterone quantification
WADA Technical Document TD2010NA  provides specific advice to laboratories on the detection of doping with nandrolone and 19-norsteroids via determination of the main urinary metabolite, 19-norandrosterone. A concentration of greater than the decision limit of 2.5 ng mL−1 of this metabolite constitutes an adverse analytical finding and thus accurate quantification of the substance is essential. TD2010NA outlines the requirements for the quantification methods being used by laboratories and includes recommendation of the use of the urine certified reference material NMIA MX002 . NMIA MX002 is a freeze-dried human urine material which is certified to contain 2.15 ± 0.15 ng mL−1 (95% coverage interval) of the sum of 19-norandrosterone and its glucuronide metabolite (expressed as equivalents of the free steroid). It has been certified by use of two independent isotope-dilution mass spectrometry methods [33, 34, 50]. The material has undergone full homogeneity testing and underwent over 12 months of stability testing before its release . In addition there is available a solution form of the substance at 192 ± 0.6 ng mL−1 in organic solvent and a pure CRM which is certified at 93.5 ± 0.8% mass fraction. Deuterated versions of the free steroid and the glucuronide are also available .
Together these pure, solution, and matrix CRMs provide a range of materials to ensure appropriate traceability and accuracy of test results for this substance, which has one of the lowest threshold limits of all substances in the WADA Prohibited List . The WADA-required maximum relative combined standard uncertainty for this analyte is 15%  and by appropriate use of these CRMs to control method performance laboratories should be able to achieve this and have the evidence to demonstrate that their method meets the uncertainty requirement.
Because adverse analytical findings for this analyte number only a few per year per laboratory it is not a huge burden to use the urine matrix CRM in such instances. Laboratories may choose to correct their sample results in accordance with the results they obtain for the urine matrix material. This provides a matrix-matched calibration of the method. Alternatively laboratories may simply check that the value obtained is within the expected range of the certified value for the urine CRM.
Reference materials for isotope-ratio measurements
Use of isotope-ratio mass spectrometry (IRMS) for confirmation of the source of endogenous steroids is a crucial part of the testing program for these analytes [51–55]. Appropriate reference materials are thus essential in order to ensure laboratories can truly distinguish between administered steroids and the urinary reference steroids with which they are compared. The requirement relies on distinguishing a difference in 13C/12C of three delta units or more for the administered steroid versus the urinary reference steroid.
The WADA technical document clearly states that the IRMS must be calibrated with an appropriate reference material . Brenna et al have produced two mixed steroid reference materials (CU/USADA 33–1 and CU/USADA 34–1) which are used for this purpose by many laboratories . The materials are characterized by gas chromatography–combustion–isotope-ratio mass spectrometry (GC–C–IRMS) using the RM8559 natural gas reference material from the National Institute of Standards and Technology in the USA (NIST); the IRMS reference gas is calibrated using the methane in RM8559 material. The materials would benefit from establishment of more rigorous measurement uncertainty estimates, because the values are simply quoted with standard deviations from replicate characterization measurements. As long as this is taken into account when using these materials they are the best fit for purpose IRMS standards currently available. The analytes in the Brenna standards are the closest structural analogues to the substances requiring routine testing and they have been certified by very thorough application of the IRMS technique . As they are actual steroids they behave the same way during GC–C–IRMS analysis and thus biases in the calibration are minimised. Other isotopic reference materials are available from the International Atomic Energy Agency (IAEA), e.g. IAEA-600, which is certified for the delta value of caffeine. Additionally, Indiana University has produced a wide range of pure materials which are traceable to NBS-19 from NIST and LSVEC from IAEA . The disadvantage of these materials is that during routine steroid IRMS analysis they can behave differently in the GC–C–IRMS system. In our laboratory we have noticed that even the Indiana University 5α-androstane reference material (which is the closest structural analogue) can behave variably as a calibrant, presumably because it does not oxidise in the same way as typical steroid molecules with more polar functionality. Thus although they are excellent materials care needs to be taken during their use in the calibration of steroid analysis.
WADA have funded the production of a matrix freeze-dried urine material that will have certified delta values for androsterone, etiocholanolone, testosterone, and epitestosterone . It will also have certified delta values for the urinary reference steroids 11-oxoetiocholanolone, 11β-hydroxyandrosterone and pregnanediol. This material will provide laboratories with the first matrix-matched reference materials for GC–C–IRMS analysis of steroids. The material is being certified by a method which has been exhaustively assessed for bias during the extraction, HPLC clean-up, and GC–C–IRMS analysis stages. The calibration of the IRMS is a crucial factor and several approaches have been tested and compared . The resulting certified delta values of the steroids in this urine material will have traceability to the Vienna PeeDee Belemnite (VPDB) international standard and will have very well defined uncertainties with full uncertainty budgets.
It is envisaged that laboratories will use this matrix CRM in the validation of their IRMS methods and that its use for every adverse sample analysis will be recommended.
Reference materials to support the WADA Athlete Biological Passport
The approval of the WADA Athlete Biological Passport program in December 2009 marks a change in approach to the fight against doping . The program involves the collection of longitudinal data from athletes for a range of biological values, for example haematological variables in blood and steroid markers in urine . The objective of the passport program is improved detection of doping by indirect detection of abnormal changes in profiles of substances, for example key steroids, which may not be detected by relying on the one-off analysis of data. The important factor is that the individual athlete becomes the benchmark against which they are judged, rather than against an average athlete population. This is important, because some athletes are known to metabolise doping agents in such a way that the effect of the agent will not be detected in their urine or blood with existing techniques or rules. For example some individuals have an unnaturally low testosterone to epitestosterone ratio and thus the normal trigger point of a T/E >4, indicating a suspicious level, is unlikely to be reached.
The longitudinal data will be collected in the web-based Anti-Doping Administrative and Management System (ADAMS). The collection of longitudinal data for athletes from laboratories around the world will place greater demand on these laboratories to produce accurate and comparable results across space and time. There is a need for CRMs to support this new program and WADA have funded two projects to this effect to provide CRMs for the steroid markers . Further details are provided under the discussion of testosterone.
The potential need for reference materials for haematological variables included in the athlete biological passport will have to be assessed. Currently, laboratories are using QC materials provided by the instrument manufacturer. However, the performance of all WADA-accredited laboratories in measuring the haematological variables is rigorously assessed by means of a monthly quality-assurance program.
The increasing need to accurately determine testosterone and related steroids
WADA Technical Document TD2004EAAS Reporting and Evaluation Guidance for Testosterone, Epitestosterone, T/E Ratio and Other Endogenous Steroids  outlines the criteria for the challenging detection of endogenous steroids. These steroids are present naturally in the human body and abuse of such substances is typically confirmed by IRMS analysis.
The WADA-funded urine matrix certified reference material NMIA MX005 and its solution equivalent, NMIA MX006, are available to support this testing . These materials are certified for testosterone, epitestosterone, and T/E ratio in freeze-dried urine and methanol, respectively. In the urine material the concentrations are 40.3 ± 1.7 ng mL−1, 10.76 ± 0.76 ng mL−1, and 3.74 ± 0.24 for testosterone, epitestosterone, and T/E ratio, respectively. The solution CRM contains 2,021 ± 81 ng mL−1, 496 ± 28 ng mL−1, and 4.08 ± 0.18, respectively. All quoted uncertainties correspond to a 95% coverage interval.
The steroid data which will be utilized in the WADA Athlete Biological Passport will typically come from the initial screening procedure applied to all urine samples to determine steroid profile concentrations. Longitudinal data will be collected and stored in ADAMS for these analytes, which adds a new dimension to the required accuracy of screening tests.
The WADA-funded matrix CRM project which supports this involves certification of two different freeze-dried materials to support the quality of longitudinal testing data . The objective is to certify these materials for the concentrations of the glucuronide conjugates of testosterone, epitestosterone (and the T/E ratio), androsterone, etiocholanolone, 5α-androstane-3α,17β-diol, and 5β-androstane-3α,17β-diol.
The routine use of RMs and the role of in-house prepared QC materials
The greatest danger laboratories face is the presence of unknown biases within their methods. These will not be detected unless laboratories have rigorous internal quality assurance systems involving independent calibrants and checks to monitor for factors such as matrix effects. One important aspect is the use of appropriate reference materials as the calibrants and the use of matrix materials to check for recovery and bias whenever results are close to threshold limits.
A simple factor which laboratories often underestimate is the importance of the storage conditions used for solutions of calibrants. These must be stored in appropriately sealed containers and monitored for changes in levels. For threshold substances which are not regularly observed, greater care is needed with calibrant solutions and so it would be recommended that when an adverse analytical finding is detected a new calibration solution be prepared (from a new portion of the solid CRM or new ampoule of the solution CRM where relevant) to check for any change in the stored working calibrant solution.
QC materials are important in a quality system. For threshold substances which are regularly detected by the laboratory there should be ongoing QC data available from matrix QC materials to enable monitoring of method performance. Most laboratories use in-house spiked QC urine materials to check for recovery . This is an excellent practice but is ideally combined with the use of matrix CRMs. Laboratories such as ours have occasionally seen a change in level of a urine QC on production, presumably resulting from some reaction with the medium. Thus the value used in the routine assessment of the QC sample is simply an observed value, not an independent spiked value. Comparison with a suitable matrix CRM enables completely independent assessment of the QC material, and of the performance of the method, and can be carried out at appropriate intervals. In-house-prepared urine QC materials are also often not subjected to rigorous stability-testing procedures and thus a matrix CRM provides a truly independent check via comparison with a material of guaranteed stability.
Use of a matrix CRM, when available, would typically be recommended at least every 3–6 months for routine measurements. For threshold substances which are not regularly observed greater care is needed, and inclusion of a matrix CRM in each batch containing an AAF would then be recommended to check for overall recovery in an independent matrix-matched sample.
Problems related to the use of CRMs
Fully certified pure substance CRMs for doping analysis have been available for over 10 years and the first matrix materials became available in 2007. Unfortunately it seems that many chemical testing laboratories still do not understand the meaning of “certified” in the context of a CRM and many still believe that a material that comes with a piece of paper with a value on it is “certified”, because this is taken to be a certificate. This is a common misconception among many testing laboratories in many fields.
It seems that laboratories are often also still unclear about the best way to utilise matrix CRMs effectively and are unsure about how to properly incorporate the results into a reported value or how to incorporate the value observed for the CRM into their measurement uncertainty estimate. The new WADA technical document  will assist to some extent but further guidance will be needed to build on these, and specific examples will be very useful .
Because the list of analytes in the WADA Prohibited List that require quantification is actually very small, laboratories must accept that these analytes require greater care. Obviously one of the greatest problems with the use of CRMs is the lack of reference materials to cover the whole set of threshold substances, but laboratories should be encouraged to make better use of the materials which are currently available.
Method performance information can also be gained from intercomparison exercises. WADA-accredited laboratories regularly participate in such exercises. WADA has an ongoing mandatory EQAS program covering a broad range of analytes . WAADS (the World Association of Anti-Doping Scientists) also coordinates an internal QA program. These programs provide valuable ongoing assessment of laboratory performance. They assess and ensure the comparability of results from different laboratories but do not establish traceability  for the results from participating laboratories and do not necessarily provide a full assessment of bias; this can be unequivocally determined only when an appropriate reference value is employed.
Reference materials should form an important part of the supporting quality systems which are in place within laboratories to ensure the traceability and accuracy of results. For threshold substances this is of particular importance. RMs are, however, no longer only important for the reporting of adverse analytical findings for threshold substances. The introduction of longitudinal data storage, which will form part of the WADA Athlete Biological Passport program, will mean screening results are being used in the detection of doping. Testing laboratories may need to change their procedures in respect of matrix CRM use to ensure they are more routinely used in order to provide appropriate control of screening data within tighter criteria than was previously required.
For many of the substances with threshold limits no matrix CRMs are available. For some of these the much higher typical concentrations makes the need for such CRMs slightly less of an issue in terms of the declaration of an AAF, but important for accuracy and precision of the value of the measurand. For example, the thresholds for 19-norandrosterone and epitestosterone are 2 and 200 ng mL−1, respectively, whereas those for salbutamol and morphine are 1,000 ng mL−1 and those for the other threshold substances are even higher. At these high concentrations there will typically be fewer recovery issues and calibration standards can be more stable. The exception to this is carboxy-THC with a threshold of 15 ng mL−1. There are two THC-9-COOH in human urine CRMs in the NIST catalogue (SRM 1507b and 1511), although both are, unfortunately, listed as out of stock at the time of writing [19–21].
There are still many areas of doping control for which no reference materials are available, and the anti-doping community will need to prioritise these and work on the technical challenges faced for their production. These include, for example, reference materials for new designer steroids and their metabolites, peptide hormone analysis, haematological variables which form part of the Athlete Biological Passport, and gene doping.
Several groups are working on the development of standards for new designer drugs and their metabolites. For these the main requirement is appropriate identity confirmation. Several groups have reported on the production of these materials and their MS analysis [42–45].
The peptide hormone area is a huge challenge. As testing methods for these analytes become increasingly sophisticated [63–71] and more is learnt about the issues in respect of the exact forms of these analytes, the time for production of appropriate reference materials is getting closer. There have been publications in the past looking at relevant QC materials for these analytes  and there have been several publications examining the currently available primary calibrants [73, 74]. Much work is being done within the clinical community to standardize such methods and provide a reference measurement system to support the accuracy and comparability of measurements of similar analytes in serum . The doping community would benefit from considering this approach although the effort involved is substantial.
As methods to detect gene doping are now being published [76, 77] groups such as ours are beginning to consider the next step with regard to the need for standards to support this testing. The need for these will depend on the approaches of the different national anti-doping organizations with regard to the implementation of testing programs.
Work carried out at NMIA in the production of reference materials for doping control has been funded by the World Anti-Doping Agency and the Australian Government Anti-Doping Research Program. The authors are very grateful for advice from the NMIA’s sports drug testing laboratory staff on the practical use of reference materials within a WADA-accredited laboratory.