GROWTH – DEDICATED CALL – 1/00
TOPIC III.29
Steroid glucuronides; development of liquid chromatography/mass
spectrometric (LC/MS) analysis in the detection of doping in sport
1. CONFORMITY WITH THE WORK PROGRAMME
This topic falls under the Competitive and Sustainable Growth Programme, generic
activity Measurement and Testing. Specifically, it is related to Objective GROW-20006.3 support to the development of certified reference materials for which expressions of
interest have been called.
2. KEYWORDS
anabolic steroids, analytical method development, doping control, LC/MS, steroid
excretion studies, steroid glucuronides, synthesis of reference material
3. SUMMARY OF OBJECTIVES AND JUSTIFICATION
The wide misuse of doping substances is a growing problem in sport. The methods to
confirm the presence of doping substances in samples of top athletes must be at level of
state-of-art. The existing methods for anabolic-androgen steroids (AAS) are indirect,
complicated, slow, and quite expensive and include several possible sources of errors.
A significant weakness of the present doping control system is that reference substances
are available only for I-phase metabolites (1) but not for II-phase metabolites although
both metabolites are excreted in urine. In addition, there are no analytical methods for
the direct detection of II-phase steroid metabolites (for example: glucuronide and
sulphate conjugates) in urine.
4. BACKGROUND
AAS are misused in competitive sports and also in non-competitive sports. Moreover,
AAS have already become "social drugs" as even young people use them as an
expression of an improved "life-style". In addition, recent findings indicate that the use
of AAS probably involves other reasons than to improve appearance or sports
performance since their use have been linked to same background factors which seem to
be of importance in the misuse of psychotropic drugs of adolescent (2).
The control of misuse of AAS is based on gas chromatographic/mass spectrometric
(GC/MS) methods after hydrolysis and derivatisation of the compounds to be analysed
(3). The presence of banned steroids and/or their metabolites in the sample is
established by comparing the mass spectra and the selected ion chromatograms of the
sample to those measured by using authentic reference substances. Improvement in the
sensitivity of analytical methods based on the use of high-resolution mass spectrometry
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(HRMS) or MS/MS Ion Trap technique has resulted in a more effective control
programme.
Nevertheless, the doping control of AAS still includes many potential problems.
Although synthesis methods to produce reference materials for several metabolites of
AAS have been developed (1,4-6), in some cases the reference material has still to be
produced by excretion studies after voluntary intake of the parent compound of AAS.
The finding that the origin of some AAS detected in human urine can be endogenous
(7,8) has increased the need for quantitative methods for AAS in urine to exclude the
possibility of false positives. The present analytical methods are time-consuming and in
some case unreliable due to incomplete enzymatic hydrolysis of AAS conjugates. The
direct analysis of AAS conjugates would provide faster and more reliable analysis
method than the present methods. However, at present direct analysis methods have
been presented only for few conjugates of endogenous androgens and of AAS in human
(9,10,11). This is at least partly due to lack of commercially available reference
materials such as AAS glucuronides and sulphates. These conjugated substances are
necessary in the development of direct analytical methods. In addition, the conjugated
AAS can be used to improve the quality of the analysis by classical methods as well.
They also make it possible to improve the quality control system in doping analysis in
general. These conjugates are also needed in steroid metabolism research in
pharmaceutical industry and research institutes, who are the end users for the
conjugated standards.
The present analytical methods based on GC/MS are not well applied to direct analysis
of AAS conjugates. Instead, it has been demonstrated that liquid chromatography-mass
spectrometry with electrospray ionisation (LC-ESI/MS) offers a rapid and sensitive
method for the direct analysis of steroid conjugates (12-16). The conjugates can be
analysed directly without enzymatic cleavage and laborious sample preparation.
Furthermore, ESI provides extremely high sensitivity. Before the method can be
accepted for doping control analysis, however, careful evaluation procedure must be
carried out for which steroid conjugate standards are required.
5. ECONOMIC AND SOCIAL BENEFITS
The development of new methods requires profound knowledge in the area of steroid
metabolic research and modern analytical techniques. The knowledge needed in the
development work is not found in one laboratory. Therefore, collaboration is needed
between laboratories in doping research and in analytical chemistry, especially in the
field of mass spectrometry. Furthermore, the experience in chemical and enzymatic
synthesis of drug conjugates as well as knowledge of steroid metabolism is necessary.
By combining this expertise, preparation of AAS conjugates as well as development of
new methods for doping control will be possible.
The research provides the following benefits:
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Reference materials i.e. AAS conjugates (mainly glucuronides) in milligram
quantities for external quality control in doping laboratories
Faster, quantitative and more reliable analysis method for the detection of
anabolic steroids
Analytical results with high juridical validity
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Assists the overall metabolic research of steroids, especially in pharmaceutical
industry
The principles of the methods can be adjusted to produce other drug conjugates
for research and routine work in other laboratories
6. SCIENTIFIC AND TECHNOLOGICAL OBJECTIVES
6.1. Synthesis of reference material and characterisation of synthesised steroid
glucuronides
Recently introduced enzymatic synthesis method offers an elegant way to produce
glucuronides (12-16). The enzymatic method will be compared to chemical method to
characterise the advantages and possible disadvantages of the methods. The main
advantage of the enzymatic method is the stereo-specificity of enzymes allowing the
synthesis of stereo-spesifically pure conjugates. The method can be applied to produce
steroid glucuronides. The work includes optimisation of synthesis procedure to
maximise yields. Therefore, enzyme kinetic studies are made in small scale before
larger scale synthesis. The goal is to produce steroid glucuronides in milligram amounts,
which is enough for thousands of analysis by LC/MS/MS. The steroid glucuronides will
be synthesised by modifying the method described earlier using rat microsomes as a
source for glucuronosyltransferase enzymes (UGT). Also a synthesis method using
UGT iso-enzymes produced by recombinant expression system will be developed.
Liquid chromatography/mass spectrometric (LC/MS) measurements offer a rapid
method for the assessment of the quality of the synthesis and therefore LC/MS will be
used during the development of synthesis methods as well as in the characterisation of
the final products. Nuclear Magnetic Resonance spectroscopy (1H and 13C spectra),
Fourier transformed infrared spectroscopy (FTIR) and high resolution MS will be used
for final characterisation of the products.
6.2. Comparison with excretion studies
The synthesised glucuronides will be compared to glucuronides isolated from urine of
persons who have been treated with anabolic-androgen steroid hormones in order to
confirm the identity of the synthesis product with those excreted in the urine. For this
purpose single excretion studies with anabolic androgen steroids with male persons are
planned. A single oral administration of anabolic-androgen steroids following the
therapeutic recommendation will be performed with e.g. boldenone, clostebol,
mesterolone, 19-nortestosterone, methandienone, methenolone, methyltestosterone,
trenbolone and stanozolol. Glucuronides of the corresponding metabolites will be
isolated from urine and purified using liquid chromatography. Conjugated metabolites
will be characterised by LC/MS and by GC/HRMS and after enzymatic hydrolysis by
GC/MS and GC/HRMS.
6.3. Development of LC/MS/MS method for AAS glucuronides
After synthesis of AAS glucuronides a LC-ESI/MS/MS screening method will be
developed to detect their presence in the urine sample. The method development
procedure both for screening and identification methods includes development of fast
and simple sample preparation method, optimisation of chromatography and MS/MS
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parameters for high sensitivity detection. Finally the methods will be validated.
6.4. Comparison of LC/MS/MS with the classical techniques: GC/MS, GC/HRMS and
GC/MS/MS Ion Trap
The suitability of LC-ESI/MS/MS method in the screening and identification of AAS
will be compared to that of the present techniques (GC/MS, GC/HRMS and GC/MS/MS
Ion Trap).
7. TIME SCALE
8. IMPORTANT ADDITIONAL INFORMATION
A larger network is later necessary when the reference material as well as the methods
shall be tested at least in doping control laboratories in Europe. It could be investigated
if similar work has been performed in the veterinary area.
References
1. Schänzer W and Donike M: Metabolism of anabolic steroids in man: Synthesis
and use of reference substances for identification of anabolic steroid metabolites.
Anal. Chim. Acta, 275 (1993) 23-48.
2. Kindlundh AMS, Isacson DGL, Berlung L, Nyberg F: Factors associated with
adolescent use of doping agents: anabolic-androgenic steroids. Addiction 94 (1999)
543-553.
3. Segure J, Ventura R, Jurado C: Derivatization procedures for gas-chromatographicmass spectrometric dtermination of xenobiotics in biological samples, with special
reference to drugs of abuse and doping agents. J Chromatogr B 713 (1998) 61-90.
4. Schänzer W: Review - Metabolism
Clin. Chem., 42 (1996) 1001-1020.
of
anabolic
androgenic
steroids.
5. Schänzer W: Abuse of androgens and detection of illegal use. In Niechlag E, Behre
HM (eds.) Testosterone - Action, Deficiency, Substitution. 2nd Edition, Springer
Verlag, Berlin-Heildelberg-New York, 1998.
6. Schänzer W: Detection of exogenous anabolic androgenic steroids. In Karch SB
(ed.) Drug abuse handbook. CRC Press, London-New York, 1998.
7. Ayotte C, Goudreault D and Charlebois A: Testing for natural and synthetic
anabolic agents in human urine. J Chromatogr B, 687 (1996) 3-25.
8. Dehennin L, Bonnaire Y, Plou Ph: Urinary excretion of 19-norandrosterone of
endogenous origin in man: quantitative analysis by gas-chromatography-mass
spectrometry. J Chromagr. B 721 (1999) 301-307.
DC 1/00/Topic III.29/ Pg 5
9. Maurer HH: Liquid chromatography-mass spectrometry in forensic and clinical
toxicology. J Chromatogr B 713 (1998) 3-25.
10. Kostiainen R, Kuuranne T, Vahermo M, Leinonen A, Kuoppasalmi K, Taskinen J:
Direct quantitative analysis of methyltestosterone metabolite 17-alfa-methyl-5beeta-androstan-3 alfa-,17-beeta-diol glucuronide by liquid chromatography mass
spectrometry, 8th International Meeting on Recent Development in Pharmaceutical
Analysis (RDPA ’99, Rome, June 29- July 3, 1999, p. 123.
11. Kuuranne T, Vahermo M, Leinonen A, Kostiainen R: Electrospray and
Atmospheric pressure Chemical Ionization Tandem Mass Spectrometic Behavior of
Seven Steroid Glucuronides. J Am Soc Mass Spectrom, submitted for publication,
1999.
12. Luukkanen L, Kilpeläinen I, Kangas H, Ottoila P, Elovaara E, Taskinen J: Enzymeasissted synthesis and structural characterization of nitrocatechol glucuronides.
Bioconjugate Chem. 10 (1999) 150-154.
13. Keski-Hynnilä H, Luukkanen L, Taskinen J, Kostiainen R: Mass Spectrometric
and tandem mass spectrometric behavior of nitrocatechol glucuronides: a
comparison of athmospheric pressure chemical ionization and electrospray
ionization. J. Am. Soc. Mass Spectrom.10 (1999) 537-545.
14. Kostiainen R, Luukkanen L, Keski-Hynnilä H, Kuuranne T, Kaivosaari S,
Taskinen J: ESI/MS/MS Spectra of Some Glucuronide Conjugates Produced by
Enzymatic Synthesis, The 46th ASMS Conference on Mass Spectrometry and
Allied Topics, May 31-June4, 1998, Orlando, Florida, p. 721.
15. Keski-Hynnilä H, Andersin R, Luukkanen L, Taskinen J, Kostiainen R: Analysis of
Catechol-type Glucuronides in Urine Samples by Liquid Chromatography
Electrospray Ionization Tandem Mass Spectrometry, J. Chromatogr. A, 794 (1998)
75.
16. Kuuranne T, Vahermo M, Leinonen A, Kuoppasalmi K, Taskinen J, Elovaara E,
Kostiainen R: ESI-MS and MS/MS Detection of the Glucuronide Conjugate of
17alfa-Methyl-5beta-Androstan-3alfa,17beta-diol and D3-Labelled Analogue.
Proceedings of the 17th Cologne Workshop on Dope Analysis 1999 (in press).
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Addendum to the supporting document for all topics related to
the development of Certified Reference Materials (CRMs)
Introduction
Research in support of the development of CRMs is an objective of the Growth
Programme. The modality for this research is a shared cost action on RTD (cf work
programme Competitive and Sustainable Growth). The guidelines for the submission of
the proposal are laid down in the Guides for Proposers.
In addition to the requirements for research on a specific topic that are laid down in the
supporting document*, there are general requirements for the development of CRMs.
These requirements are described below and are in addition to the general requirements
for shared cost actions, as described in the guides for proposers and the model contract.
The requirements have been divided in 2 major parts:
I) Description of the work
This part concentrates on the technical requirements of the research as well as
the reporting requirements in view of the future certification.
II) Implementation of the objectives of the research
The research on CRMs consists of the development of the ability to produce and
certify reference materials that are fit for purpose. The planned exploitation of
the results has to aim at the production and certification of the reference
material(s) in accordance with prevailing international quality standards.
*
The dedicated call system enables the Commission to identify priority topics that have
been forwarded under the call for the expressions of interest. The supporting document
that has been used to evaluate the topic now serves as a reference document to the
proposers for the preparation of the proposal.
DC 1/00/Topic III.29/ Pg 7
I) Description of the work
Research on CRMs consists of the development of the ability to produce and certify
reference materials. The actual production of the CRM is not part of the research but is
part of the exploitation of the result(s).
In the first phase of the research project the certification strategy should be elaborated
(development of the materials and methods for characterisation), in the second phase of
the research project the ability to produce and certify should be demonstrated and
finally the specifications for the final CRM should be set.
The development of a CRM normally consists of 4 major parts:
1) development of the know-how required to produce (and package) the future CRM
2) development of the methods needed for future certification of the material
3) demonstration of the feasibility of the CRM production (production of a small batch
+ mimicking its certification)
4) reporting of detailed and realistic specifications for the future CRM
1. Development of the know-how required to produce (and package) the future
CRM

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specify the sort of material that is most likely to meet both the targets laid down in
the supporting document and be suitably homogeneous, stable, safe to handle, and
acceptable in cost
identify the options available for selecting / preparing such a material
take into account special storage and / or transport requirements existing for certain
types of materials
develop of methods for production / selection and preparation, homogenisation,
stabilisation, protection and packaging of material as required, to enable the
production of a CRM which will be fit for purpose
if a material needs to be altered substantially (e.g. freeze drying), or when it is
artificially spiked with the substance of interest, it is necessary to check if the material
behaves in the same manner as the routine samples when applying the relevant
methods of measurement.
Ensure the availability of a suitable reconstitution procedure if a material will need
reconstitution
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2. Development of the methods needed for future certification of the material
Certification can be performed according to different criteria. The research will
define which certification route, following internationally recognised quality
requirements and standards, will be followed.
Certification on the basis of pure substances

If the CRM is a pure substance the preparation methods should be such that the
presence of impurities are reduced to a minimum. Additionally the substance should
be tested for impurities by carefully selected methods at least in two laboratories.
Certification on the basis of preparation data

When dealing with a synthetic CRM and a quantity which is directly related to (or
depends in a well-established way on) composition, examine if preparation under
metrological conditions and certification on that basis (e.g. using gravimetric data) is
possible
 Identify nevertheless a method of measurement that can be used as an independent
check for large errors
Certification on the basis of measurements - method-dependent quantities

If the quantity to be certified is defined on the basis of a specific (standard) sample
treatment or method of measurement, certification will be based on statistical
processing of results produced by a sufficient number of laboratories (e.g. 12 - 15),
which follow strictly the standard protocol but include all variability which is
allowed by the protocol (do not use common sources of calibrants, reagents, ...
unless these will be available to the future CRM users and prescribed in the
protocol). Such protocol may have to be developed
 Work may be necessary to achieve an acceptable level of reproducibility
Certification on the basis of measurements - method-independent quantities

As error-free, matrix-independent, calibration-independent methods are exceptional,
the basis for certification will usually be an agreement between different methods and
different laboratories, applied under conditions of independence which rule out the
risk of a common systematic error. Methods with a high potential for matrixindependence and/or calibration-independence (e.g. IDMS for determining
polyisotopic elements) should be included if available
 it may be necessary to perform a substantial collaborative method improvement (to
achieve the required level of agreement between methods and between laboratories),
or even research (method development) to achieve the required ability to measure.
Guidance for collaborative method improvement is given in the BCR Guidelines*
*
Guidelines for the production and certification of BCR reference materials, available from the
Commission on request (E. Michel, tel: 32/2-295.11.33, fax: 32/2-295.80.72, email:
ellen.michel@cec.eu.int )
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3. Demonstration of the feasibility of the CRM production

prepare a small batch of material, similar to that intended to be prepared as a CRM,
using the method intended to be used to prepare the actual reference material
 Check the fitness for purpose:
 form, shape or texture of the material; if applicable, contamination (air, moisture,
vapours, dust and dirt, micro-organisms etc.)
 homogeneity
 stability (the analyte / measurand as well as the matrix)
 safety of packaging when exposed to shocks, extreme temperatures and other possible
causes of damage during storage and transport
 ease of opening and use
 lyophilisation and reconstitution procedures
Guidance for homogeneity and stability testing is given in the BCR Guidelines.

Use the test batch to mimic the certification of a future CRM in a measurement
campaign, designed as a certification exercise (see BCR Guidelines)

If the results show that a CRM prepared and certified as in the test round is not good
enough, then the development work (1. or 2. above) must be recommended
4. Reporting of detailed and realistic specifications for the future CRM
The reporting requirements are set in the guidelines for reporting. In addition to the
Final Technical Report the coordinator should prepare an overview of all the results,
as foreseen in the Technical Implementation Plan. In this report the outcome of the
research that sets the boundaries for the future CRM production should be given
preferably in the form of a technical data sheet. It should contain the following
information:
 form / type of samples (with preference for individually packed portions for single
use)
 lyophilisation and reconstitution procedures
 specify bottling, labelling, packing, storage and transport requirements
 identify any legal restrictions on the transport of such material and need for safety
labelling, collect data for material safety data sheets
 magnitude of the quantity of interest (acceptable range)
 acceptable uncertainty on the certified value for an individual sample (this includes
at least: acceptable uncertainty on the mean value for the entire batch, and
acceptable level of inhomogeneity of the batch)
 method requirements (if the CRM is method dependent)
 acceptable magnitude of other quantities
 an estimate of the quantity of CRM that should be produced as well as an estimate
of the production costs.
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II) Implementation of the objectives of the research
As for all shared cost contracts the contractors are obliged to exploit the knowledge
arising from the project. In the case of research on CRMs the contractors are, among
others, obliged to assure that the CRM is produced and certified within a reasonable
time limit (article 10 of Annex II General Conditions of model contract).
The "Technological Implementation Plan" (TIP), which has to be prepared in any
RTD project of the 5th Framework Programme, shall explain the actions planned to
assure that results of the project will be exploited. In case of CRMs this TIP has to
outline the strategy for the CRM production, certification and marketing within a
reasonable time frame, at a reasonable cost and according to internationally
recognised quality requirements. A draft TIP has to be available at the project midterm. Options, ranging from production by the consortium to production by a third
party or the Commission, will be explored in consultation with the Commission
Services.