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Sample Prep: Performance Enhancing Drugs

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    Purpose: To introduce students to the processes required to prepare samples for various separation/detection systems.  This includes sample purification, concentration and derivatization.

    Learning Outcomes:

    By the end of the assignment students will be able to:

    • Identify interfering matrix components
    • Be familiar with different extraction processes
    • Understand the derivatization process for increasing sample volatility

    The samples that will be collected from athletes will not be pure steroids in pure liquid solutions.  There will invariably be other components to the sample and these will vary depending on the sample itself (hair, blood or urine).  Urine is the most commonly used sample for steroid testing, as it can be easily obtained from an athlete and contains both steroids and related compounds/metabolites.  Because of the complexity of urine, a separation step is necessary prior to the detection of any steroids.  Gas chromatography, coupled with a mass spectrometer detection system (GC-MS) is most frequently used in the analysis of steroid samples.

    1. What compounds do you expect to find in urine? Do you think it is possible to directly inject urine samples into a GC-MS?
    2. How would you go about removing unwanted salts and proteins from urine samples (or, alternatively, removing the steroids from the salts and proteins)?
    3. Given that the World Anti-Doping Agency classifies urine samples with greater than 200 ng/mL of urinary testosterone to be evidence of doping, what concentration of an exogenous steroid (e.g. nandrolone) would you expect to see in the urine sample of a doping athlete?
    4. What is the boiling point of testosterone? Is this compound volatile enough to be analyzed by GC?
    5. What other analytical method could be used to detect the presence of steroids in urine? What factors must be considered in the selection of an alternate method?  Would the sample prep for this method be different from that needed for GC-MS?
    6. An alternative to GC-MS for the analysis of steroids is the use of LC-MS. Though LC-MS is more compatible with aqueous samples why is it less frequently used for the analysis of steroids?
    7. What can be done to improve the ionization efficiency of steroids to allow for better analysis by LC-MS?


    The following urine sample preparation method is reported in U. Mareck, M. Thevis, S. Guddat, A. Gotzmann, M. Bredehöft, H. Geyer, W. Schänzer, Comprehensive sample preparation for anabolic steroids, gluco- corticosteroids, beta-receptor blocking agents, selected anabolic androgenic steroids and buprenorphine in human urine, in: W. Schänzer, H. Geyer, A. Gotzmann, U. Mareck (Eds.), Recent Advances in Doping Analysis (12), Sport und Buch Strauß, Köln, 2004, pp. 65–69.


    1. Use 3 mL of urine if the density is greater than 1.01 g/cm3, 6 mL if the density is less than 1.01 g/cm3.
    2. To the urine sample add 60 µL of known quantities of internal standards.
    3. Add 750 µL of phosphate buffer (0.8 M, pH 7.0) and 25 µL of β-glucuronidase from E. Coli - heat the mixture for 1 hour at 50°C.
    4. Add 750 µL of 20% K2CO3/KHCO3 (1:1, pH ~9.5).
    5. Add 6 mL of t-butyl methyl ether (TBME) - shake for 5 minutes and centrifuge for 5 minutes at 2500 rpm.
    6. Collect 4 mL of the organic layer and evaporate to dryness.
    7. To the dried sample, add 100 µL of liquid N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide, and ethanethiol (1000:2:5, v/w/v) - only 50 µL of the mixture are used if the specific gravity of the sample was less than 1.005 g/cm3. Heat the solution for 20 minutes at 60°C.
    8. Once the above steps have been completed inject 3 µL samples into the GC-MS for analysis.
    1. Why is a greater volume of urine used for the samples of lower density?
    2. What is the purpose of the internal standard? What would be a suitable internal standard for this analysis?
    3. What other standardizations could be used? What is the principal advantage of an internal standard in this analysis?
    4. What is the purpose of adding the β-glucuronidase to the urine sample?
    5. Why was the sample buffered to pH 7.0 in step 3 then altered to pH ~9.5 in step 4?
    6. What are some of the limitations of the solvent extraction process?
    7. What alternative extraction process(es) could be used?
    8. If the recovery of a Stanozolol standard in the urine was found to be 31%, will this method be acceptable for the analysis of steroids?
    9. A urine sample, with a density of 1.03 g/mL is to be analyzed by the procedure outlined above. A 20 ng/mL solution of Stanozolol is used as the internal standard. Assuming that there is no loss of the internal standard during the processing, what will be the final concentration of Stanozolol when the sample is ready to be injected into the GC-MS?   What would this amount be if the recovery was only 31%?
    10. For both scenarios above, what mass of Stanozolol is injected into the GC-MS?
    11. What is the purpose of the addition of MSFTA to the extracted sample?
    12. Why was MSFTA not added to the samples prior to step 7?
    13. Where, in the structure of the steroids listed below, would the derivatization with MSFTA take place?


    14. Given that higher molecular weight compounds typically have higher boiling points, explain how the derivatization of a steroid with MSTFA, which increases its mass, will result in a reduction of the boiling point temperature for the molecule.



    As synthetic chemists continue to develop novel steroids for medical purposes, detecting the use of steroids becomes more challenging.

    1. How does an analyst identify a compound that is completely novel?

    In one instance, analysts were presented with a used syringe that was suspected of containing a new synthetic steroid.  Analyses ultimately revealed that this syringe contained a new “designer drug”, a steroid known as Tetrahydrogestrinone (THG).

    Rapid Commun. Mass Spectrom. 2004; 18: 1245–1249

    1. If presented with a used syringe to analyze for steroid residue, what sample preparation steps would you take?
    2. Is the quantification of the steroid important in this analysis?
    3. Are there contaminants that could be problematic in this analysis?

    Contributors and Attributions

    This page titled Sample Prep: Performance Enhancing Drugs is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by Contributor.

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