100 Relative abundance
10 20 30 40 50 60 70 80 90
143.2
136.
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg
Thr y2
129.2 217.3 120.2
Gly Phe Phe Tyr Thr Lys Pro [M+5H]5+ = 1162.4
197.2
249.3 270.3 345.3 489.3 598.4 626.3 739. 900.5 1086.4 1155.4
10 20 30 40 50 60 70 80 90 100 110 1200 m/z
Figure 3. Mass spectral information on Humalog LisPro, a synthetic derivative of human insulin
There are several options to approach the problem of unknown, modified steroidal agents, and the one including mass spectrometry focuses on conserved steroid nuclei. Those generate common product ions under ESI/CID conditions, and screening for steroid-typical fragments allows to ‘profile’ urine samples, for example, if unusual signals are found further studies on the analyte behind are required to prove whether it is a new steroid derivative or not. [2]
BM: Athletes are frequently led by their coach/trainers in to taking the performance enhancing drugs in cocktails. These cases must be difficult to analyse. Are their any high profile success stories where you have identified these cocktails?
MT: The most prominent case of designer steroid detection was probably the so-called BALCO affair. The compound tetrahydrogestrinone (THG) was discovered and identified by the doping control laboratory in Los Angeles, CA, which was provided with a syringe containing the, at that time unknown, substance THG. [3] Based on the knowledge that this compound exists, detection assays were adjusted and numerous elite athletes were convicted of having used the designer steroid.
Another finding was remarkable and concerned the steroid methyltrienolone (or methyltrenbolone), a drug that was developed in the 1960s and immediately discontinued due to severe side effects, particularly liver toxicity. It was never approved, neither for veterinary nor for human use; however, it was detected in 11 elite weight lifters’ urine samples in 2008 and further to that in an Olympic doping control sample in Beijing. [4]
BM: What are the next generation drugs that the cheats are using and are you already able to tackle this trend?
MT: There are numerous new drug candidates that possess potential for misuse in sports. These include for instance so-called selective androgen receptor modulators (SARMs), which could be referred to as the anabolic agents of the future. Although structurally different from steroids, as
can be seen in Figure 4, they selectively stimulate the steroid receptor and have several beneficial effects for the ageing person; in addition, they are certainly drugs of interest for cheating athletes as a gain in muscle mass and performance is very likely. Other therapeutics developed to treat the metabolic syndrome such as GW1516 might be misused in sports due to their ability to stimulate fat utilisation and to mimic exercise on a genetic level. In both cases, methods to detect these drugs and/or metabolites have been established and are further optimised to ensure utmost retrospective. [5]
BM: There appears to be room for much more research into methods of testing and screening. What are you currently working on?
MT:We have focused our work on various new classes of compounds such as releasing hormones, for example, luteinizing hormone releasing hormone, growth hormone releasing hormone), hypoxia-inducible factor (HIF)-stabilisers, and drugs stimulating mitochondrial biogenesis (e.g., AICAR). These include the peptide hormones LH-RH (or gonadorelin) 6 and Geref (sermorelin), the prolyl hydroxylase inhibitor FG- 2216, and the endogenous compound AICAR.5 LH-RH triggers the production and release of endogenous testosterone and might increase plasma levels of testosterone that a) increases athletic
NC CF3 O2N CF3
N H
N H
O O HO CH3 1 O O HO CH3 2 F CF3
N H
O2N F CF3
N H
O2N O O
HO CH3 3
O O
HO CH3 4
N H
O H C3 Cl
pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
b b3 2 y5 100 y3 328.25 Relative Abundance (%) b3 435.00 b4 522.17 476.83 171.92 y2 200 249.08 b2 552.75 300.33 300 393.58 614.50 400 500 600 m/z 700 800 900 1000 1100 y6 a7 685.08 b5 1161.67 1200 827.33 y7
b5-H2O 748.42 667.00
582.92 b7 855.25 y8 934.42 498.33
b b5 4
b b8 7
pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly- y y2
y y7 8 y y5 6 b8 1011.33 3
Figure 5. Gonadorelin (LH-RH) and its analysis from urine (3 pg/mL).
is there still to be made in the hyphenated sequence you have used? Is the sensitivity and ruggedness of your methods enough to keep you ahead of the cheats?
MT: Chromatographic-mass spectrometric methods will still be the most important tool in doping control laboratories but might be complemented by other techniques. With the increasing number of analytes, faster analyses without loss of sensitivity and specificity would be desirable, and the instrumental developments seem to support these needs with ultrahigh performance LC and constantly reduced duty cycle times of modern mass spectrometers. This should allow us to efficiently conduct our research and routine doping controls.
REFERENCES
1. Thevis M, Thomas A, Schänzer W. Mass spectrometric determination of insulins and their degradation products in sports drug testing. Mass Spectrometry Reviews 2008;27: 35-50.
5.0 0. 5.5 4. 5. 5. 5. 5. 5. 2.8 0. 4. 2.0 0. 4. 84 0
SARM 1 381 SARM 3 401 SARM 2 417
5. 5. 5. 5. 5. 5. 5. 5. 4. 5. 5. 5. 5. ISTD 429 - 6. 6. 61 5. 6. 6. 6. 63 5. 6. 6. 6. 6. 6. 6.6 5. 6. Time, 6. 6. 6. 6. 7.7.7.
Figure 4. Model aryl-propionamide derived SARMs
performance, and b) interferes with detection assays developed to test for administered synthetic testosterone. The analysis of LH-RH in urine is shown in Figure 5. Geref stimulates the secretion of human growth hormone, which is also desirable for athletes, FG- 2216 mimics hypoxia and causes an increased production of erythropoietin with subsequently increased amounts of erythrocytes, and AICAR was shown to improve endurance in laboratory rodents due to the elevated amount of mitochondria in skeletal muscle tissue.
BM: How do you see the
spectroscopic methods of detection advancing in, say, the next 3 – 5 years? What room for improvement
3. Catlin DH, Sekera MH, Ahrens BD, Starcevic B, Chang Y-C, Hatton CK. Tetrahydrogestrinone: discovery, synthesis, and detection in urine. Rapid Communications in Mass Spectometry 2004;18: 1245-1249.
4. Thevis M, Guddat S, Schanzer W. Doping control analysis of trenbolone and related compounds using liquid chromatography- tandem mass spectrometry. Steroids 2009;74: 315-321.
5. Thevis M, Thomas A, Kohler M, Beuck S, Schänzer W. Emerging drugs: mechanism of action, mass spectrometry and doping control analysis. Journal of Mass Spectrometry 2009;44: 442-460.
6. Thomas A, Geyer H, Kamber M, Schänzer W, Thevis M. Mass spectrometric determination of gonadotrophin-releasing hormone (GnRH) in human urine for doping control purposes by means of LC-ESI-MS/MS. Journal of Mass Spectrometry 2008;43: 908-915.
7. 7. 7. 6. 6. 7. 7. 7. 6. 6. 6.7. 7. 7.
Urine spiked to 1 ng/mL of SARMs
2. Thevis M, Geyer H, Mareck U, Schänzer W. Screening for unknown synthetic steroids in human urine by liquid chromatography- tandem mass spectrometry. Journal of Mass Spectrometry 2005;40: 955-962.
Spectroscopy Focus
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