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LAAN-A-LM-E069

Application Liquid Chromatography Mass Spectrometry

News Analysis of Steroids and NSAIDs Using the Shimadzu

LCMS-8050 Triple Quadrupole Mass Spectrometer
No.C98

With performance enhancing drug use considered (NSAIDs) are drugs used to treat pain and inflammation
contrary to fair play, along with the adverse effects they as well as fever, and although they are not specified as
may have on the health and social welfare of athletes, prohibited drugs, their abuse by athletes is being
sports doping testing is increasing and has been viewed as a problem due to their side effects.
conducted according to the provisions of WADA (World Since doping tests provide information for making
Anti-Doping Agency). critical decisions that actually affect athletes’ lives,
Drugs that are registered as prohibited substances accuracy at the time of testing, as well fairness, are
mainly fall into the categories of anabolic steroids (AAS) necessary. In this Application News, we introduce an
used primarily for building muscle strength, steroidal accurate identification method for typical steroidal and
anti-inflammatory drugs for their anti-inflammatory and non-steroidal anti-inflammatory drugs using multiple
immunosuppressive effects, and narcotic and designer reference ion ratios, in addition to an example of high-
drugs. Also, non-steroidal anti-inflammatory drugs sensitivity measurement.

n MRM Analysis of Standards and Matrix-Matched
Calibration Curves
We conducted MRM measurement of a mixed standard their respective LOQs. Table 1 shows minimum and
solution consisting of 14 typical steroids and non- maximum concentrations used for generating the
steroidal anti-inflammatory drugs. Fig. 1 shows the respective calibration curves. The lower limits of
MRM chromatograms obtained using the mixed quantitation ranged from 10 to 100 pg/mL (20 – 200 fg
standard solution (each component at 50 ng/mL), and on column), and excellent linearity was obtained over a
Fig. 2 shows MRM chromatograms obtained from wide range of more than 3 orders of magnitude for
analysis of typical compounds at concentrations near each substance.

Table 1 Calibration Curve Min. /Max. Concentrations
Max.
Min.
4500000 1 2 8. Zeranol Compounds Conc. Conc.
9. Ketoprofen
4000000 3 4 10. Testosterone Clenbuterol 0.01 10
3500000 6 9 11. Clostebol Prednisolone 0.05 20
12. Diclofenac
5 8 13. Melengestrol acetate Hydrocortisone acetate 0.1 50
3000000 14. Closantel
7 Methylprednisolone 0.5 50
2500000 10 11 Dexamethasone 0.5 50
12
2000000 Ћ , Ќ Trenbolone 0.1 50
1. Clenbuterol 13
1500000 2. Prednisolone Zeranol 0.1 50
3. Hydrocortisone acetate Ketoprofen 0.05 50
1000000 4. Methylprednisolone 14 Testosterone 0.05 10
5. Dexamethasone
500000 6. β-Trenbolone Clostebol 0.05 50
0 7. α-Trenbolone Diclofenac 0.01 50
Melengestrol acetate 0.05 50
3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 min Closantel 0.01 10
Unit: ng/mL
Fig. 1 Chromatograms of Steroids and NSAIDs
(×1,000) (×1,000) (×100) (×100)
Clenbuterol 1.25 α-, β-Trenbolone Melengestrol Closantel
1.50 10 pg/mL 1.00 50 pg/mL 4.0 acetate 7.5 10 pg/mL
50 pg/mL
1.25
1.00 0.75 3.0 5.0
0.75 0.50 2.0
0.50 1.0 2.5
0.25 0.25
0.00 0.00 0.0 0.0
3.0 3.5 7.5 8.0 8.5 9.5 10.0 11.5 12.0 12.5

Fig. 2 MRM Chromatograms Near the LOQ of Typical Compounds