Fig. 1.
Fig. 2.
LC-MS/MS ion acquisition parameters used for the identification of 17β-clostebol (17β-CLOS), 17α-clostebol (17α-CLOS) and 4-chloroandrostenedione (CLAD)
| Compound | MRM transition (m/z) | Collision energy(V) | Declustering potential(V) | Entrance potential(V) | Collision cell exit potential (V) | ± standard deviation | Samples fulfilling the confirmation criteria (%) | |
|---|---|---|---|---|---|---|---|---|
| CCα – 2.50 μg L−1 | ||||||||
| 17β-CLOS | 323.2 > 143.1a | 32 | - | - | ||||
| 323.2 > 131.0 | 33 | 70 | 10 | 24 | 0.532 ± 0.028 | 100.0 | ||
| 323.2 > 95.2 | 52 | 0.048 ± 0.009 | 99.1 | |||||
| 17α-CLOS | 323.3 > 143.0a | 40 | - | - | ||||
| 323.3 > 131.0 | 31 | 70 | 11 | 20 | 0.381 ± 0.021 | 100.0 | ||
| 323.3 > 95.0 | 53 | 0.141 ± 0.018 | 97.2 | |||||
| CLAD | 321.0 > 143.0a | 31 | - | - | ||||
| 321.0 > 131.0 | 32 | 70 | 10 | 20 | 0.551 ± 0.015 | 100.0 | ||
| 321.0 > 95.2 | 53 | 0.056 ± 0.008 | 99.1 | |||||
| 17β-CLOS-D3 | 326.4 > 143.0a | 31 | 75 | 9 | 22 | - | - | - |
Validation parameters of the liquid chromatography–tandem mass spectrometry method for the determination of 17β-clostebol (17β-CLOS), 17α-clostebol (17α-CLOS) and 4-chloroandrostenedione (CLAD) in animal urine
| Parameter | Number of samples | Spiking level (μg L−1) | Compound | ||
|---|---|---|---|---|---|
| 17β-CLOS | 17α-CLOS | CLAD | |||
| Mean concentration (μg L−1)/Apparent recovery (%) | n = 21 | 0.25 | 0.24/95.1 | 0.24/96.8 | 0.25/100.8 |
| 0.50 | 0.54/107.4 | 0.52/103.2 | 0.53/106.4 | ||
| 0.75 | 0.77/102.9 | 0.75/100.5 | 0.78/104.4 | ||
| n = 9 | 1.00 | 0.97/96.9 | 0.99/98.5 | 0.96/96.3 | |
| 2.50 | 2.49/99.6 | 2.50/100.1 | 2.50/100.0 | ||
| n = 10 | CCα | 0.31/112.4 | 0.27/94.5 | 0.32/99.1 | |
| Repeatability (sr, μg L−1/RSD, %) | n = 21 | 0.25 | 0.01/4.2 | 0.01/5.3 | 0.02/8.0 |
| 0.50 | 0.03/6.4 | 0.03/5.8 | 0.02/4.3 | ||
| 0.75 | 0.06/7.3 | 0.04/5.4 | 0.05/6.1 | ||
| n = 9 | 1.00 | 0.05/5.0 | 0.05/4.8 | 0.03/3.5 | |
| 2.50 | 0.04/1.5 | 0.11/4.3 | 0.10/3.9 | ||
| n = 10 | CCα | 0.03/9.1 | 0.03/10.0 | 0.03/10.3 | |
| Within-lab reproducibility (sR, μg L−1/RSD, %) | n = 21 | 0.25 | 0.02/6.3 | 0.02/8.0 | 0.03/12.3 |
| 0.50 | 0.05/9.7 | 0.05/8.8 | 0.03/6.4 | ||
| 0.75 | 0.08/10.9 | 0.06/8.0 | 0.07/9.2 | ||
| n = 9 | 1.00 | 0.07/7.6 | 0.07/7.2 | 0.05/5.2 | |
| 2.50 | 0.06/2.3 | 0.16/6.5 | 0.15/5.9 | ||
| Decision limit (CCα, μg L−1) | 0.28 | 0.29 | 0.32 | ||
| Expanded measurement uncertainty (U, k = 2, %) | 0.25 | 13 | 16 | 24 | |
| 0.50 | 18 | 17 | 13 | ||
| 0.75 | 21 | 16 | 17 | ||
| 1.00 | 15 | 15 | 11 | ||
| 2.50 | 9 | 13 | 12 | ||
| Matrix effect (%) | 14 | 14 | 19 | ||
| Ruggedness | A/a factors | tA/a | 0.0600 | 0.5250 | 1.8583 |
| B/b factors | tB/b | 0.3650 | 0.0550 | 1.1417 | |
| C/c factors | tC/c | 0.3950 | 0.2400 | 0.3000 | |
| D/d factors | tD/d | 0.0850 | 0.0250 | 0.3833 | |
| E/e factors | tE/e | 0.3950 | 0.0200 | 0.1917 | |
| F/f factors | tF/f | 0.3200 | 0.2200 | 0.7750 | |
| G/g factors | tG/g | 0.1200 | 0.1600 | 0.5500 | |
| All factors | SI/i | 0.01 | 0.01 | 0.02 | |
| Standard calibration curve | Slope ± sb | 1.0014 ± 0.2316 | 0.8378 ± 0.3434 | 0.8479 ± 0.2464 | |
| y-intercept ± sa | -0.0808 ± 0.0658 | -0.1093 ± 0.0530 | -0.0767 ± 0.0390 | ||
| Correlation coefficient | 0.9975 | 0.9935 | 0.9969 | ||
| Standard error | 0.1819 | 0.2462 | 0.1714 | ||
| Matrix-matched calibration curve | Slope ± sb | 0.8586 ± 0.2401 | 0.7704 ± 1.2009 | 0.7997 ± 0.2704 | |
| y-intercept ± sa | 0.0293 ± 0.0263 | -0.0007 ± 0.0444 | 0.0131 ± 0.0208 | ||
| Correlation coefficient | 0.9992 | 0.9999 | 0.9997 | ||
| Standard error | 0.0324 | 0.0121 | 0.0188 | ||