Quantification of 17 Endogenous and Exogenous Steroidal Hormones in Equine and Bovine Blood for Doping Control with UHPLC-MS/MS
Abstract
:1. Introduction
2. Results and Discussion
2.1. Setup of the Chromatographic and Mass Analyzer Conditions
2.2. Method Validation
2.3. Evaluation of the Stability of Steroids in Glass and Plastic
2.4. Precision and Linearity
2.5. Accuracy
2.6. Recovery
2.7. Matrix Effect
2.8. Specificity
2.9. Application and Testing of the Developed Method to Equine and Bovine Blood
3. Materials and Methods
3.1. Disposable Chemicals and Materials
3.2. Collection of Equine Blood
3.3. Preparation of Steroids-Free Blood and Sample Preparation
3.4. UHPLC-ESI-MS/MS
3.5. Method Validation Settings
3.6. Internal Standards
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Compound | Abbreviation | Time Window (Minute) | Precursor Ion a (m/z) | Product Ion (m/z) | Fragmentor (V) | Collision Energy (V) | Dwell Time (Milli- Second) |
---|---|---|---|---|---|---|---|
Dexamethasone Sodium Phosphate | Desa-NA-P | 2.0–3.7 | 473.11 | 435.2 355.2 | 97 | 8 | 200 |
Cortisol | CORT | 2.0–3.7 | 363.01 | 121.1 327.2 | 136 | 24 | 200 |
Aldosterone | ALDO | 2.0–3.7 | 361.41 | 343.2 315.2 | 116 | 16 | 200 |
Pregnenolone | PRE | 2.0–3.7 | 361.41 | 343.2 105.0 | 87 | 4 | 200 |
Methylprednisolone | ME-PRE | 3.7–5.5 | 375.01 | 357.2 323.2 | 92 | 4 | 180 |
11-Deoxycortisol | 11-DOC | 3.7–5.5 | 347.51 | 109.10 97.2 | 141 | 32 | 180 |
Corticosterone | COCO | 3.7–5.5 | 347.01 | 329.2 329.2 | 111 | 12 | 180 |
Stanozolol | STA | 3.7–5.5 | 329.51 | 81.10 95.10 | 170 | 50 | 180 |
Boldenone | BOL | 3.7–5.5 | 287.41 | 121.00 135.00 | 107 | 24 | 180 |
Nandrolone | NAN | 3.7–5.5 | 275.10 | 109.10 82.90 | 100 | 28 | 180 |
Dexamethasone isonicotinate | DESA-ISO | 5.5–6.8 | 498.61 | 47.20 124.0 | 121 | 8 | 200 |
11-Deoxycorticosterone | 11-DCC | 5.5–6.8 | 331.01 | 97.10 109.1 | 117 | 20 | 200 |
Dihydrotestosterone | DHT | 6.8–9.0 | 273.10 | 255.30 147.0 | 159 | 15 | 200 |
Testosterone | TESTO | 5.5–6.8 | 289.01 | 97.10 109.1 | 131 | 20 | 100 |
Androstenedione | ANDD | 5.5–6.8 | 287.01 | 97.10 109.1 | 131 | 24 | 200 |
Dehydroepiandrosterone | DHEA | 5.5–6.8 | 271.01 | 253.10 253.2 | 92 | 8 | 200 |
Androsterone | ANDRO | 6.8–9.0 | 291.41 | 273.20 255.2 | 78 | 4 | 200 |
Testosterone–d3 | d3-TESTO | 5.5–6.8 | 292.00 | 97.00 | 135 | 25 | 120 |
Compound | LOD | LOQ | C1 | CM | C2 | CU | U1 |
---|---|---|---|---|---|---|---|
ppb | |||||||
DESA-NA-P | 0.333 | 1.00 | 10 | 50 | 100 | 500 | 1000 |
COR | 0.183 | 0.55 | 11 | 55 | 110 | 550 | 1100 |
ALDO | 0.183 | 0.55 | 11 | 55 | 110 | 550 | 1100 |
PRE | 0.033 | 0.10 | 10 | 50 | 100 | 500 | 1000 |
ME-PRE | 0.067 | 0.20 | 20 | 100 | 200 | 1000 | 2000 |
11-DOC | 0.037 | 0.11 | 11 | 55 | 110 | 550 | 1100 |
COCO | 0.167 | 0.50 | 10 | 50 | 100 | 500 | 1000 |
STA | 0.033 | 0.10 | 10 | 50 | 100 | 500 | 1000 |
BOL | 0.167 | 0.50 | 10 | 50 | 100 | 500 | 1000 |
NAN | 0.333 | 1.00 | 10 | 50 | 100 | 500 | 1000 |
DESA-ISO | 0.023 | 0.069 | 6.9 | 34.5 | 69 | 345 | 690 |
11-DCC | 0.037 | 0.11 | 11 | 55 | 110 | 550 | 1100 |
TESTO | 0.037 | 0.05 | 11 | 55 | 110 | 550 | 1100 |
ANDD | 0.333 | 1.00 | 10 | 50 | 100 | 500 | 1000 |
DHEA | 1.833 | 5.50 | 11 | 55 | 110 | 550 | 1100 |
ANDRO | 0.733 | 2.20 | 22 | 110 | 220 | 1100 | 2200 |
DHT | 1.833 | 5.50 | 11 | 55 | 110 | 550 | 1100 |
DAY 1 | DAY 2 | |||
---|---|---|---|---|
Loss in Plastic (%) | Loss in Glass (%) | Loss in Plastic (%) | Loss in Glass (%) | |
COR | <1 | 99.63 | <2% | >99.90 |
ALDO | <1 | 94.29 | <2% | >99.90 |
11-DOC | <1 | 99.94 | <2% | >99.90 |
COCO | <1 | 99.76 | <2% | >99.90 |
11-DCC | <1 | 99.88 | <2% | >99.90 |
PRE | <1 | 99.97 | <2% | >99.90 |
ANDRO | <1 | 99.52 | <2% | >99.90 |
TESTO | <1 | 99.85 | <2% | >99.90 |
ANDD | <1 | 99.83 | <2% | >99.90 |
DHT | <1 | 99.12 | <2% | >99.90 |
DHEA | <1 | 99.65 | <2% | >99.90 |
Compound | Limit of Quantification | Medium Concentration | CU | U1 | Linearity | ||||
---|---|---|---|---|---|---|---|---|---|
Intra-Day (CV%) | Inter-Day (CV%) | Intra-Day (CV%) | Inter-Day (CV%) | Intra-Day (CV%) | Inter-Day (CV%) | Intra-Day (CV%) | Inter-Day (CV%) | R2 | |
DESA-NA-P | 10.86 | 14.16 | 6.74 | 7.58 | 2.62 | 4.12 | 1.02 | 1.48 | 0.99326 |
COR | 16.47 | 18.49 | 9.50 | 10.35 | 6.49 | 8.24 | 3.56 | 4.49 | 0.99939 |
ALDO | 14.45 | 17.52 | 6.95 | 8.40 | 6.79 | 7.63 | 6.07 | 6.85 | 0.99995 |
PRE | 15.84 | 17.41 | 10.07 | 10.91 | 2.78 | 3.69 | 1.18 | 2.37 | 0.99922 |
ME-PRE | 15.93 | 17.45 | 15.04 | 17.85 | 6.32 | 18.40 | 3.26 | 8.12 | 0.99912 |
11-DOC | 17.10 | 17.87 | 6.63 | 7.72 | 6.93 | 7.53 | 5.48 | 6.73 | 0.99954 |
COCO | 10.34 | 14.57 | 7.97 | 10.63 | 1.56 | 4.51 | 0.48 | 2.28 | 0.99991 |
STA | 13.25 | 14.95 | 5.73 | 9.11 | 3.15 | 5.24 | 1.18 | 4.20 | 0.99941 |
BOL | 15.83 | 17.89 | 11.85 | 16.66 | 3.71 | 18.05 | 2.03 | 6.97 | 0.99658 |
NAN | 17.35 | 18.62 | 7.12 | 14.75 | 5.49 | 8.13 | 2.26 | 5.83 | 0.99861 |
DESA-ISO | 17.43 | 18.37 | 9.77 | 18.99 | 7.88 | 9.65 | 4.49 | 6.41 | 0.99981 |
11-DCC | 16.60 | 18.07 | 16.06 | 18.78 | 12.21 | 18.27 | 7.21 | 9.41 | 0.99953 |
TESTO | 11.76 | 18.17 | 11.40 | 16.89 | 6.35 | 15.06 | 4.39 | 5.72 | 0.99841 |
ANDD | 10.08 | 16.66 | 14.04 | 18.46 | 8.56 | 9.88 | 6.29 | 8.06 | 0.99987 |
DHEA | 14.21 | 17.84 | 9.99 | 18.64 | 4.67 | 8.49 | 2.18 | 3.26 | 0.99970 |
ANDRO | 16.81 | 17.77 | 5.34 | 8.23 | 4.30 | 4.80 | 2.93 | 3.21 | 0.99940 |
DHT | 13.97 | 17.10 | 9.07 | 9.19 | 6.31 | 6.57 | 4.29 | 5.30 | 0.99997 |
Exogenous Steroids | Endogenous Steroids | ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Animal | DESA-NA-P | PRE | ME-PRE | STA | BOL | NAN | DESA-ISO | 11-DCC | TESTO | ANDD | DHEA | ANDRO | DHT | COR | ALDO | 11-DOC | COCO |
ppb | |||||||||||||||||
A 01 | 0.152 | - | 0.470 | 0.395 | - | 0.237 | 0.245 | - | 0.145 | 0.481 | - | - | - | 4.207 | 1.038 | 0.190 | 0.319 |
A 02 | 0.134 | - | 0.478 | 0.397 | - | - | 0.243 | - | 0.142 | 0.482 | - | - | - | 0.668 | 1.095 | - | 0.330 |
A 03 | - | 0.444 | 0.558 | 0.392 | - | - | 0.242 | 0.183 | 0.138 | 0.497 | - | - | - | 3.631 | - | 0.179 | 0.288 |
A 04 | - | - | - | 0.419 | - | - | 0.247 | - | - | 0.478 | - | - | - | 6.578 | 0.846 | 0.201 | 0.587 |
A 05 | 0.130 | - | - | 0.393 | - | - | 0.246 | - | - | 0.484 | - | - | - | 2.363 | - | - | - |
A 06 | 0.100 | - | - | 0.390 | - | - | 0.242 | 0.178 | - | 0.461 | - | - | - | 3.247 | - | 0.180 | - |
A 07 | - | - | - | - | - | - | 0.241 | 0.187 | - | 0.497 | - | - | - | - | - | - | |
A 08 | - | - | - | - | - | - | - | 0.194 | 0.142 | 0.436 | - | - | - | 1.670 | - | - | - |
A 09 | 0.071 | - | - | - | - | - | - | - | - | 0.479 | - | - | - | 0.444 | - | - | 0.334 |
A 10 | - | - | - | 0.462 | - | - | 0.250 | 0.202 | 0.146 | 0.500 | - | - | - | 0.465 | - | - | 0.351 |
A 11 | - | - | - | - | - | - | 0.241 | - | 0.137 | 0.532 | - | - | - | 2.082 | - | - | 0.315 |
A 12 | 0.094 | - | - | - | - | - | 0.243 | - | - | 0.571 | - | - | - | 0.435 | - | - | 0.393 |
A 13 | 0.170 | - | - | 0.393 | - | - | 0.244 | - | - | 0.561 | - | - | - | 1.956 | - | - | 0.375 |
A 14 | 0.121 | - | - | 0.387 | - | - | 0.247 | - | - | 0.443 | - | - | - | 3.935 | - | - | 0.491 |
A 15 | 0.196 | 0,472 | - | - | 0.064 | 0.291 | 0.258 | 0.229 | - | 0.515 | - | - | - | 3.486 | - | 0.197 | 0.383 |
B 01 | - | - | 0.87 | - | 0.02 | - | - | 0.39 | - | - | - | - | 0.98 | 5.96 | - | 0.53 | 0.49 |
B 02 | - | - | - | - | - | - | - | 0.39 | 0.23 | 0.60 | - | - | 1.87 | 1.29 | 0.70 | - | 0.40 |
B 03 | - | - | - | - | - | - | 0.47 | 0.39 | 0.22 | - | - | - | 1.95 | 0.99 | - | - | 0.44 |
B 04 | - | - | 0.83 | - | - | - | - | - | 0.22 | - | - | - | 1.06 | 5.62 | - | - | 0.41 |
B 05 | - | - | 0.95 | - | - | - | 0.47 | - | 0.21 | - | - | - | 3.26 | 1.67 | - | - | - |
B 06 | - | - | 0.86 | - | - | - | 0.47 | - | - | - | - | - | - | 1.35 | - | - | - |
C 01 | - | - | 1.33 | - | - | - | 0.39 | - | 0.31 | 0.62 | - | - | - | 10.00 | 0.79 | 0.48 | 0.60 |
C 02 | - | - | 1.05 | - | - | - | 0.40 | - | - | 0.64 | - | - | - | 3.61 | - | 0.45 | 0.54 |
C 03 | - | - | 1.07 | 0.27 | - | - | 0.40 | - | - | - | - | - | - | 1.17 | - | - | - |
C 04 | - | - | 1.08 | - | - | - | - | - | 0.31 | - | - | - | - | 2.36 | - | - | 0.54 |
C 05 | - | - | 1.08 | - | - | - | 0.40 | - | - | - | - | - | - | 0.88 | - | - | 0.45 |
C 06 | - | - | 1.02 | - | - | - | 0.40 | - | - | 0.62 | - | - | - | 4.96 | - | 0.46 | 0.58 |
D 01 | - | - | 0.876 | - | 0.024 | 0.531 | - | 0.397 | - | - | - | - | 0.986 | 5.967 | - | 0.531 | 0.497 |
D 02 | - | - | - | - | - | - | - | 0.396 | 0.236 | 0.604 | - | - | 1.872 | 1.296 | 0.702 | - | 0.401 |
D 03 | - | - | - | - | - | - | 0.3 | 0.394 | 0.221 | - | - | - | 0.959 | 0.992 | - | - | 0.448 |
D 04 | - | - | 0.834 | - | - | - | - | - | 0.226 | - | - | - | 1.060 | 5.62 | - | - | 0.418 |
D 05 | - | - | 0.953 | - | - | - | - | - | 0.219 | - | - | - | - | 1.671 | - | - | - |
D 06 | - | - | 0.866 | - | - | - | 0.4 | - | 0.213 | - | - | - | - | 1.356 | - | - | - |
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Caprioli, G.; Genangeli, M.; Mustafa, A.M.; Petrelli, R.; Ricciutelli, M.; Sagratini, G.; Sartori, S.; Laus, F.; Vittori, S.; Cortese, M. Quantification of 17 Endogenous and Exogenous Steroidal Hormones in Equine and Bovine Blood for Doping Control with UHPLC-MS/MS. Pharmaceuticals 2021, 14, 393. https://doi.org/10.3390/ph14050393
Caprioli G, Genangeli M, Mustafa AM, Petrelli R, Ricciutelli M, Sagratini G, Sartori S, Laus F, Vittori S, Cortese M. Quantification of 17 Endogenous and Exogenous Steroidal Hormones in Equine and Bovine Blood for Doping Control with UHPLC-MS/MS. Pharmaceuticals. 2021; 14(5):393. https://doi.org/10.3390/ph14050393
Chicago/Turabian StyleCaprioli, Giovanni, Michele Genangeli, Ahmed M. Mustafa, Riccardo Petrelli, Massimo Ricciutelli, Gianni Sagratini, Stefano Sartori, Fulvio Laus, Sauro Vittori, and Manuela Cortese. 2021. "Quantification of 17 Endogenous and Exogenous Steroidal Hormones in Equine and Bovine Blood for Doping Control with UHPLC-MS/MS" Pharmaceuticals 14, no. 5: 393. https://doi.org/10.3390/ph14050393