Variation of Sequential Ligandrol (LGD-4033) Metabolite Levels in Routine Anti-Doping Urine Samples Detected with or without Other Xenobiotics
Abstract
:1. Introduction
2. Results and Discussion
2.1. LGD-4033 Metabolites
2.2. Real Samples and Other Prohibited Substances in Sport
2.3. Analysis of Dietary Supplements for the Purposes of Disciplinary Proceedings
3. Materials and Methods
3.1. Chemical and Materials
3.2. Sample Preparation
3.2.1. Urine Samples
3.2.2. Dietary Supplements
3.3. Instrumental Analysis
3.3.1. Liquid Chromatography
3.3.2. Mass Spectrometry
3.3.3. Assay Characterization
3.3.4. Linearity, LOD, LOQ
3.3.5. Relative Extraction Efficiency
3.3.6. Selectivity, Sample Carry-Over, and Method Robustness Control
3.3.7. Intra-Laboratory Repeatability and Reproducibility of the Method
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Sample Availability
References
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Compound | RE (%) (Mean) | Precision (%RSD) |
---|---|---|
LGD-4033 | 89.91 | 7.40 |
Compound | Urine | ||||||
---|---|---|---|---|---|---|---|
1C | 2C | 3C | 4C | 5C | 6C | %RSD | |
R2 | |||||||
LD-4033 | 0.9993 | 0.9978 | 0.9996 | 0.9943 | 0.9982 | 0.9969 | 0.19 |
Substance | LOD (ng/mL) | LOQ (ng/mL) |
---|---|---|
LGD-4033 | 0.5 | 0.80 |
Measurements over One Day | |||
---|---|---|---|
Compound | Concentration (ng/mL) | Precision (%RSD) | Accuracy (%) |
LGD-4033 | 0.500 | 10.73 | 8.33 |
0.750 | 5.96 | 0.00 | |
1 | 4.29 | 2.50 | |
2 | 2.12 | 1.25 | |
5 | 5.72 | 2.50 | |
10 | 1.21 | 0.33 |
Analyte | Proposed Chemical Structure | Metabolic Transformation | Precursor Ion (m/z) [M]+ Parent Compound and [M]− Metabolites | Product Ion (m/z) | Retention Times (min) Determined Experimentally |
---|---|---|---|---|---|
LGD-4033 | 339.10 | 220.00; 240.02; 213.03; 184.96 | 9.05 | ||
M2-a | Hydroxylation, Dehydrogenation | 351.05 | 237.06; 253.02; 281.05 | 4.90 | |
M2-b | 237.06; 253.02; 281.05 | 5.11 | |||
M2-c | 237.06; 253.02; 281.05 | 5.40 | |||
M2-d | 237.06; 253.02; 281.05 | 5.93 | |||
M3-a | Hydroxylation | 353.07 | 255.07; 199.04; 185.03 | 6.25 | |
M3-b | 255.07; 185.03 | 6.40 | |||
M4-a | Hydroxylation, Ring cleavage | 355.08 | 285.08; 257.09; 185.03 | 5.00 | |
M4-b M4-c | 285.08; 257.09; 185.03 | 5.19 | |||
285.08; 185.03 | 5.67 | ||||
M4-d | 285.08; 185.03 | 5.93 | |||
M5-a | bis-Hydroxylation | 369.06 | 281.05; 237.06; 253.05 | 4.89 | |
M5-b | 281.05; 237.06; 255.07 | 5.10 | |||
M6-b | tri-Hydroxylation | 385.06 | 225.06; 227.04 | 4.48 |
Analyte | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 |
---|---|---|---|---|---|
LGD-4033 | 45.5 | 1.45 | 324 | 125 | 545 |
M2-a | + | + | + | + | + |
M2-b | + | + | + | + | + |
M2-c | + | + | + | + | + |
M2-d | + | + | + | + | + |
M3-a | - | - | - | + | + |
M3-b | - | - | - | - | + |
M4-a | - | - | + | + | + |
M4-b | + | + | + | + | + |
M4-c | + | + | + | + | + |
M4-d | + | + | + | + | + |
M5-a | + | + | + | + | + |
M5-b | + | + | + | + | + |
M6-b | + | - | + | + | + |
Analyte | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Reference Material | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
TC/PC | TC/ISTD | TC/PC | TC/ISTD | TC/PC | TC/ISTD | TC/PC | TC/ISTD | TC/PC | TC/ISTD | TC/PC | TC/ISTD | |
LGD-4033 | 1 | 1.7625 | 1 | 0.1498 | 1 | 28.0490 | 1 | 6.8591 | 1 | 101.9872 | 1 | 0.0831 |
M2-a | 0.0594 | 0.0886 | 0.1624 | 0.0213 | 0.0199 | 0.4629 | 0.0434 | 0.2437 | 0.0756 | 4.5054 | 0.0514 | 0.0042 |
M2-b | 0.0534 | 0.0796 | 0.1464 | 0.0192 | 0.0246 | 0.5727 | 0.0462 | 0.2594 | 0.0764 | 4.5539 | 0.1406 | 0.0116 |
M2-c | 0.0338 | 0.0504 | 0.0628 | 0.0082 | 0.0075 | 0.1734 | 0.0358 | 0.2012 | 0.0313 | 1.8633 | 0.0330 | 0.0027 |
M2-d | 0.0069 | 0.0103 | 0.0073 | 0.0010 | 0.0023 | 0.0539 | 0.0106 | 0.0595 | 0.0086 | 0.5103 | - | - |
M3-a | - | - | - | - | - | - | 0.0109 | 0.0610 | 0.0168 | 1.0008 | - | - |
M3-b | - | - | - | - | - | - | - | - | 0.0010 | 0.0621 | - | - |
M4-a | - | - | - | - | 0.0020 | 0.0458 | 0.0082 | 0.0462 | 0.0079 | 0.4706 | - | - |
M4-b | 0.2818 | 0.4205 | 0.2131 | 0.0279 | 0.1189 | 2.7653 | 0.2082 | 1.1704 | 0.1968 | 11.7233 | 0.6973 | 0.0574 |
M4-c | 0.0467 | 0.0697 | 0.0918 | 0.0120 | 0.0317 | 0.7381 | 0.0492 | 0.2766 | 0.0865 | 5.1555 | 0.0943 | 0.0078 |
M4-d | 0.0245 | 0.0365 | 0.0643 | 0.0084 | 0.0216 | 0.5023 | 0.0303 | 0.1704 | 0.0536 | 3.1921 | 0.0718 | 0.0059 |
M5-a | 0.1902 | 0.2838 | 0.4915 | 0.0644 | 0.0588 | 1.3668 | 0.1207 | 0.6784 | 0.1479 | 8.8091 | 0.1684 | 0.0139 |
M5-b | 1.5928 | 2.3765 | 4.5244 | 0.5928 | 0.6918 | 16.0886 | 1.2134 | 6.8203 | 0.9451 | 56.3030 | 4.2050 | 0.3463 |
M6-b | 0.0031 | 0.0047 | - | - | 0.0011 | 0.0256 | 0.0014 | 0.0081 | 0.0035 | 0.2115 | - | - |
Compounds Detected | Estimated Concentration (ng/mL) |
---|---|
Sample 1 | |
Arimistane (Androst-3,5-diene-7,17-dione) [15] | 110.65 |
Arimistane M (Androst-3,5-diene-7β-ol-17-one) [16] | 454.38 |
Ibutamoren (MK-677) | 44.46 |
Hydroxyibutamoren M1 [17] | not determined |
Hydroxyibutamoren M2 [17] | not determined |
Dihydroxyibutamoren M3 [17] | not determined |
Desbenzylibutamoren M4 [17] | not determined |
Ostarine | 924.42 |
o-Dephenylostarine | 11.14 |
Hydroxyostarine M1a [18] | not determined |
Hydroxyostarine M1b [18] | not determined |
Sample 3 | |
Ibutamoren (MK-677) | 0.13 |
Sample 5 | |
Ibutamoren (MK-677) | 203.25 |
Dehydrochlormethyltestosterone M3 (4-chloro-18-nor-17β-hydroxymethyl,17α-methyl-5α-androst-13-en-3α-ol) | 1.1 |
LGD-4033 | 544.86 |
LGD-4033 M5a (bis-hydroxylation) | not determined |
Ostarine | 1.03 |
RAD140 | 844.97 |
Nandrolone M (19-norandrosterone) | 3.71 |
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Kwiatkowska, D.; Wicka, M.; Grucza, K.; Konarski, P.; Drapała, A.; Kaliszewski, P. Variation of Sequential Ligandrol (LGD-4033) Metabolite Levels in Routine Anti-Doping Urine Samples Detected with or without Other Xenobiotics. Molecules 2023, 28, 6486. https://doi.org/10.3390/molecules28186486
Kwiatkowska D, Wicka M, Grucza K, Konarski P, Drapała A, Kaliszewski P. Variation of Sequential Ligandrol (LGD-4033) Metabolite Levels in Routine Anti-Doping Urine Samples Detected with or without Other Xenobiotics. Molecules. 2023; 28(18):6486. https://doi.org/10.3390/molecules28186486
Chicago/Turabian StyleKwiatkowska, Dorota, Mariola Wicka, Krzysztof Grucza, Patryk Konarski, Aleksandra Drapała, and Paweł Kaliszewski. 2023. "Variation of Sequential Ligandrol (LGD-4033) Metabolite Levels in Routine Anti-Doping Urine Samples Detected with or without Other Xenobiotics" Molecules 28, no. 18: 6486. https://doi.org/10.3390/molecules28186486
APA StyleKwiatkowska, D., Wicka, M., Grucza, K., Konarski, P., Drapała, A., & Kaliszewski, P. (2023). Variation of Sequential Ligandrol (LGD-4033) Metabolite Levels in Routine Anti-Doping Urine Samples Detected with or without Other Xenobiotics. Molecules, 28(18), 6486. https://doi.org/10.3390/molecules28186486