Abstract
Background: Detection and identification of a wide range of drugs, including small peptides, corticosteroids, selective androgen receptor modulators (SARMs), and quaternary ammonium drugs (QADs), are imperative across several domains, particularly in anti-doping analysis, given the potential misuse of these substances in animal sports, there is an urgent demand for an allencompassing screening method to effectively identify these compounds.
Objective: To develop a robust and sensitive high-resolution method for simultaneous screening of small peptides, corticosteroids, SARMs, and QADs using liquid chromatography accurate mass spectrometry in post-race urine samples. This method integrates a streamlined single-stage extraction approach, significantly enhancing efficiency and adaptability for screening drugs across various classes.
Methods: The method development and validation involved a comprehensive solid-phase extraction protocol, which included a sequential elution for corticosteroids, small peptides, SARMs, and QADs. Chromatographic separation was achieved using a reverse-phase C18 column, and the analysis was performed using liquid chromatography - high-resolution accurate mass spectrometry.
Results: The developed method was validated using a selection of drugs representing each class. The method exhibited robustness and sensitivity, enabling the simultaneous screening of the specified drug classes. The flexibility inherent in the proposed extraction and analysis method allows for seamless integration of new drug candidates eliminating the need for method redevelopment.
Conclusion: A versatile and effective screening method was successfully developed and validated for the simultaneous detection of small peptides, corticosteroids, SARMs, and QADs. The method's ability for retrospective analysis of emerging drugs using full scan HRMS enhances its utility. This method holds great promise across various fields where accurate and comprehensive drug screening is imperative.
Graphical Abstract
[1]
Delcourt, V.; Garcia, P.; Chabot, B.; Loup, B.; Remy, P.; Popot, M.A.; Bailly-Chouriberry, L. Screening and confirmatory analysis of recombinant human erythropoietin for racing camels’ doping control. Drug Test. Anal., 2020, 12(6), 763-770.
[http://dx.doi.org/10.1002/dta.2772] [PMID: 31984676]
[http://dx.doi.org/10.1002/dta.2772] [PMID: 31984676]
[2]
Ashraf, S.S.; El-Gahany, W.; Alraeesi, A.; Al-Hajj, L.; Al-Maidalli, A.; Shah, I. Analysis of illicit glucocorticoid levels in camel hair using competitive ELISA - Comparison with LC-MS/MS. Drug Test. Anal., 2020, 12(4), 458-464.
[http://dx.doi.org/10.1002/dta.2750] [PMID: 31840435]
[http://dx.doi.org/10.1002/dta.2750] [PMID: 31840435]
[3]
World antidoping agency WADA List of probited drugs. 2022. Available from: https://www.wada-ama.org/sites/default/files/2022-01/2022list_final_en_0.pdf (Accessed May 3, 2023).
[4]
FEI Equine Prohibited Substances List. 2022. Available from: https://inside.fei.org/sites/default/files/2022 Prohibited Substances List.pdf (Accessed May 3, 2023).
[5]
Judák, P.; Grainger, J. Goebel, Catrin DMSO assisted electrospray ionization for the detection of small peptide hormones in urine by dilute-and-shoot-liquid-chromatography-high resolution mass spectrometry. J. Am. Soc. Mass Spectrom., 2017, 28(8), 1657-1665.
[http://dx.doi.org/10.1007/s13361-017-1670-7]
[http://dx.doi.org/10.1007/s13361-017-1670-7]
[6]
Emmie, N.M. Doping control analysis of TB-500, a synthetic version of an active region of thymosin 4, in equine urine and plasma by liquid chromatography–mass spectrometry. J. Chromatogr. A, 2012, 1265, 57-69.
[http://dx.doi.org/10.1016/j.chroma.2012.09.043] [PMID: 23084823]
[http://dx.doi.org/10.1016/j.chroma.2012.09.043] [PMID: 23084823]
[7]
Esposito, S.; Deventer, K.; Goeman, J.; Van der Eycken, J.; Van Eenoo, P. Synthesis and characterization of the N‐terminal acetylated 17‐23 fragment of thymosin beta 4 identified in TB‐500, a product suspected to possess doping potential. Drug Test. Anal., 2012, 4(9), 733-738.
[http://dx.doi.org/10.1002/dta.1402] [PMID: 22962027]
[http://dx.doi.org/10.1002/dta.1402] [PMID: 22962027]
[8]
Argente, J.; García-Segura, L.M.; Pozo, J.; Chowen, J.A. Growth hormone-releasing peptides: Clinical and basic aspects. Horm. Res., 1996, 46(4-5), 155-159.
[http://dx.doi.org/10.1159/000185015] [PMID: 8950613]
[http://dx.doi.org/10.1159/000185015] [PMID: 8950613]
[9]
Sanchis-Gomar, F.; Martinez-Bello, V.E.; Nascimento, A.L.; Perez-Quilis, C.; Garcia-Gimenez, J.L.; Viña, J.; Gomez-Cabrera, M.C. Desmopresssin and hemodilution: Implications in doping. Int. J. Sports Med., 2010, 31(1), 5-9.
[http://dx.doi.org/10.1055/s-0029-1239500] [PMID: 19885778]
[http://dx.doi.org/10.1055/s-0029-1239500] [PMID: 19885778]
[10]
Deventer, K.; Pozo, O.J.; Verstraete, A.G.; Van Eenoo, P. Dilute-and-shoot-liquid chromatography-mass spectrometry for urine analysis in doping control and analytical toxicology. Trends Analyt. Chem., 2014, 55, 1-13.
[http://dx.doi.org/10.1016/j.trac.2013.10.012]
[http://dx.doi.org/10.1016/j.trac.2013.10.012]
[11]
Kwok, W.H.; Ho, E.N.M.; Lau, M.Y.; Leung, G.N.W.; Wong, A.S.Y.; Wan, T.S.M. Doping control analysis of seven bioactive peptides in horse plasma by liquid chromatography-mass spectrometry. Anal. Bioanal. Chem., 2013, 405(8), 2595-2606.
[http://dx.doi.org/10.1007/s00216-012-6697-9] [PMID: 23318763]
[http://dx.doi.org/10.1007/s00216-012-6697-9] [PMID: 23318763]
[12]
Guan, F.; Robinson, M.A. Comprehensive solid-phase extraction of multitudinous bioactive peptides from equine plasma and urine for doping detection. Anal. Chim. Acta, 2017, 985, 79-90.
[http://dx.doi.org/10.1016/j.aca.2017.07.005] [PMID: 28864198]
[http://dx.doi.org/10.1016/j.aca.2017.07.005] [PMID: 28864198]
[13]
Guan, F.; You, Y.; Li, X.; Robinson, M.A. A comprehensive approach to detecting multitudinous bioactive peptides in equine plasma and urine using hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry. Drug Test. Anal., 2019, 11(9), 1308-1325.
[http://dx.doi.org/10.1002/dta.2671] [PMID: 31250565]
[http://dx.doi.org/10.1002/dta.2671] [PMID: 31250565]
[14]
Yu, N.H.; Ho, E.N.M.; Wan, T.S.M.; Wong, A.S.Y. Doping control analysis of recombinant human erythropoietin, darbepoetin alfa and methoxy polyethylene glycol-epoetin beta in equine plasma by nano-liquid chromatography-tandem mass spectrometry. Anal. Bioanal. Chem., 2010, 396(7), 2513-2521.
[http://dx.doi.org/10.1007/s00216-010-3455-8] [PMID: 20148243]
[http://dx.doi.org/10.1007/s00216-010-3455-8] [PMID: 20148243]
[15]
Esposito, S.; Vanni, D.; Menta, S.; Orsatti, L.; Monteagudo, E. Comparison of different protein precipitation and solid‐phase extraction protocols for the study of the catabolism of peptide drugs by LC‐HRMS. J. Pept. Sci., 2020, 26(9), e3272.
[http://dx.doi.org/10.1002/psc.3272] [PMID: 32633064]
[http://dx.doi.org/10.1002/psc.3272] [PMID: 32633064]
[16]
Karakka Kal, A.K.; Karatt, T.K.; Nalakath, J. Simultaneous analysis of 44 frequently abused corticosteroid drugs using polysaccharide‐based chiral column‐HRMS approach. Anal. Sci. Adv., 2021, 2(9-10), 427-439.
[http://dx.doi.org/10.1002/ansa.202000166]
[http://dx.doi.org/10.1002/ansa.202000166]
[17]
Knoop, A.; Thomas, A.; Fichant, E.; Delahaut, P.; Schänzer, W.; Thevis, M. Qualitative identification of growth hormone-releasing hormones in human plasma by means of immunoaffinity purification and LC-HRMS/MS. Anal. Bioanal. Chem., 2016, 408(12), 3145-3153.
[http://dx.doi.org/10.1007/s00216-016-9377-3] [PMID: 26879649]
[http://dx.doi.org/10.1007/s00216-016-9377-3] [PMID: 26879649]
[18]
Kwok, K.Y.; Choi, T.L.S.; Kwok, W.H.; Lau, M.Y.; Leung, E.M.K.; Leung, G.N.W.; Wong, J.K.Y.; Wan, T.S.M.; Adrian, F.F.; Prabhu, A.; Ho, E.N.M. Detection of bioactive peptides including gonadotrophin‐releasing factors (GnRHs) in horse urine using ultra‐high performance liquid chromatography–high resolution mass spectrometry (UHPLC/HRMS). Drug Test. Anal., 2020, 12(9), 1274-1286.
[http://dx.doi.org/10.1002/dta.2880] [PMID: 32558326]
[http://dx.doi.org/10.1002/dta.2880] [PMID: 32558326]
[19]
Broberg, M.N.; Knych, H.; Bondesson, U.; Pettersson, C.; Stanley, S.; Thevis, M.; Hedeland, M. Investigation of equine in vivo and in vitro derived metabolites of the selective androgen receptor modulator (Sarm) acp-105 for improved doping control. Metabolites, 2021, 11(2), 85.
[http://dx.doi.org/10.3390/metabo11020085] [PMID: 33535528]
[http://dx.doi.org/10.3390/metabo11020085] [PMID: 33535528]
[20]
Ondern Komathu, P.; Padusha, M.K.S.A.; Laya, S.; Nalakath, J.; Palathinkal, A.B.; Nelliyott, I. Investigation of in vitro generated metabolites of LGD‐4033, a selective androgen receptor modulator, in homogenized camel liver for anti‐doping applications. Rapid Commun. Mass Spectrom., 2023, 37(22), e9633.
[http://dx.doi.org/10.1002/rcm.9633] [PMID: 37817338]
[http://dx.doi.org/10.1002/rcm.9633] [PMID: 37817338]
[21]
Görgens, C.; Guddat, S.; Thomas, A.; Wachsmuth, P.; Orlovius, A.K.; Sigmund, G.; Thevis, M.; Schänzer, W. Simplifying and expanding analytical capabilities for various classes of doping agents by means of direct urine injection high performance liquid chromatography high resolution/high accuracy mass spectrometry. J. Pharm. Biomed. Anal., 2016, 131, 482-496.
[http://dx.doi.org/10.1016/j.jpba.2016.09.015] [PMID: 27693991]
[http://dx.doi.org/10.1016/j.jpba.2016.09.015] [PMID: 27693991]
[22]
Ahrens, B.D.; Starcevic, B.; Butch, A.W. Detection of prohibited substances by liquid chromatography tandem mass spectrometry for sports doping control. Methods Mol. Biol., 2012, 902, 115-128.
[http://dx.doi.org/10.1007/978-1-61779-934-1_10] [PMID: 22767112]
[http://dx.doi.org/10.1007/978-1-61779-934-1_10] [PMID: 22767112]
[23]
Hsu, K.F.; Chien, K.Y.; Chang-Chien, G.P.; Lin, S.F.; Hsu, P.H.; Hsu, M.C. Liquid chromatography-tandem mass spectrometry screening method for the simultaneous detection of stimulants and diuretics in urine. J. Anal. Toxicol., 2011, 35(9), 665-674.
[http://dx.doi.org/10.1093/anatox/35.9.665] [PMID: 22080905]
[http://dx.doi.org/10.1093/anatox/35.9.665] [PMID: 22080905]
[24]
Mazzarino, M.; Fiacco, I.; de la Torre, X.; Botrè, F. Screening and confirmation analysis of stimulants, narcotics and beta-adrenergic agents in human urine by hydrophilic interaction liquid chromatography coupled to mass spectrometry. J. Chromatogr. A, 2011, 1218(45), 8156-8167.
[http://dx.doi.org/10.1016/j.chroma.2011.09.020] [PMID: 21978955]
[http://dx.doi.org/10.1016/j.chroma.2011.09.020] [PMID: 21978955]
[25]
Jiménez Girón, A.; Deventer, K.; Roels, K.; Van Eenoo, P. Development and validation of an open screening method for diuretics, stimulants and selected compounds in human urine by UHPLC–HRMS for doping control. Anal. Chim. Acta, 2012, 721, 137-146.
[http://dx.doi.org/10.1016/j.aca.2012.02.002] [PMID: 22405312]
[http://dx.doi.org/10.1016/j.aca.2012.02.002] [PMID: 22405312]
[26]
Musenga, A.; Cowan, D.A. Use of ultra-high pressure liquid chromatography coupled to high resolution mass spectrometry for fast screening in high throughput doping control. J. Chromatogr. A, 2013, 1288, 82-95.
[http://dx.doi.org/10.1016/j.chroma.2013.03.006] [PMID: 23540767]
[http://dx.doi.org/10.1016/j.chroma.2013.03.006] [PMID: 23540767]
[27]
Tudela, E.; Deventer, K.; Geldof, L.; Van Eenoo, P. Urinary detection of conjugated and unconjugated anabolic steroids by dilute‐and‐shoot liquid chromatography‐high resolution mass spectrometry. Drug Test. Anal., 2015, 7(2), 95-108.
[http://dx.doi.org/10.1002/dta.1650] [PMID: 24753397]
[http://dx.doi.org/10.1002/dta.1650] [PMID: 24753397]
[28]
Badoud, F.; Grata, E.; Perrenoud, L.; Avois, L.; Saugy, M.; Rudaz, S.; Veuthey, J.L. Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography–quadrupole time-of-flight mass spectrometry. J. Chromatogr. A, 2009, 1216(20), 4423-4433.
[http://dx.doi.org/10.1016/j.chroma.2009.03.033] [PMID: 19342059]
[http://dx.doi.org/10.1016/j.chroma.2009.03.033] [PMID: 19342059]
[29]
Vonaparti, A.; Lyris, E.; Angelis, Y.S.; Panderi, I.; Koupparis, M.; Tsantili-Kakoulidou, A.; Peters, R.J.B.; Nielen, M.W.F.; Georgakopoulos, C. Preventive doping control screening analysis of prohibited substances in human urine using rapid‐resolution liquid chromatography/high‐resolution time‐of‐flight mass spectrometry. Rapid Commun. Mass Spectrom., 2010, 24(11), 1595-1609.
[http://dx.doi.org/10.1002/rcm.4554] [PMID: 20486255]
[http://dx.doi.org/10.1002/rcm.4554] [PMID: 20486255]
[30]
Kolmonen, M.; Leinonen, A.; Kuuranne, T.; Pelander, A.; Ojanperä, I. Generic sample preparation and dual polarity liquid chromatography-time‐of‐flight mass spectrometry for high‐throughput screening in doping analysis. Drug Test. Anal., 2009, 1(6), 250-266.
[http://dx.doi.org/10.1002/dta.50] [PMID: 20355204]
[http://dx.doi.org/10.1002/dta.50] [PMID: 20355204]
[31]
Hørning, O.B.; Theodorsen, S.; Vorm, O.; Jensen, O.N. Solid phase extraction-liquid chromatography (SPE-LC) interface for automated peptide separation and identification by tandem mass spectrometry. Int. J. Mass Spectrom., 2007, 268(2-3), 147-157.
[http://dx.doi.org/10.1016/j.ijms.2007.06.017]
[http://dx.doi.org/10.1016/j.ijms.2007.06.017]
[32]
Thomas, A.; Höppner, S.; Geyer, H.; Schänzer, W.; Petrou, M.; Kwiatkowska, D.; Pokrywka, A.; Thevis, M. Determination of growth hormone releasing peptides (GHRP) and their major metabolites in human urine for doping controls by means of liquid chromatography mass spectrometry. Anal. Bioanal. Chem., 2011, 401(2), 507-516.
[http://dx.doi.org/10.1007/s00216-011-4702-3] [PMID: 21298258]
[http://dx.doi.org/10.1007/s00216-011-4702-3] [PMID: 21298258]
[33]
Okano, M.; Sato, M.; Ikekita, A.; Kageyama, S. Determination of growth hormone secretagogue pralmorelin (GHRP‐2) and its metabolite in human urine by liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom., 2010, 24(14), 2046-2056.
[http://dx.doi.org/10.1002/rcm.4619] [PMID: 20552695]
[http://dx.doi.org/10.1002/rcm.4619] [PMID: 20552695]
[34]
Gansbeke, W.V.; Polet, M.; Hooghe, F.; Devos, C.; Eenoo, P.V. Improved sensitivity by use of gas chromatography—positive chemical ionization triple quadrupole mass spectrometry for the analysis of drug related substances. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 2015, 1001, 221-240.
[http://dx.doi.org/10.1016/j.jchromb.2015.07.052] [PMID: 26296082]
[http://dx.doi.org/10.1016/j.jchromb.2015.07.052] [PMID: 26296082]
[35]
Albertsdóttir, A.D.; Van Gansbeke, W.; Coppieters, G.; Balgimbekova, K.; Van Eenoo, P.; Polet, M. Searching for new long‐term urinary metabolites of metenolone and drostanolone using gas chromatography-mass spectrometry with a focus on non‐hydrolysed sulfates. Drug Test. Anal., 2020, 12(8), 1041-1053.
[http://dx.doi.org/10.1002/dta.2818] [PMID: 32386339]
[http://dx.doi.org/10.1002/dta.2818] [PMID: 32386339]
[36]
The World Anti-Doping Code International Standard for Laboratories. Available from: https://www.wada-ama.org/sites/default/files/resources/files/isl_nov2019.pdf (Accessed March 1,2023).
[37]
General Accreditation Guidance - Validation and verification of quantitative and qualitative test methods. 2018. Available from: https://nata.com.au/files/2021/05/Validation-and-Verification-of-Quantitative-and-Qualitative-Test- Methods.pdf (Accessed March 01,2023).
