Doping control analysis of seven bioactive peptides in horse plasma by liquid chromatography–mass spectrometry
详细信息 查看全文
- 作者：Wai Him Kwok (1)
Emmie N. M. Ho (1)
Ming Yip Lau (1)
Gary N. W. Leung (1)
April S. Y. Wong (1)
Terence S. M. Wan (1) - 关键词：Bioactive peptides ; Horse plasma ; Doping control ; Liquid chromatography–mass spectrometry.
- 刊名：Analytical and Bioanalytical Chemistry
- 出版年：2013
- 出版时间：March 2013
- 年：2013
- 卷：405
- 期：8
- 页码：2595-2606
- 全文大小：1619KB
- 参考文献：1. Kuuranne T, Thomas A, Leinonen A, Delahaut P, Bosseloir A, Sch?nzer W, Thevis M (2008) Insulins in equine urine: qualitative analysis by immunoaffinity purification and liquid chromatography/tandem mass spectrometry for doping control purposes in horse-racing. Rapid Commun Mass Spectrom 3:355-62 CrossRef
2. Ho ENM, Wan TSM, Wong ASY, Lam KKH, Stewart BD (2008) Doping control analysis of insulin and its analogues in equine plasma by liquid chromatography. J Chromatogr A 1201:183-90 CrossRef
3. Ho ENM, Wan TSM, Wong ASY, Lam KKH, Stewart BD (2011) Doping control analysis of insulin and its analogues in equine urine by liquid chromatography–tandem mass spectrometry. J Chromatogr A 1218:1139-146 CrossRef
4. Thomas A, Sch?nzer W, Delahaut P, Thevis M (2012) Immunoaffinity purification of peptide hormones prior to liquid chromatography–mass spectrometry in doping controls. Methods 56:230-35 CrossRef
5. De Kock SS, Rodgers JP, Swanepoel BC (2001) Growth hormone abuse in the horse: preliminary assessment of a mass spectrometric procedure for IGF-1 identification and quantitation. Rapid Commun Mass Spectrom 15:1191-197 CrossRef
6. Popot MA, Woolfitt AR, Garcia P, Tabet JC (2008) Determination of IGF-I in horse plasma by LC electrospray ionisation mass spectrometry. Anal Bioanal Chem 390:1843-852 CrossRef
7. Bredeh?ft M, Sch?nzer W, Thevis M (2008) Quantification of human insulin-like growth factor-1 and qualitative detection of its analogues in plasma using liquid chromatography/electrospray ionisation tandem mass spectrometry. Rapid Commun Mass Spectrom 22:477-85 CrossRef
8. Guan F, Uboh CE, Soma LR, Birks E (2007) LC-MS/MS method for confirmation of recombinant human erythropoietin and darbepoetin alpha in equine plasma. Anal Chem 79:4627-635 CrossRef
9. Guan F, Uboh CE, Soma LR, Maylin G, Jiang Z, Chen J (2010) Confirmatory analysis of continuous erythropoietin receptor activator and erythropoietin analogues in equine plasma by LC-MS for doping control. Anal Chem 82:9074-081 CrossRef
10. Yu NH, Ho ENM, Wan TSM, Wong ASY (2010) 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 396:2513-521 CrossRef
11. Kwok WH, Ho ENM, Leung GNW, Wong ASY, Yue SK, Wan TSM (2012) Identification of recombinant human relaxin-2 in equine plasma by liquid chromatography/high resolution mass spectrometry. Drug Test Anal. doi:10.1002/dta.1427
12. Okano M, Sato M, Ikekita A, Kageyama S (2010) 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 24:2046-056 CrossRef
13. Thomas A, H?ppner S, Geyer H, Sch?nzer W, Petrou M, Kwiatkowska D, Pokrywka A, Thevis M (2011) 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 401:507-16 CrossRef
14. Thomas A, Kohler M, Sch?nzer W, Kamber M, Delahaut P, Thevis M (2009) Determination of Synacthen in urine for sports drug testing by means of nano-ultra-performance liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 23:2669-4 CrossRef
15. Esposito S, Deventer K, Goeman J, Van der Eycken J, Van Eenoo P (2012) Synthesis and characterization of the N-terminal acetylated 17-3 fragment of thymosin beta 4 identified in TB-500, a product suspected to possess doping potential. Drug Test Anal 4:733-38 CrossRef
16. Ho ENM, Kwok WH, Wong ASY, Wan TSM (2012) Doping control analysis of TB-500, a synthetic version of an active region of thymosin β4, in equine urine and plasma by liquid chromatography/mass spectrometry. J Chromatogr A 1265:57-9 CrossRef
17. Smith RG (2005) Development of growth hormone secretagogues. Endocr Rev 26:346-60 CrossRef
18. Ghigo E, Arvat E, Muccioli G, Camanni F (1997) Growth hormone-releasing peptides. Eur J Endocrinol 136:445-60 CrossRef
19. Jetté L, Léger R, Thibaudeau K, Benquet C, Robitaille M, Pellerin I, Paradis V, van Wyk P, Pham K, Bridon DP (2005) Human growth hormone-releasing factor (hGRF)1-29-albumin bioconjugates activate the GRF receptor on the anterior pituitary in rats: identification of CJC-1295 as a long-lasting GRF analog. Endocrinology 146:3052-058 CrossRef
20. Teichman SL, Neale A, Lawrence B, Gagnon C, Castaigne JP, Frohman LA (2006) Prolonged stimulation of growth hormone (GH) and insulin-like growth factor I secretion by CJC-1295, a long-acting analog of GH-releasing hormone, in healthy adults. J Clin Endocrinol Metab 91:799-05 CrossRef
21. Sackmann-Sala L, Ding J, Frohman LA, Kopchick JJ (2009) Activation of the GH/IGF-1 axis by CJC-1295, a long-acting GHRH analog, results in serum protein profile changes in normal adult subjects. Growth Horm IGF Res 19:471-77 CrossRef
22. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K (1999) Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656-60 CrossRef
23. Blake AD, Smith RG (1991) Desensitization studies using perifused rat pituitary cells show that growth hormone-releasing hormone and His-d -Trp-Ala-Trp-d -Phe-Lys-NH2 stimulate growth hormone release through distinct receptor sites. J Endocrinol 129:11-9 CrossRef
24. Free Press Release (2011) http://www.free-press-release.com/news-do-you-know-how-to-use-tb-500-read-on-1317211858.html Assessed 24 April 2012
25. van den Broek I, Sparidans RW, Schellens JHM, Beijnen JH (2008) Quantitative bioanalysis of peptides by liquid chromatography coupled to (tandem) mass spectrometry. J Chromatogr B 872:1-2 CrossRef
26. Chambers E, Wagrowski-Diehl DM, Lu Z, Mazzeo JR (2007) Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses. J Chromatogr B Anal Technol Biomed Life Sci 852:22-4 CrossRef
27. Smith EL (1949) Proteolytic Enzymes. Annu Rev Biochem 18:35-8 CrossRef
28. Frohman LA, Downs TR, Heimer EP, Felix AM (1989) Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human growth hormone-releasing hormone in plasma. J Clin Invest 83:1533-540 CrossRef
29. Wong JKY, Tang FPW, Wan TSM (2011) / In vitro metabolic studies using homogenised horse liver in place of horse liver microsomes. Drug Test Anal 3:393-99 CrossRef
30. Duffus JH, Nordberg M, Templeton DM (2007) Glossary of terms used in toxicology, 2nd edition. Pure Appl Chem 79:1153-344 CrossRef - 作者单位：Wai Him Kwok (1)
Emmie N. M. Ho (1)
Ming Yip Lau (1)
Gary N. W. Leung (1)
April S. Y. Wong (1)
Terence S. M. Wan (1)
1. Racing Laboratory, The Hong Kong Jockey Club, 6/F Central Complex, Sha Tin Racecourse, Sha Tin, Hong Kong, China - ISSN：1618-2650
文摘
In recent years, there has been an ongoing focus for both human and equine doping control laboratories on developing detection methods to control the misuse of peptide therapeutics. Immunoaffinity purification is a common extraction method to isolate peptides from biological matrices and obtain sufficient detectability in subsequent instrumental analysis. However, monoclonal or polyclonal antibodies for immunoaffinity purification may not be commercially available, and even if available, such antibodies are usually very costly. In our study, a simple mixed-mode anion exchange solid-phase extraction cartridge was employed for the extraction of seven target peptides (GHRP-1, GHRP-2, GHRP-6, ipamorelin, hexarelin, CJC-1295, and N-acetylated LKKTETQ (active ingredient of TB-500)) and their in vitro metabolites from horse plasma. The final extract was subject to ultra-high-performance liquid chromatographic separation and analysed with a hybrid high-resolution mass spectrometer. The limits of detection for all seven peptides were estimated to be less than 50?pg/mL. Method validation was performed with respect to specificity, precision, and recovery. The applicability of this multi-analyte method was demonstrated by the detection of N-acetylated LKKTETQ and its metabolite N-acetylated LK from plasma samples obtained after subcutaneous administration of TB-500 (10?mg N-acetylated LKKTETQ) to two thoroughbred geldings. This method could easily be modified to cover more bioactive peptides, such as dermorphin, β-casomorphin, and desmopressin. With the use of high-resolution mass spectrometry, the full-scan data acquired can also be re-processed retrospectively to search for peptides and their metabolites that have not been targeted at the time of analysis. To our knowledge, this is the first identification of in vitro metabolites of all the studied peptides other than TB-500 in horses. Figure Product-ion scans and mass spectral assignments of the fragment ions of the seven target peptides