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Doping control analysis of seven bioactive peptides in horse plasma by liquid chromatography-mass spectrometry.
MedLine Citation:
PMID:  23318763     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
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.
Authors:
Wai Him Kwok; Emmie N M Ho; Ming Yip Lau; Gary N W Leung; April S Y Wong; Terence S M Wan
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-1-15
Journal Detail:
Title:  Analytical and bioanalytical chemistry     Volume:  -     ISSN:  1618-2650     ISO Abbreviation:  Anal Bioanal Chem     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-1-15     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101134327     Medline TA:  Anal Bioanal Chem     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Racing Laboratory, The Hong Kong Jockey Club, 6/F Central Complex, Sha Tin Racecourse, Sha Tin, Hong Kong, China, wh.kwok@hkjc.org.hk.
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