Quantification of insulin-like growth factor-1 in dried blood spots for detection of growth hormone abuse in sport

Abstract

There is significant evidence that athletes are using recombinant human growth hormone (rhGH) to enhance performance, and its use is banned by the World Anti-Doping Agency and professional sports leagues. Insulin-like growth factor-1 (IGF-1) is the primary mediator of growth hormone action and is used as a biomarker for the detection of rhGH abuse. The current biomarker-based method requires collection and expedited shipment of venous blood which is costly and may decrease the number of tests performed. Measurement of GH biomarkers in dried blood spots (DBS) would considerably simplify sample collection and shipping methods to allow testing of a greater number of samples regardless of location. A method was developed to quantify intact IGF-1 protein in DBS by liquid chromatography-tandem mass spectrometry. A step-wise acid-acetonitrile extraction was optimized to achieve a sensitive assay with a lower limit of quantification of 50 ng/mL. IGF-1 remained stable at room temperature for up to 8 days, which would allow shipment of DBS cards at ambient temperature. In a comparison between plasma concentrations of IGF-1 and concentrations measured from venous and finger prick DBS, there was good correlation and agreement, r(2) of 0.8551 and accuracy of 86-113 % for venous DBS and r(2) of 0.9586 and accuracy of 89-122 % for finger prick DBS. The method is intended for use as a rapid screening method for IGF-1 to be used in the biomarker method of rhGH abuse detection.

Multiple Reaction Monitoring Enables Precise Quantification of 97 Proteins in Dried Blood Spots

“…For example, we were not able to detect tryptic peptides from insulin-like growth factor-1(P05019) in digested DBS samples. However, Cox and coworkers developed an elegant analytical method for this 7.6 kDa protein by using an acetonitrile precipitation step to remove most background proteins (29,30). In addition, detectability could be improved by using multidimensional separations or immunoaffinity enrichment prior to LC/MRM-MS (45)(46)(47).…”

“…deWilde et al used LC/MRM-MS to quantify ceruloplasmin as a screen for Wilson's disease (28). Recently, Cox et al reported LC/ MRM-MS methods for quantifying insulin-like growth facter-1 for the detection of human growth hormone abuse in sports (29,30).…”

Development and validation of a liquid chromatography–mass spectrometry procedure after solid-phase extraction for detection of 19 doping peptides in human urine

“…Finally, novel, previously unknown macromolecules (i.e., TB500 17-23 fragment), with no current approval for human therapeutic use (i.e., agents under preclinical or clinical development, designer drugs, or compounds approved only for veterinary use), but illegally marketed (e.g., via Internet websites) [5] are included in section S0 ''Non-approved substances''. Different analytical approaches have already been developed and published to detect these compounds in nutritional supplements [6][7][8][9][10] and in doping control samples (either blood [11][12][13][14][15][16][17][18] or urine [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37]), employing immunological [26,[35][36][37], electrophoretic [16][17][18][19]27], or liquid chromatography-mass spectrometry techniques [6-15, 20-25, 28-34] or a combination of different analytical technologies. In general, the most effective analytical approach (combined sample pretreatment and the instrumental method) has to take into ...…”

Water-Soluble Dried Blood Spot in Protein Analysis: A Proof-of-Concept Study

“…whole blood spotted on CMC. Both different temperatures [room temperature (RT), 8 and −25°C for 45 days] and storage lengths were tested (7,14, and 45 days for RT samples). All samples were kept in airtight zip-bags during the storage period.…”