Determination of IGF-1 and IGF-2, their degradation products and synthetic analogues in urine by LC-MS/MS

Abstract

Peptide analysis in doping controls by means of nano-UPLC coupled high resolution/high mass accuracy mass spectrometry provides the state-of-the-art technique in modern sports drug testing. The present study describes a recent application of this technique for the qualitative determination of different urinary insulin-like growth factor (IGF) related peptides. After simultaneous isolation by solid phase extraction and magnetic particle-based immunoaffinity purification, target analytes (IGF-1, IGF-2, Des1-3-IGF-1, R(3)-IGF-1 and longR(3)-IGF-1) were separated by nano-liquid chromatography prior to mass spectrometric detection. Endogenously produced IGF-1 and IGF-2, as well as the degradation product Des1-3-IGF-1, were frequently detected in urine samples from healthy volunteers in a concentration range of 20-400 pg mL(-1). The impact of IGF binding proteins (IGFBPs), being also present in urine, was potentially estimated by an additional ultrafiltration step in the sample preparation procedure. The synthetic analogue longR(3)-IGF-1, which is assumed to be subject to misuse by cheating athletes, was also analysed and detected in fortified urine samples. Besides the intact molecule, an N-terminally truncated degradation product Des1-10-longR(3)-IGF-1 was identified as the more stable target for doping controls using urine samples. The method was validated for qualitative purposes considering the parameters specificity, limit of detection (20-50 pg mL(-1)), recovery (10-35%), precision (<20%), linearity, robustness and stability.

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 ...…”

New Detection Methods of Growth Hormone and Growth Factors

“…Adoption of the isoform approach to mass spectrometry would also require the availability of a measurement technique for other (pituitary derived) isoforms. In case of the marker approach, the major progress has been made regarding the measurement of IGF-1 by mass spectrometry [37,38]. However, similar progress has not been made for the analysis of P-III-P concentrations.…”

Using Mass Spectrometry to Detect, Differentiate, and Semiquantitate Closely Related Peptide Hormones in Complex Milieu: Measurement of IGF-II and Vesiculin

“…If these variant peptides are differentially expressed in disease and have differences in activity, measurements of expression changes in these peptides need to be specific. This technique has been used to distinguish highly related peptides in biological samples for the purpose of detecting doping in the sports world but appears to have been mostly overlooked in basic biological research (27)(28)(29). The islet secretome is relatively underexplored; proteomic analysis has revealed ϳ100 granule-associated proteins, the majority within a 2 to 9 kDa mass range, and most of these have yet to be identified and characterized (30).…”