Abstract
RATIONALE
Insulin-like growth factor-I is one of the biomarkers used to detect growth hormone administration prohibited in human sport. Current testing approaches for IGF-I rely on commercial immunoassays, which may change from time to time requiring complex revalidation. Mass spectrometry (MS)-based approaches often rely on enzymatically digesting the protein and measuring specific peptide concentrations. In order to reinforce the current available methodology for IGF-I testing, a reliable and equally sensitive MS method is required for the analysis of intact protein using small sample volumes (<25 μL).
METHODS
IGF-I was extracted from human serum samples by a simple protein precipitation procedure. Separation was achieved via nano-ultrahigh-performance liquid chromatography and MS analysis was conducted by nano-electrospray ionisation triple-quadrupole mass spectrometry in the selected reaction monitoring mode using a stable-isotope-labelled internal standard.
RESULTS
A six-point calibration curve ranging from 50 to 1000 ng/mL of human IGF-I in rat serum was used to establish instrument response. The method provided a limit of quantification of 50 ng/mL, with intra- and inter-day precision ≤5% and intra- and inter-day accuracy ≥95%.
CONCLUSIONS
A quantitative method was developed for the quantification of intact IGF-I in human serum samples. The data generated provided important information for the development of a new reference method for the growth hormone biomarker test and helped create a reliable system for monitoring peptide hormones in individual athletes, a possible extension to the athlete biological passport system. Nano-electrospray has here been shown to be sufficiently robust for routine use in an analytical laboratory, allowing for the analysis of minute sample volumes.
Insulin-like growth factor-I is one of the biomarkers used to detect growth hormone administration prohibited in human sport. Current testing approaches for IGF-I rely on commercial immunoassays, which may change from time to time requiring complex revalidation. Mass spectrometry (MS)-based approaches often rely on enzymatically digesting the protein and measuring specific peptide concentrations. In order to reinforce the current available methodology for IGF-I testing, a reliable and equally sensitive MS method is required for the analysis of intact protein using small sample volumes (<25 μL).
METHODS
IGF-I was extracted from human serum samples by a simple protein precipitation procedure. Separation was achieved via nano-ultrahigh-performance liquid chromatography and MS analysis was conducted by nano-electrospray ionisation triple-quadrupole mass spectrometry in the selected reaction monitoring mode using a stable-isotope-labelled internal standard.
RESULTS
A six-point calibration curve ranging from 50 to 1000 ng/mL of human IGF-I in rat serum was used to establish instrument response. The method provided a limit of quantification of 50 ng/mL, with intra- and inter-day precision ≤5% and intra- and inter-day accuracy ≥95%.
CONCLUSIONS
A quantitative method was developed for the quantification of intact IGF-I in human serum samples. The data generated provided important information for the development of a new reference method for the growth hormone biomarker test and helped create a reliable system for monitoring peptide hormones in individual athletes, a possible extension to the athlete biological passport system. Nano-electrospray has here been shown to be sufficiently robust for routine use in an analytical laboratory, allowing for the analysis of minute sample volumes.
| Original language | English |
|---|---|
| Pages (from-to) | 1426-1432 |
| Number of pages | 7 |
| Journal | Rapid Communications in Mass Spectrometry |
| Volume | 28 |
| Issue number | 13 |
| DOIs | |
| Publication status | Published - 15 Jul 2014 |