High‐throughput ultra‐high‐performance liquid chromatography/tandem mass spectrometry quantitation of insulin‐like growth factor‐I and leucine‐rich α‐2‐glycoprotein in serum as biomarkers of recombinant human growth hormone administration |
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| Authors: | R G Kay C Barton C P Velloso P R Brown C Bartlett A J Blazevich R J Godfrey G Goldspink R Rees G R Ball D A Cowan S D Harridge J Roberts P Teale C S Creaser |
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| Institution: | 1. Drug Development Services, Quotient Bioresearch Ltd., Fordham CB7 5WW, UK;2. Department of Surgery, University College London, London WC1E 6AU, UK;3. HFL Sport Science, Quotient Bioresearch Ltd., Fordham CB7 5WW, UK;4. Drug Control Centre, Kings College London, London SE1 9NH, UK;5. School of Exercise, Biomedical and Health Sciences, Edith Cowan University, Joondalup WA 6027, Western Australia, Australia;6. Centre for Sports Medicine and Human Performance, Brunel University, Uxbridge UB8 3PH, UK;7. School of Science and Technology, Nottingham Trent University, Nottingham NG11 8NS, UK;8. Department of Physiology, University College London, London WC1E 6BT, UK;9. Department of Chemistry, Loughborough University, Loughborough LE11 3TU, UK |
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| Abstract: | Insulin‐like growth factor‐I (IGF‐I) is a known biomarker of recombinant human growth hormone (rhGH) abuse, and is also used clinically to confirm acromegaly. The protein leucine‐rich α‐2‐glycoprotein (LRG) was recently identified as a putative biomarker of rhGH administration. The combination of an ACN depletion method and a 5‐min ultra‐high‐performance liquid chromatography/tandem mass spectrometry (uHPLC/MS/MS)‐based selected reaction monitoring (SRM) assay detected both IGF‐I and LRG at endogenous concentrations. Four eight‐point standard addition curves of IGF‐I (16–2000 ng/mL) demonstrated good linearity (r2 = 0.9991 and coefficients of variance (CVs) <13%). Serum samples from two rhGH administrations were extracted and their uHPLC/MS/MS‐derived IGF‐I concentrations correlated well against immunochemistry‐derived values. Combining IGF‐I and LRG data improved the separation of treated and placebo states compared with IGF‐I alone, further strengthening the hypothesis that LRG is a biomarker of rhGH administration. Artificial neural networks (ANNs) analysis of the LRG and IGF‐I data demonstrated an improved model over that developed using IGF‐I alone, with a predictive accuracy of 97%, specificity of 96% and sensitivity of 100%. Receiver operator characteristic (ROC) analysis gave an AUC value of 0.98. This study demonstrates the first large scale and high throughput uHPLC/MS/MS‐based quantitation of a medium abundance protein (IGF‐I) in human serum. Furthermore, the data we have presented for the quantitative analysis of IGF‐I suggest that, in this case, monitoring a single SRM transition to a trypsin peptide surrogate is a valid approach to protein quantitation by LC/MS/MS. Copyright © 2009 John Wiley & Sons, Ltd. |
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