Comparison of magnetic bead surface functionalities for the immunopurification of growth hormone-releasing hormones prior to liquid chromatography-high resolution mass spectrometry

Abstract

Growth hormone-releasing hormone and its analogues sermorelin, tesamorelin and CJC-1295 are included in the prohibited list of the World Antidoping Agency. These target peptides are found at very low concentrations in urine (at the pg/mL level). For this reason, hyphenated enrichment and purification steps prior to mass spectrometric detection are required. Among different strategies, immunopurification based on magnetic beads is an excellent alternative, as it offers improved selectivity when the immunoreactivity and orientation of the antibody are optimum and non-specific adsorption is minimized. However, choosing the magnetic bead surface functionalities that provide the best recoveries is not so straightforward. In this work, we have evaluated the suitability of magnetic beads with different supports, binding capacities and affinity chemistries prior analysis of human urine samples by liquid chromatography coupled to high resolution mass spectrometry using a Quadrupole-Orbitrap instrument. After optimization of the immunopurification protocol with the magnetic beads that provided better recoveries, the method was fully validated and found to be adequate considering the parameters specificity, intra- and inter-day precision (lower than 15 and 25%, respectively), matrix effect, limit of detection (0.2 ng/mL) and limit of identification (0.5 ng/mL).