An antibody-free, ultrafiltration-based assay for the detection of growth hormone-releasing hormones in urine at low pg/mL concentrations using nanoLC-HRMS/MS.

This study developed and validated an antibody-free analytical method for detecting prohibited growth hormone-releasing hormone (GHRH) analogues — specifically sermorelin, CJC-1293, a sermorelin metabolite, CJC-1295, and tesamorelin — in human urine samples. Instead of the conventional, labor-intensive immunoaffinity purification approach, the researchers used ultrafiltration alone to preconcentrate urine samples before analysis by nano liquid chromatography coupled with high-resolution tandem mass spectrometry (nanoLC-HRMS/MS). The method achieved limits of detection between 5 and 25 pg/mL and limits of identification between 25 and 50 pg/mL, with analyte recoveries of 59–115%. Robustness was demonstrated across over 200 injections. When compared directly to immunoaffinity purification, the ultrafiltration approach yielded similar sensitivity at lower cost and without requiring specialized antibodies. Stability experiments revealed that sermorelin and its metabolite degrade rapidly at temperatures above 4°C and at pH below 7, highlighting the critical importance of proper sample handling. The authors note the method could be extended to other emerging peptide drugs (≥ ~3 kDa) and their metabolites. A key limitation is that the study is an analytical methods validation paper rather than a clinical or pharmacological study; it does not assess biological effects or pharmacokinetics in human subjects.

Why this grade: This is an analytical chemistry/methods validation study focused on detection of GHRH analogues in urine; it provides no clinical, pharmacological, or efficacy data in humans or animals.