Cationic exchange SPE combined with triple quadrupole UHPLC-MS/MS for detection of GHRHs in urine samples.
This study, conducted by a doping control laboratory, describes the development and analytical validation of a method for detecting growth hormone-releasing hormones (GHRHs) — specifically tesamorelin, CJC-1295, sermorelin (GRF 1-29), sermorelin (3-29)-NH₂, and somatorelin — in human urine samples. GHRHs are prohibited in sport under World Anti-Doping Agency (WADA) regulations. The method combines weak cation exchange solid-phase extraction (SPE) with ultra-high-performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry (UHPLC-MS/MS). The researchers validated the method according to WADA technical documents, evaluating selectivity, limit of detection (LOD), carryover, reliability, stability, and recovery. The method achieved an LOD of 0.2 ng/mL, a limit of quantification (LOQ) of 0.6 ng/mL, and linearity from 0.1 to 1.2 ng/mL. The study reports adequate recovery and sensitivity for routine anti-doping screening. A key limitation is that this is purely an analytical/method-development study; it does not investigate pharmacological effects, clinical outcomes, or administer any compound to human or animal subjects.
Why this grade: This is an analytical method-development and validation study with no human or animal subjects, pharmacological intervention, or clinical outcomes, making it insufficient for grading evidence of any compound's effects in humans.
The use of growth hormone-releasing hormones (GHRHs) is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). Considering the complexity of urine samples and the low concentrations at which these analytes should be detected, analyzing GHRHs is a challenging task. In most of the studies, GHRHs are analyzed using UHPLC-HRMS with an orbitrap. The present developed and validated method for some GHRHs (tesamorelin, CJC-1295, sermorelin (GRF 1-29), sermorelin (3-29)-NH 2 , somatorelin) is based on the triple quadrupole UHPLC/MS-MS method with solid phase extraction (SPE) with weak cation exchange and is able to detect concentrations as low as 0.2 ng/mL (LOD), a limit of quantification (LOQ) at 0.6 ng/mL, and linearity across the range of 0.1 ng/mL to 1.2 ng/mL. The present method developed by our doping control laboratory was validated according to WADA technical documents for selectivity, limit of detection (LOD), carryover, reliability of detection, stability and recovery. The results show that the method has adequate recoveries and sensitivity, hence, it can be employed for routine screening in anti-doping laboratories.
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