Quantitative analysis and clinical evaluation of 20- and 22-kDa growth hormone isoforms in somatotroph adenoma using immunocapture coupled with LC-MS/MS

Abstract Objectives The 20- and 22-kDa isoforms of growth hormone 1 (GH1) play potentially distinct physiological and pathological roles, but their structural similarity makes quantification challenging. Conventional immunoassays lack sufficient specificity, highlighting the need for more precise analytical methods. Methods We developed a method combining immunocapture with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for precise quantification of GH1 isoforms, with evaluation of sensitivity, accuracy, precision, and linearity. GH1 was isolated from 1-mL serum samples by immunocapture using antibody-coated magnetic beads, eluted, and quantified by LC-MS/MS. Stable isotope-labeled 22-kDa GH1 was used as an internal standard (IS). Clinical applicability was evaluated by analyzing serum GH1 isoforms in 63 patients with somatotroph adenoma. Results The assay demonstrated linear dynamic ranges of 0.2–20.0 μg/L for 20-kDa GH1 and 1.0–100.0 μg/L for 22-kDa GH1. Intra- and inter-assay coefficients of variation were <10 %, and recoveries ranged from 95.0 to 102.5 % after IS correction. Although total GH1 concentrations measured by LC-MS/MS were systematically lower than those obtained by immunoassay, the two methods were strongly correlated ( r =0.991, p<0.01). Most patients with somatotroph adenoma (80.0 %; 51/63) exhibited increased 22/20-kDa GH1 ratios, while six patients with unfavorable clinical features and endocrine prognosis exhibited elevated 20-kDa GH1 levels. Conclusions This immunocapture LC-MS/MS method enables simultaneous quantification of 20- and 22-kDa GH1 isoforms without enzymatic digestion. The implementation of this method may refine the assessment of biochemical remission and prognostic stratification in GH-related disorders.