A Novel High‐Throughput Immunoaffinity LC–MS/MS Assay for P‐III‐NP and Other Fragments of Type III Procollagen in Human Serum

ABSTRACT The amino‐terminal propeptide of type III procollagen (P‐III‐NP) is used with IGF‐I to detect the illicit use of growth hormone and to monitor growth hormone therapy. However, the only currently available assays for P‐III‐NP are immunoassays, which are not well harmonized. In addition, other fragments of type III procollagen may better evaluate collagen turnover. We aimed to develop a high‐throughput assay using immunoaffinity enrichment coupled to ultra‐high‐performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) to quantify peptides belonging to three different regions of type III procollagen in human serum simultaneously. To facilitate higher throughput, we transferred the assay from microcentrifuge tubes to a 96‐well plate format with partially automated pipetting. The method was linear (Pearson's R ≥ 0.994) over an estimated concentration range of 1.35–13.3 nM, 0.04–2.28 nM, and 0.26–5.1 nM for each surrogate peptide of P‐III‐NP, collagen degradation products, and the carboxyl‐terminal propeptide, respectively. Intra‐day and inter‐day imprecision were both < 13.6%, and the results of robustness testing were also encouraging. The method was successfully applied to capillary blood samples obtained using Tasso+ microsampling devices. Modest correlation of P‐III‐NP concentration was observed between our new method and a WADA‐approved immunoassay ( N = 40, Pearson's R = 0.789) with a significant bias of −87.8%. Our method simultaneously quantifies four peptides belonging to three regions of type III procollagen in human serum. High bias between assays highlights the need for common higher‐order calibrators or reference materials to help improve the comparability of results across laboratories.