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

Autor: Huynh HH; Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA., Barahona-Carrillo L; Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA., Moncrieffe D; Drug Control Centre, Department of Analytical, Environmental and Forensic Science, King's College London, London, UK.; Department of Analytical, Environmental & Forensic Sciences, King's College London, London, UK., Cowan DA; Drug Control Centre, Department of Analytical, Environmental and Forensic Science, King's College London, London, UK., Forrest K; Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA., Becker JO; Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA., Emrick MA; Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA., Thomas A; Center for Preventive Doping Research (ZePraeDo), Institute of Biochemistry, German Sport University, Cologne, Germany., Thevis M; Center for Preventive Doping Research (ZePraeDo), Institute of Biochemistry, German Sport University, Cologne, Germany., Eichner D; Sport Medicine Research and Testing Laboratory, Salt Lake City, Utah, USA., Byers PH; Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA., Miller GD; Sport Medicine Research and Testing Laboratory, Salt Lake City, Utah, USA., Hoofnagle AN; Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.; Department of Medicine, University of Washington, Seattle, Washngton, USA.
Jazyk: angličtina
Zdroj: Drug testing and analysis [Drug Test Anal] 2024 Oct 27. Date of Electronic Publication: 2024 Oct 27.
DOI: 10.1002/dta.3814
Abstrakt: 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.
(© 2024 John Wiley & Sons, Ltd.)
Databáze: MEDLINE
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