B-169 A Novel Immunoprecipitation-based Liquid Chromatography–Tandem Mass Spectrometry Analysis for Accurate Determination for Copeptin in Human Serum

Abstract Background Copeptin, a glycosylated peptide of 39 amino acids, is derived from the C-terminal segment of the arginine vasopressin (AVP) precursor peptide. It is released in an equimolar mode with AVP from the pituitary gland and has emerged as a stable and simple-to-measure surrogate marker for AVP. Copeptin demonstrates excellent clinical values, particularly in diagnosing polyuria-polydipsia syndromes such as diabetes insipidus. However, conventional immunoassay methods are limited and lack comparability. Therefore, our aim was to establish a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for quantifying copeptin levels in serum. Methods Copeptin was extracted from serum using immunoprecipitation with antibody-conjugated magnetic beads. The elution of the beads was then digested with trypsin. Then the samples were prepared using anion exchange solid-phase extraction before being detected by LC–MS/MS. The analytical performance of the method was validated in accordance with the recommendations of the Clinical and Laboratory Standards Institute C62-A and C64. Additionally, the method was compared to an immunofluorescent assay on the B.R.A.H.M.S Copeptin-proAVP KRYPTOR platform. A total of 221 residual serum samples were retrospectively collected from healthy individuals who underwent physical examinations and reference interval for copeptin was established. Diagnostic value of copeptin in identifying patients with diabetes insipidus was evaluated. Results A total of nine types of antibodies were obtained and their ability to capture serum copeptin was compared. The most effective antibody was subsequently utilized in this method after being conjugated to magnetic beads. The LC-MS/MS method had a total runtime of 8.5 minutes. Within-run precision ranged from 5.2 to 12.1%, between-run precision from 7.6 to 12.1%, and total variable coefficient from 8.1 to 13.5%. Copeptin quantitation showed linearity within the range of 5 to 1000 pg/mL, with a limit of detection at 2.5 pg/mL. Recovery rates ranged from 95.2% to 103.1%, and no significant matrix effect was observed with internal standard correction. The LC-MS/MS method had a good consistency with the immunoassay (r= 0.926, slope = 0.989). Additionally, the reference interval for copeptin was 3.66–58.25 pg/mL. A total of 44 patients diagnosed with central diabetes insipidus (CDI) were included and the diagnostic accuracy of copeptin for identifying CDI was evaluated by ROC curve analysis, yielding an area under the ROC curve of 0.922 (95% CI 0.882–0.952), with a sensitivity of 81.4% and a specificity of 89.7%. The cut-off value for CDI identification was determined to be 6.59 pg/mL. Conclusion We have demonstrated the accuracy and dependability of this LC-MS/MS method for quantifying copeptin. This innovative application showed satisfactory precision, a wide linear range, and a low limit of detection. Clinical studies are anticipated to be conducted to assess diagnostic accuracy using this method.