Fully validated SRM-MS-based method for absolute quantification of PIVKA-II in human serum: Clinical applications for patients with HCC

[Display omitted] •An SRM-MS method was developed for determining the absolute quantities of PIVKA-II in human serum.•The method was fully validated according to FDA, EMA, KFDA, and CLSI guidelines.•This method was compared with the conventional approach using 400 clinical samples.•Novel development and validation approaches were adopted to ensure the establishment of a robust and reproducible method. Protein induced by vitamin K absence or antagonist-II (PIVKA-II), an abnormal form of prothrombin, is used as a serological biomarker that aids in the diagnosis of hepatocellular carcinoma (HCC). PIVKA-II is typically measured by liquid binding assay (LiBA). However, without an internal standard, it is difficult to obtain accurate results. Thus, we aimed to develop a selected reaction monitoring-mass spectrometry (SRM-MS)-based assay to quantify PIVKA-II in serum. Our SRM-MS assay entailed the addition of a protein analog as an internal standard, the enrichment of PIVKA-II using a monoclonal antibody, chymotrypsin digestion, online desalting, and SRM-MS analysis. The performance of the SRM-MS assay was compared with that of LiBA in 400 human serum samples (100 chronic hepatitis, 100 liver cirrhosis, and 200 HCC). Integrated multinational guidelines were followed to validate the assay for clinical implementation. The linearity ranged from 1.28 to 100,000 ng/mL, and the use of a labeled protein analog as an internal standard allowed the error from the sample preparation to be corrected, improving the precision and accuracy. The SRM-MS assay was validated to meet all of the criteria of the compliance with guidelines per the US Food and Drug Administration (FDA), European medicines agency (EMA), Korea FDA (KFDA), and Clinical & Laboratory Standards Institute (CLSI). We have developed and validated a robust and reproducible SRM-MS assay that is superior to the conventional method of distinguishing HCC from noncancer patients, based on PIVKA-II levels, and satisfies clinical standards. This method has potential applications in quantifying other protein biomarkers.