Analytical and Bioanalytical Chemistry

, Volume 409, Issue 11, pp 2829–2838 | Cite as

A quantitative analytical method for PIVKA-II using multiple reaction monitoring-mass spectrometry for early diagnosis of hepatocellular carcinoma

  • Areum Sohn
  • Hyunsoo Kim
  • Su Jong Yu
  • Jung-Hwan Yoon
  • Youngsoo KimEmail author
Research Paper


Prothrombin induced by vitamin K absence-II (PIVKA-II) is an effective tumor marker for hepatocellular carcinoma (HCC). We have developed a novel targeted mass spectrometric (MS) assay for quantifying PIVKA-II in human serum. The ideal signature peptide was selected to measure PIVKA-II concentrations on a triple quadrupole (QqQ) mass spectrometer, and the chromatography gradient was optimized for the peptide separation to minimize elution interference. Using multiple reaction monitoring-mass spectrometry (MRM-MS), good linearity (R 2 = 0.9988) was obtained for PIVKA-II over a range of 3 orders. We achieved a limit of detection (LOD) of 0.45 nM (31.72 ng/mL), a limit of quantification (LOQ) of 0.93 nM (65.31 ng/mL), a lower limit of quantification (LLOQ) of 0.49 nM (34.32 ng/mL), and an upper limit of quantification (ULOQ) of 1000.00 nM (70,037.00 ng/mL). The intra-day and inter-day precisions were within ±14.96%, and the accuracy ranged from 87.66 to 114.29% for QC samples at four concentrations. Compared with an established immunoassay, the correlation (R = 0.8335) was good for the measurements of PIVKA-II concentrations. This method was successfully applied to the analysis of clinical samples for normal control (n = 50), chronic hepatitis (n = 50), liver cirrhosis (n = 50), HCC (n = 50), and recovery (n = 50) serum.

Graphical Abstract

MRM-MS assay development for determining concentration of PIVKA-II in serum and a comparison between MRM-MS assay and immunoassay with high correlation


Multiple Reaction Monitoring (MRM) Mass spectrometry Hepatocellular carcinoma (HCC) Prothrombin induced by vitamin K absence-II (PIVKA-II) Absolute quantification 



Ammonium bicarbonate




Coefficient of variation

DCP (also known as PIVKA-II)

Des-gamma-carboxy prothrombin




Formic acid


Gamma-carboxy glutamic acid


Glutamic acid


Hepatocellular carcinoma




Liquid chromatography


Lower limit of quantification


Limit of detection


Limit of quantification


Multiple reaction monitoring-mass spectrometry


Triple quadrupole


Stable isotope-labeled internal standard


Upper limit of quantification



This work was supported by the Multi-omics Research Program through the National Research Foundation and a National Research Foundation grant (No. 2011-0030740), funded by the Korean government [MSIP, Korea]. This work was also supported by the Industrial Strategic Technology Development Program (#10045352), funded by the Ministry of Knowledge Economy (MKE, Korea), and a grant from the Korea Health Technology R&D Project, funded by the Ministry of Health and Welfare (No. HI14C2640). It was also supported by the grant No. 34-2013-005 from the SK Telecom Research Fund (Seoul National University Hospital).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2017_227_MOESM1_ESM.pdf (2 mb)
ESM 1 (PDF 2056 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Areum Sohn
    • 1
    • 2
  • Hyunsoo Kim
    • 1
    • 2
    • 3
  • Su Jong Yu
    • 4
    • 5
  • Jung-Hwan Yoon
    • 4
    • 5
  • Youngsoo Kim
    • 1
    • 2
    • 3
    Email author
  1. 1.Department of Biomedical SciencesSeoul National University College of MedicineSeoulKorea
  2. 2.Department of Biomedical EngineeringSeoul National University College of MedicineSeoulKorea
  3. 3.Institute of Medical and Biological Engineering, Medical Research CenterSeoul National University College of MedicineSeoulKorea
  4. 4.Department of Internal MedicineSeoul National University College of MedicineSeoulKorea
  5. 5.Liver Research InstituteSeoul National University College of MedicineSeoulKorea

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