A quantitative analytical method for PIVKA-II using multiple reaction monitoring-mass spectrometry for early diagnosis of hepatocellular carcinoma
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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.
KeywordsMultiple Reaction Monitoring (MRM) Mass spectrometry Hepatocellular carcinoma (HCC) Prothrombin induced by vitamin K absence-II (PIVKA-II) Absolute quantification
Coefficient of variation
- DCP (also known as PIVKA-II)
Gamma-carboxy glutamic acid
Lower limit of quantification
Limit of detection
Limit of quantification
Multiple reaction monitoring-mass spectrometry
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.
- 5.Beale G, Chattopadhyay D, Gray J, Stewart S, Hudson M, Day C, et al. AFP, PIVKAII, GP3, SCCA-1 and follisatin as surveillance biomarkers for hepatocellular cancer in non-alcoholic and alcoholic fatty liver disease. BMC Canc. 2008;8:200. doi: 10.1186/1471-2407-8-200.
- 18.Suzuki K, Tamano M, Kuniyoshi T, Katayama Y, Takada H, Suzuki K. Positioning of novel tumor marker NX-PVKA-R in the diagnosis of hepatocellular carcinoma in comparison with PIVKA-II. Dokkyo J Med Sci. 2013;40(3):163–8.Google Scholar
- 29.Yu R, Xiang X, Tan Z, Zhou Y, Wang H, Deng G. Efficacy of PIVKA-II in prediction and early detection of hepatocellular carcinoma: a nested case-control study in Chinese patients. Sci Rep. 2016;6:35050. doi: 10.1038/srep35050.