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

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Abstract

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.

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

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Abbreviations

ABC:

Ammonium bicarbonate

ACN:

Acetonitrile

CV:

Coefficient of variation

DCP (also known as PIVKA-II):

Des-gamma-carboxy prothrombin

DTT:

Dithiothreitol

FA:

Formic acid

Gla:

Gamma-carboxy glutamic acid

Glu:

Glutamic acid

HCC:

Hepatocellular carcinoma

IAA:

Iodoacetamid

LC:

Liquid chromatography

LLOQ:

Lower limit of quantification

LOD:

Limit of detection

LOQ:

Limit of quantification

MRM-MS:

Multiple reaction monitoring-mass spectrometry

QqQ:

Triple quadrupole

SIS:

Stable isotope-labeled internal standard

ULOQ:

Upper limit of quantification

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Acknowledgements

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).

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    Authors and Affiliations

    1. Department of Biomedical Sciences, Seoul National University College of Medicine, Yongon-Dong, Seoul, 110-799, Korea

      Areum Sohn, Hyunsoo Kim & Youngsoo Kim

    2. Department of Biomedical Engineering, Seoul National University College of Medicine, Yongon-Dong, Seoul, 110-799, Korea

      Areum Sohn, Hyunsoo Kim & Youngsoo Kim

    3. Institute of Medical and Biological Engineering, Medical Research Center, Seoul National University College of Medicine, Yongon-Dong, Seoul, 110-799, Korea

      Hyunsoo Kim & Youngsoo Kim

    4. Department of Internal Medicine, Seoul National University College of Medicine, Yongon-Dong, Seoul, 110-799, Korea

      Su Jong Yu & Jung-Hwan Yoon

    5. Liver Research Institute, Seoul National University College of Medicine, Yongon-Dong, Seoul, 110-799, Korea

      Su Jong Yu & Jung-Hwan Yoon

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    1. Areum Sohn
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    Areum Sohn and Hyunsoo Kim contributed equally to this work.

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    Sohn, A., Kim, H., Yu, S.J. et al. A quantitative analytical method for PIVKA-II using multiple reaction monitoring-mass spectrometry for early diagnosis of hepatocellular carcinoma. Anal Bioanal Chem 409, 2829–2838 (2017). https://doi.org/10.1007/s00216-017-0227-8

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