Liquid Chromatography-Tandem Mass Spectrometry Method for Quantification of Thymidine Kinase Activity in Human Serum by Monitoring the Conversion of 3'-deoxy-3'-fluorothymidine to 3'-deoxy-3'-fluorothymidine Monophosphate

J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Oct 15;907:13-20. doi: 10.1016/j.jchromb.2012.08.024. Epub 2012 Aug 24.

Abstract

Thymidine kinase 1 (TK1) is an enzyme involved in DNA synthesis whose activity in serum is indicative of tumor proliferation and the severity of blood malignancies. 3'-deoxy-3'-fluorothymidine (FLT), a specific exogenous substrate for TK1, is phosphorylated by TK1 in the presence of a phosphorylating buffer, therefore the conversion of FLT to 3'-deoxy-3'-fluorothymidine monophosphate (FLT-MP) can be measured to assess serum TK1 activity. Here we describe a liquid chromatography-MS/MS (LC-MS/MS) method for quantification of FLT and FLT-MP from serum using protein precipitation and column switching followed by detection on an Applied Biosystems SCIEX API 4000 QTrap mass spectrometer. The method was linear over the range of 0.5-500 ng/mL for FLT and 2.5-2000 ng/mL for FLT-MP with a mean correlation coefficient of 0.9964 and 0.9935 for FLT and FLT-MP, respectively. The lower limit of quantification was 0.5 ng/mL for FLT and 2.5 ng/mL for FLT-MP. Intra-assay accuracy and inter-assay accuracy was within ±12% for both FLT and FLT-MP. Intra-assay precision was 2.8% to 7.7% for FLT and 3.3% to 5.8% for FLT-MP. Inter-assay precision was 4.6% to 14.9% for FLT and 4.9% to 14.6% for FLT-MP. Serum TK1 activity was measured in serum from hepatocellular carcinoma patients and age-matched controls under standardized conditions. Elevated TK1 activity was detected in 26.3% of hepatocellular carcinoma samples compared to controls. This method provides a robust alternative to radiometric and immunochemical assays of serum TK1 activity.

MeSH terms

  • Carcinoma, Hepatocellular / blood
  • Carcinoma, Hepatocellular / enzymology
  • Case-Control Studies
  • Chromatography, High Pressure Liquid / methods*
  • Dideoxynucleosides / blood*
  • Dideoxynucleosides / metabolism
  • Drug Stability
  • Female
  • Humans
  • Linear Models
  • Liver Neoplasms / blood
  • Liver Neoplasms / enzymology
  • Male
  • Middle Aged
  • Phosphorylation
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Tandem Mass Spectrometry / methods*
  • Thymidine Kinase / blood*
  • Thymidine Kinase / metabolism
  • Thymidine Monophosphate / analogs & derivatives
  • Thymidine Monophosphate / blood*
  • Thymidine Monophosphate / metabolism

Substances

  • Dideoxynucleosides
  • Thymidine Monophosphate
  • Thymidine Kinase
  • thymidine kinase 1
  • alovudine