title: 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.
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.
authors.affiliation: Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Morse
authors.initials: M
authors.lastname: Faria
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Halquist
authors.forename: Erick
authors.initials: E
authors.lastname: Kindt
authors.forename: Wenlin
authors.initials: W
authors.lastname: Li
authors.forename: H Thomas
authors.initials: HT
authors.lastname: Karnes
authors.forename: Peter J
authors.initials: PJ
authors.lastname: O'Brien
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000201:enzymology; D016022:Case-Control Studies; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015224:Dideoxynucleosides / Q000097:blood* / Q000378:metabolism; D004355:Drug Stability; D005260:Female; D006801:Humans; D016014:Linear Models; D008113:Liver Neoplasms / Q000097:blood / Q000201:enzymology; D008297:Male; D008875:Middle Aged; D010766:Phosphorylation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013937:Thymidine Kinase / Q000097:blood* / Q000378:metabolism; D013938:Thymidine Monophosphate / Q000031:analogs & derivatives / Q000097:blood* / Q000378:metabolism
chemical_list: D015224:Dideoxynucleosides; D013938:Thymidine Monophosphate; D013937:Thymidine Kinase; C092717:thymidine kinase 1; C002854:alovudine