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Abstract: Accurate measurement of in vitro cell growth is critical for oncology drug development, but cell counting and the most accurate indirect proliferation assays are impractical. Here, we describe a robust alternative method that monitors proliferating cell thymidine kinase 1 (TK1) activity via LC–MS/MS quantification of 3′-deoxy-3′-fluorothymidine (FLT) and its monophosphate metabolite FLT-MP. LNCaP prostate cancer cells were cultured at four densities (20,000; 10,000; 5000; and 500cells/well) and incubated with 2000ng/mL FLT in multi-well plates. Internal standards were FLT-d3 for FLT and d4-thymidine for FLT-MP. In culture medium, peak area ratios of FLT to FLT-d3 and FLT-MP to d4-thymidine were linear over the range 0.25–100ng/mL (r 2 ≥0.998). Accuracy for quality controls was between −7.3% and 6.3% for FLT, and from −3.3% to 1.7% for FLT-MP. Quality control precision was from 2.4% to 5.7% for FLT and 3.2% to 7.5% for FLT-MP. The limit of quantification was 0.25ng/mL, with good control results (precision of 9.6% for FLT and 14.8% for FLT-MP). FLT-MP formation was linearly proportional to cell number from 500 to 20,000 cells/well 1h after FLT addition. FLT-MP and ATP generation were comparable in LNCaP cells exposed to cell cycle inhibitor drugs (Spearman r =0.925, p <0.0001), demonstrating assay suitability for drug screening. This fit for purpose method is amenable to analysis of tumor tissue extracts, and should enable direct assessment of in vitro–in vivo relationships in animal models of cancer. [Copyright &y& Elsevier]

Abstract: The degradation of type II collagen has been associated with the pathology of osteoarthritis (OA). Matrix metalloproteinases (MMPs) are enzymes that are responsible for catalyzing the degradation of collagen and, therefore, are pursued as potential targets for the treatment of OA. Collagen-derived peptides identified as a reflection of in vivo MMP activity have been investigated as target biomarkers of MMP activity as well as potential biomarkers of OA disease state and/or progression. An immunoaffinity liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay developed for the quantification of the most abundant urinary type II collagen neoepitope (uTIINE) peptide, a 45-mer with 5 HO-proline residues resulting from MMP-13-catalyzed degradation, was validated for clinical use. Validation experiments were designed with attention to specific challenges related to quantification of endogenous analytes. The validated method is sensitive, selective, accurate (<15% relative error) and precise (<15% coefficient of variation) over a linear range of 0.156–7.50ng/ml. Sample stability and inter- and intrasubject variability were evaluated in the urine of normal and OA populations. The method was applied to analyze human urine samples from clinical studies investigating the utility of uTIINE as a potential biomarker for OA. [Copyright &y& Elsevier]

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 0.5–500ng/mL for FLT and 2.5–2000ng/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 0.5ng/mL for FLT and 2.5ng/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. [Copyright &y& Elsevier]

Abstract: An LC–MS/MS method was developed and validated to quantify endogenous cyclic guanosine 3′,5′-monophosphate (cGMP) in human plasma. The LC–MS/MS and competitive enzyme immunoassay (EIA) assays were compared. cGMP concentrations of 20 human plasma samples were measured by both methods. For the MS-based assay, plasma samples were subjected to a simple protein precipitation procedure by acetonitrile prior to analysis by electrospray ionization LC–MS/MS. De-protonated analytes generated in negative ionization mode were monitored through multiple reaction monitoring (MRM). A stable isotope-labeled internal standard, 13C10,15N5 -cGMP, which was biosynthesized in-house, was used in the LC–MS/MS method. The competitive EIA was validated using a commercially available cGMP fluorescence assay kit. The intra-assay accuracy and precision for MS-based assay for cGMP were 6–10.1% CV and −3.6% to 7.3% relative error (RE), respectively, while inter-assay precision and accuracy were 5.6–8.1% CV and −2.1% to 6.3% RE, respectively. The intra-assay accuracy and precision for EIA were 17.9–27.1% CV and −4.9% to 24.5% RE, respectively, while inter-assay precision and accuracy were 15.1–39.5% CV and −30.8% to 4.37% RE, respectively. Near the lower limits of detection, there was little correlation between the cGMP concentration values in human plasma generated by these two methods (R 2 =0.197, P =0.05). Overall, the MS-based assay offered better selectivity, recovery, precision and accuracy over a linear range of 0.5–20ng/mL. The LC–MS/MS method provides an effective tool for the quantitation of cGMP to support clinical mechanistic studies of curative pharmaceuticals. [Copyright &y& Elsevier]