Determination of dabigatran, rivaroxaban and apixaban by ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) and coagulation assays for therapy monitoring of novel direct oral anticoagulants.

BACKGROUND: Three novel direct oral anticoagulants (DOACs) have recently been registered by the Food and Drug Administration and European Medicines Agency Commission: dabigatran, rivaroxaban, and apixaban. To quantify DOACs in plasma, various dedicated coagulation assays have been developed. OBJECTIVE: To develop and validate a reference ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) method and to evaluate the analytical performance of several coagulation assays for quantification of dabigatran, rivaroxaban, and apixaban. METHODS: The developed UPLC-MS/MS method was validated by determination of precision, accuracy, specificity, matrix effects, lower limits of detection, carry-over, recovery, stability, and robustness. The following coagulation assays were evaluated for accuracy and precision: laboratory-developed (LD) diluted thrombin time (dTT), Hemoclot dTT, Pefakit PiCT, ECA, Liquid anti-Xa, Biophen Heparin (LRT), and Biophen DiXal anti-Xa. Agreement between the various coagulation assays and UPLC-MS/MS was determined with random samples from patients using dabigatran or rivaroxaban. RESULTS: The UPLC-MS/MS method was shown to be accurate, precise, sensitive, stable, and robust. The dabigatran coagulation assay showing the best precision, accuracy and agreement with the UPLC-MS/MS method was the LD dTT test. For rivaroxaban, the anti-factor Xa assays were superior to the PiCT-Xa assay with regard to precision, accuracy, and agreement with the reference method. For apixaban, the Liquid anti-Xa assay was superior to the PiCT-Xa assay. CONCLUSIONS: Statistically significant differences were observed between the various coagulation assays as compared with the UPLC-MS/MS reference method. It is currently unknown whether these differences are clinically relevant. When DOACs are quantified with coagulation assays, comparison with a reference method as part of proficiency testing is therefore pivotal.

Autocrine production of prostaglandin F2alpha enhances phenotypic transformation of normal rat kidney fibroblasts.

We have used normal rat kidney (NRK) fibroblasts as an in vitro model system to study cell transformation. These cells obtain a transformed phenotype upon stimulation with growth-modulating factors such as retinoic acid (RA) or transforming growth factor-beta (TGF-beta). Patch-clamp experiments showed that transformation is paralleled by a profound membrane depolarization from around -70 to -20 mV. This depolarization is caused by a compound in the medium conditioned by transformed NRK cells, which enhances intracellular Ca2+ levels and thereby activates Ca2+-dependent Cl- channels. This compound was identified as prostaglandin F2alpha (PGF2alpha) using electrospray ionization mass spectrometry. The active concentration in the medium conditioned by transformed NRK cells as determined using an enzyme immunoassay was 19.7 +/- 2.5 nM (n = 6), compared with 1.5 +/- 0.1 nM (n = 3) conditioned by nontransformed NRK cells. Externally added PGF2alpha was able to trigger NRK cells that had grown to density arrest to restart their proliferation. This proliferation was inhibited when the FP receptor (i.e., natural receptor for PGF2alpha) was blocked by AL-8810. RA-induced phenotypic transformation of NRK cells was partially (approximately 25%) suppressed by AL-8810. Our results demonstrate that PGF2alpha acts as an autocrine enhancer and paracrine inducer of cell transformation and suggest that it may play a crucial role in carcinogenesis in general.