HPLC DETECTION OF ANTITHROMBITIC CALIX[4]ARENE IN BLOOD PLASMA OF ANIMALS

Abstract

Previously sodium salt of 5,11,17,23-bis (dihydroxyphosphoryl) methylcalix[4]arene (C-145) was shown to be promising antithrombotic agent. Aim. This work was focused on the development of the method for the direct detection of this substance in blood plasma and estimation of pharmacokinetics of this compound. Methods. C-145 was injected into the Wistar rat’s lateral tail vein and into rabbit’s marginal vein of the ear (12 mg/kg) or was administrated per-oral. The anticoagulant effects of C-145 in blood plasma were confirmed by activated partial thromboplastin time (APTT) test. HPLC was performed using Agilent 1100 series (Agilent, USA) on the phase cyano ZorbaxCN Column which parameters were L×I.D. 25 cm×4.6 mm. Results. The maximal antithrombotic effect after the intravenous or per-oral administration of C-145 was observed after 4-6 hours. In particular clotting time in APTT-test in these blood plasma samples was prolonged trice and more (120 s against 46 s in control). Normalization of blood clotting was achieved after 24 hours after the injection. To develop a method for direct C-145 detection in blood plasma we selected samples with maximal prolongation of clotting time. For accurate analysis of blood plasma samples proteins were saturated by 10 % trichloroacetic acid. After neutralization by NaHCO3 samples were prepared using 12-port vacuum unit for solid-phase extraction (Agilent, USA) with a Bond-Elut C18 cartridge. Samples that contained C-145 were eluted by 100% methanol for the HPLC analysis performed on the phase cyano ZorbaxCN Column equilibrated with an acetonitrile solution (ddH2O:AcCN 99:1). Elution was performed using a combined gradient of acetonitrile (100 %) and citrate buffer (0.1 M, pH 6.0). The elution zone of C-145 was detected on the 128th minute at 280 nm. Conclusion. Application of the developed methods allowed us to confirm the direct antithrombotic effect of calix[4]arene C-145 on blood of experimental animals during intravenous administration. Also HPLC technique enabled to detect this substance in blood plasma and most likely could be applied for other biological solutions and could be modified for the quantitative analysis in the pharmacokinetic studies as well.