Determination of heparin using terbium-danofloxacin as a luminescent probe

Introduction. Heparin (Hep) is a naturally occurring anticoagulant produced by basophiles and mast cells, with an average molecular weight of 15,000 Da. It consists of repeating units of uronic/glucuronic acid and glucosamine residues. The most common disaccharide unit is composed of a 2-O-sulfated iduronic acid and 6-O-sulfated N-sulfated glucosamine. Hep is widely used as an injectable anticoagulant. Hep is negatively charged in an aqueous solution with the average charge of –70 [1]. It can also be used as an anticoagulant in various experimental and medical devices such as test tubes and renal dialysis machines. Pharmaceutical grade Hep is derived from mucosal tissues of slaughtered meat of animals such as porcine (pig) intestine or bovine (cow) lung. It acts as an anticoagulant, preventing the formation of clots and extension of existing clots within the bloodstream. It is one of the oldest drugs with widespread clinical applications. Hep and its derivatives have a variety of biological activities including antilipemic, antithrombotic, immunoregulatory, antiphlogistic, and antianaphylactic actions [2]. So, the Hep level in the patient’s blood needs to be carefully and accurately monitored during surgery and recovery processes. The reported methods for Hep determination are focused on: fl ow injection analysis [3], ion-channel sensors [4], resonance Rayleigh scattering spectra [5, 6], capillary chromatography [7], high-performance liquid chromatography [8], surface plasmon resonance sensor analysis [9], rotating electrode potentiometry [10], piezoelectric quartz crystal sensor [11], protamine titration using a membrane electrode [12], and extracorporeal membrane oxygenation [13]. To date, some researchers have reported the detection of Hep using rare earth fl uorescent probes such as Tb 3+ [14, 15] or Eu 3+ [16]. But there has been no report on the determination of Hep using Tb 3+ -Danofl oxacin (Dano) as a fl uorescent probe. Dano, an antibacterial agent containing the α-carbonyl carboxylic acid confi guration, is an ideal ligand for Tb 3+ . In this work, Dano is chosen as the ligand of Tb 3+ , and the possibility to enhance the Tb 3+ fl uorescence sensitized by using Hep as a co-ligand has been investigated. Experimental results showed that the characteristic peak of Tb 3+ at 545 nm can be enhanced, with the fl uorescence intensity being proportional to the concentration of Hep. Therefore, a new highly sensitive method for the spectrofl uorimetric determination of Hep was developed and validated. Experimental. Materials and methods. Analytical-grade ethanol, hydrochloric acid (HCl), methanol, 2-propanol, acetonitrile, tris-(hydroxymethyl)aminomethane (Tris) and other buffers were obtained from Merck (Darmstadt, Germany), terbium(III) chloride hexahydrate (TbCl3·6H2O) from Acros Organics (Geel, Belgium), Dano powder from Jamedat Afagh Pharmaceutical Company (Tehran, Iran), and Hep powder from Caspian Tamin Pharmaceutical Co. (Rasht, Iran). Double