Development of alternative HPLC method for determination of tirofiban in rat serum

Marija Darkovska-Serafimovska, Emilija Janevik-Ivanovska, Trajan Balkanov, Nenad Ugresic


Tirofiban hydrochloride is a reversible antagonist of fibrinogen binding to the GPIIb/IIIa receptor, used for the treatment of acute coronary syndrome. A novel RP-HPLC method has been developed and validated for the determination of Tirofiban in serum of Wistar rats with and without deep acute venous thrombosis. The chromatographic separation was carried out using a reverse-phase HPLC column Purospher® RP-18e (150 mm ´ 4.6 mm i.d.; 5 μm) coupled with a guard column LiChrosorb® (4 mm ´ 4 mm i.d.; 7 μm) and mobile phase consisting of the mixture of 1-octane sulfonic acid in water (pH 3.0, adjusted with orthophosphoric acid) and acetonitrile, with a ratio of 60 : 40 (v/v) and a flow rate of 1.0 ml/min, at a wavelength of 277 nm. The serum concentrations of Tirofiban in the group of rats with DVT were lower than those in the control group, and it could be explained with the binding of Tirofiban with the GPIIb/IIIa receptors. 


Tirofiban; Deep acute venous thrombosis (DVT); HPLC Determination; Rat serum

Full Text:



A. Giordano, A. D'Angelillo , S. Romano, P. D'Arrig, N. Corcione, R. Bisogni, S. Messina, M. Polimeno, P. Pepino, P. Ferraro, M. F. Romano, Tirofiban induces VEGF production and stimulates migration and prolifer-ation of endothelial cells, Vascul Pharmacol., 61(2–3), 63–71 (2014).

M. Hashemzadeh, M. Furukawa, S. Goldsberry, M. R. Movahed, Chemical structures and mode of action of in-travenous glycoprotein IIb/IIIa receptor blockers: A re-view, Exp Clin Cardiol., Winter; 13(4), 192–197 (2008).

L. L. Brunton, J. S. Lazo, K. Parker, Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 11th ed.; The McGraw Hill Co., 2006.

J. I. Weitz. Blood Coagulation and Anticoagulant, Fibrinolytic, and Antiplatelet Drugs. In: Goodman and Gilman's The Pharmacological Basis of Therapeutics, 12th ed., New York; The McGraw Hill Co, 2011, 849–877.

A. Kumar, H. C. Herrmann, Tirofiban: an investigational platelet glycoprotein IIb/IIIa receptor antagonist, Exp Opin Invest Drugs, 6, 1257–67 (1997).

The Platelet Receptor Inhibition in Ischemic Syndrome Management in Patients Limited by Unstable Signs and Symptoms (PRISM-PLUS) Study Investigators. Inhibi-tion of the platelet glycoprotein IIb/IIIa receptor with Tirofiban in unstable angina and non-Q-wave myocardial infarction. N Engl J Med, 338, 1488–97 (1998).

The Study Investigators. Effects of platelet glycoprotein IIb/IIIa blockade with Tirofiban on adverse cardiac events in patients with unstable angina or acute myocardial infarction undergoing coronary angioplasty. Circulation, 96, 1445–53 (1997).

L. Brass, Understanding and evaluating platelet function, Hematology Am Soc Hematol Educ Program, 387–396 (2010).

/020912s018,0209 13s017lbl.pdf assessed on 24th of October, 2014.

Anon, Tirofiban hydrochloride, Drugs Future, 20, 897–901 (1995).

K. Kondo, K. Umemura, Clinical pharmacokinetics of Tirofiban, a nonpeptide glycoprotein IIb/IIIa receptor antagonist: comparison with the monoclonal antibody abciximab, Clin Pharmacokinet, 41(3), 187–95 (2002).

Medicure Pharma, Aggrastat (Tirofiban hydrochloride) injection premixed and injection prescribing information, Somerset, NJ; Nov. (2007).

/020912s018,0209 13s017lbl. pdf, assessed on 14th of July 2012.

D. W. Bougie, P. R. Wilker, E. D. Wuitschick, B. R. Curtis, M. Malik, S. Levine, R. N. Lind, J. Pereira, R. H. Aster, Acute thrombocytopenia after treatment with Tirofiban or eptifibatide is associated with antibodies spe-cific for ligandoccupied GPIIb/IIIa, Journal of the Ameri-can Society of Hematology, 100(6), 2071–2076 (2002).

R. Oertel, A. Köhler, A. Koster, W. Kirch, Determination of Tirofiban in human plasma by liquid chromato¬graphy-tandem mass spectrometry, J Chromatogr B Analyt Technol Biomed Life Sci., 805(1), 181–5 (2004).

M. Darkovska Serafimovska, E. Janevik-Ivanovska, Z. Arsova-Sarafinovska, I. Djorgoski, N. Ugresic, Develop-ment and validation of reversed phase high performance liquid chromatographic method for determination of Tirofiban in serum, Int J Pharm, 4(4), 115–120 (2014).

K. Sridevi Ranjitha, A. Lakshmana Rao, Development and validation of new RP-HPLC method for the deter-mination of Tirofiban in pharmaceutical formulation, IJPCBS, 1(1), 43–47 (2011).

ICH Q2R1: Validation of Analytical Procedures: Text and Methodology. Proceeding of the International Con-ference on Harmonization of Technical Requirements for the Registration of Drugs for Human Use, Geneva, Switzerland, 1996.

ICH, Guideline on Analytical Method Validation, Pro-ceeding of International Convention on Quality for the Pharmaceutical Industry, Toronto, Canada, 2002.

J. Lister-James, A. Mauer, Thrombus imaging with a technetium 99m labeled, activated platelet receptor binding peptide, J. Nucl. Med., 213, 207 (1996).

J. W. Callas, D. D. J. Fareed, A survey of animal models to develop new and novel antithrombotic agents, New Therapeutic Agents in Thrombosis and Thrombolysis (Sasahara, A. A., Loscalzo, J. L., Eds), 1997, 9–28.

J. M. Herbert, A. Bernat, J. P. Maffrand, Importance of platelets in experimental venous thrombosis in the rat, Blood, 80 (9), 2281–2286 (1992).

M. Darkovska Serafimovska, E. Janevik-Ivanovska, N. Ugresic, I. Djorgoski, Imaging of deep venous throm-bosis using radioactive-labeled tirofiban. Bratisl Med J, 116 (10), 621–626 (2015).

M. Gibaldi, D. Perrier, Pharmacokinetics, 2nd edition, Marcel Dekker, Inc. New York, USA, 1982.



  • There are currently no refbacks.

Copyright (c) 2016 Marija Darkovska-Serafimovska

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.