Development of analytical method for identification of azathioprine, 6-thioguanine and folic acid in tablets and plasma of patients

Upalne bolesti crijeva, u koje spadaju Chronova bolest i ulcerozni kolitis, su autoimune bolesti nepoznate etiologije, karakterizirane kroničnom upalom u području probavnog trakta. Imunosupresivi su uz aminosalicilate, kortikosteroide, biološku terapiju jedna od glavnih skupina lijekova koji se koriste u liječenju IBD-a. Među njima se najčešće koristi azatioprin i 6-tiogvanin. To su lijekovi koji smanjuju upalnu reakciju, suprimirajući pretjerani imunološki odgovor. Upalne bolesti crijeva dokazano uzrokuju deficit folne kiseline u organizmu oboljelih, koji se povezuje s povećanim rizikom od razvoja kolorektalnog karcinoma. Stoga pacijenti trebaju uzimati nadomjestke folne kiseline. U cilju postizanja veće suradljivosti i adherencije pacijenata, u radu je predložen razvoj jedinstvene analitičke metode za istovremenu identifikaciju azatioprina, 6- tiogvanina i folne kiseline u uzorcima tableta i plazmi pacijenata primjenom vezanog sustava tekućinske kromatografije visoke djelotvornosti i masene spektrometrije. Korištena je binarna gradijentna metoda eluacije budući da su ispitani analiti bili strukturno različiti spojevi. Mjerenja su provedena primjenom instrumenta Agilent 1100 Series LC/MSD Trap. Korištena je C18 analitička kolona Symmetry. Mobilna faza sastojala se od 0,1% mravlje kiseline u metanolu (otapalo A) i 0,1% mravlje kiseline u pročišćenoj vodi (otapalo B). Za snimanje masenih spektara korištena je ionizacija elektroraspršenjem te je dušik korišten kao plin za sušenje i raspršivanje mobilne faze. Zbog veće količine vode u pokretnoj fazi pojačan je protok plina nebulizatora. Na kraju smo dobili relativno kratku metodu za određivanje sva tri analita i ispitana je primjenjivost metode za identifikaciju analita u lijekovima i ˝umjetnoj plazmi˝. Daljnja istraživanja bit će usmjerena na validaciju predložene analitičke metode.Inflammatory bowel diseases, including Chron disease and ulcerative colitis, are autoimmune diseases of unknown etiology characterized by chronic inflammation in digestive tract. Immunosuppressant’s are with amino salicylate, corticosteroids and biological therapy one of the major groups of medicines used in the treatment of IBD. Azathioprine and 6-thioguanine are most frequently used by immunosuppressant. These are drugs that reduce the inflammatory reaction, suppressing an over-immune response. Inflammatory bowel disease can cause folate deficiency. Low folate status may increase the risk of developing cancer, especially cancer of the colorectal. Therefore, patients should take folic acid supplement daily. We proposed the development of a new analytical method for identification of azathioprine, 6-thioguanine and folic acid in tablets and plasma control samples. The aim of this work was to develop a unique method for the determination these drugs using high-performance liquid chromatography with diode-array detector and electrospray ionization mass spectrometry. A binary gradient elution method was used as investigated compounds are structurally different compounds. All experiments were performed using Agilent Series LC/MSD Trap system. A Symmetry C18 analytical column and the mobile phase consisted of 0.1% Formic acid in methanol (eluent A) and of 0.1% Formic acid in purified water were used. The MS system was operated with electrospray ionization source in the positive ion mode. Nitrogen is used both as drying and nebulizing gas. The flow of nebulizer gas was increased because of the higher amount of water in the mobile phase. Finally, a relatively short method for determining all three analytes was proposed and the applicability of the analytical drug identification method was tested. Further studies will be focuses on the validation of the proposed method