Optimization of the liquid chromatography procedure for measurement of the tramadol and its metabolites in plasma

Uvod: Tramadol je često korišten analgetik pri različitim stanjima boli u svakoj životnoj dobi. Njegova upotreba osim same analgezije može uzrokovati razne nuspojave. Tramadol se u organizmu metablizira u aktivni metabolit O-desmetiltramadol i neaktivni metabolit N-desmetiltramadol. Zbog njihove sve češće uporabe i različitih komplikacija koje se javljaju prilikom uzimanja, kliničaru je ponekad važno znati koncentraciju tramadola, a često i aktivnog metabolita. Cilj: Optimizacija HPLC metode s FLD-om za određivanje koncentracije tramadol i njegova dva metabolita u svrhu postizanja što bolje kvantifikacije svih triju analita. Materijal i metode: Isprobane su četiri kolone s različitim mobilnim fazama pri različitim temperaturama separacije i različitim postavkama fluorescentnog detektora. Nakon optimizacije HPLC separacije metoda je djelomično validirana ispitivanjem linearnosti, limita detekcije i kvantifikacije, preciznosti i točnosti. Rezultati: LOQ za sva tri analita nalazi se u intervalu od 3,52 μg/l do 4,52 μg/l. RSD je kod mjerenja ponovljivosti za sve analite bio < 10 %. Iskorištenje za sva tri analita bilo je u intervalu 90 – 110 %. Zaključak: Nakon optimizacije metode i dobivenih rezultata djelomične validacije možemo zaključiti da je metoda prihvatljiva za određivanje tramadola i njegovih metabolita u uzorcima plazme.Introduction: Tramadol is often used as an analgesic to relieve pain symptoms and other conditions. It is appropriate and safe for all ages groups. Besides analgesia, it can cause various adverse effects in organism. Tramadol is metabolized in two main metabolites, active O-desmetyltramadol and inactive N-desmetyltramadol. Because of their more frequent usage and different adverse effects which occur during treatment with tramadol, sometimes it is important for the clinicant to know the concentration of tramadol and its active metabolite. Goals: HPLC method optimization with FLD for determing plasma tramadol concentration and its two main metabolites with a view to achieve optimal quantification of all three analytes. Materials and methods: Four columns were tested with two different mobile phases at different separation temperatures and with different FLD settings. After the optimization of HPLC separation, method is partially validated by the examining the linearity, detection limit range and quantification as well as precision and accuracy. Results: LOQ for all three analytes is in the range 3,52 μg/l – 4,52 μg/l. The RSD for repeatability of method was for all analytes < 10 %. Accuracy of method for all three analytes was in range 90 – 110 %. Conclusion: After the optimization of method and performed partial validation of the results, it can be concluded that the method is suitable for the determination of tramadol and its two main metabolites in plasma samples