Simultaneous determination of antiepileptics in biological samples using HPLC method : diploma thesis

Ciljevi istraživanja: Razviti metodu za simultano određivanje karbamazepina, lamotrigina i fenobarbitona koristeći visoko djelotvornu tekućinsku kromatografiju, odrediti optimalne uvjete metode za određivanje antiepileptika visoko djelotvornom tekućinskom kromatografijom, te ispitati valjanost razvijene metode na biološkim uzorcima krvi. Ustroj istraživanja: eksperimentalna studija Mjesto istraživanja: Kemijsko-toksikološki laboratorij Kliničkog odjela za sudsku medicinu, Zavoda za patologiju, citologiju i sudsku medicinu, Kliničkog bolničkog centra u Splitu Materijali i metode: Pripremljene su otopine lamotrigina, fenobarbitona i karbamazepina te odgovarajuća mobilna faza, miješanjem pufera, metanola i acetonitrila u različitim omjerima. Ispitivani su parametri za odabir optimalne metode za simultano određivanje sva tri antiepileptika koristeći HPLC metodu, uključujući promjenu valne duljine (306 nm, 270 nm i 220 nm), temperature (30 °C i 40 °C) i protoka mobilne faze (0,5- 1,4 mL/min). Izuzeti biološki uzorci krvi, s dodatkom otopina lijekova, ekstrahirani su metodom tekuće-tekuće (LLE), te su injektirani u HPLC uređaj. Rezultati: HPLC analizom je uspješno dokazano prisustvo lamotrigina na sve tri ispitivane valne duljine, s najizraženijim odzivom na valnoj duljini od 270 nm, dok je prisustvo fenobarbitona dokazano samo pri mjerenju na valnoj duljini 270 nm. Iz tog razloga valna duljina od 270 nm odabrana je za daljnje ispitivanje karbamazepina, čije je prisustvo pod tim uvjetima uspješno dokazano. Prilagodbom protoka mobilne faze uspješno je skraćeno mrtvo vrijeme analize. Primjenom razvijene metode na realne biološke uzorke krvi simultano su određena sva tri ispitivana antiepileptika. Zaključak: Analizom provedenom visokodjelotvornom tekućinskom kromatografijom uspješno je simultano dokazano prisustvo tri antiepileptika: lamotrigina, fenobarbitona i karbamazepina. Razvijena optimalna metoda i parametri za kvalitativnu i kvantitativnu analizu tri antiepileptika uspješno je primijenjena na realne biološke uzorke. Od velike je važnosti razviti metode za određivanje koncentracije antiepileptika ponajviše zbog prilagođavanja terapija pacijenata. Za daljnje istraživanje preporučuje se analizirati veći broj antiepileptika za određivanje što učinkovitije i brže metode primjenjive u svakodnevnom laboratorijskom radu.Objectives: Development of a method for the simultaneous determination of carbamazepine, lamotrigine and phenobarbital by using highly effective liquid chromatography, to determine the optimal conditions for the determination of antiepileptics by high performance liquid chromatography and to investigate the efficacy of the chromatographic method on biological samples of blood. Design: Experimental study Settings: Laboratory of toxicology, Department of pathology, medicine and cytology, University Hospital of Split Materials and methods: Lamotrigine, phenobarbital and carbamazepine solutions were prepared and the appropriate mobile phase was mixed, by mixing the buffer, methanol and acetonitrile in various ratios. The parameters for selecting the optimal method for simultaneous determination of all three antiepileptics using the HPLC method were investigated, including a change of wavelength (306 nm, 270 nm and 220 nm), temperature (30 ° C and 40 ° C) and mobile phase flow (0.5 - 1.4 mL / min). Biological blood samples, supplemented with drug solutions, were extracted by liquid-liquid (LLE) method and injected into an HPLC device. Results: HPLC analysis has successfully demonstrated the presence of lamotrigine on all three wavelengths investigated, with the most pronounced response at wavelength of 270 nm, while the presence of phenobarbital is only proved when measuring at wavelength 270 nm. For that reason, the wavelength of 270 nm was selected for further testing of carbamazepine, whose presence under these conditions has been successfully proved. Adapting the mobile phase flow shortened the dead time of the analysis. By applying the developed method to real biological blood samples simultaneously, all three antiepileptics tested were determined . Conclusion: High performance liquid chromatography was successfully used for simultaneous determintaion of three antiepileptics: lamotrigine, phenobarbital and carbamazepine. An optimal method and parameters for qualitative and quantitative analysis of three antiepileptics were determined separately and in biological samples. It is of great importance to develop methods for determining the concentration of antiepileptics mainly due to adaptation of patient therapy. For further research it is recommended to use a greater number of antiepileptics to determine the most efficient and quickest method applicable to daily laboratory work