Development of new micellar electrokinetic chromatography method for the analysis of febuxostat and its impurities

Analiza onečišćenja sastavni je dio kontrole kakvoće lijekova zbog čega je nužan razvoj jednostavnih i selektivnih analitičkih metoda za analizu potencijalnih onečišćenja. Febuksostat je novi lijek za giht koji se u kliničkim istraživanjima pokazao znatno učinkovitijim u snižavanju razine mokraćne kiseline u serumu u odnosu na liječenje uobičajeno korištenim dozama alopurinola. Za analizu febuksostata i njegovih onečišćenja (DEE, amidno i etilno onečišćenje), uz pravastatin kao unutarnji standard, odabrana je micelarna elektrokinetička kromatografija zbog kratkog vremena analize i ekološke prihvatljivosti. Tijekom razvoja MEKC metode optimizirani su uobičajeni parametri MECK metode (vrsta i pH pufera, napon i temperatura, valna duljina detektora, vrsta i koncentracija surfaktanta) kako bi se osiguralo razdvajanje svih analita u što kraćem vremenu analize. Optimalni uvjeti analize bili su 20 mM boratni pufer pH 9,3, 50 mM SDS, primijenjeni napon 30 kV, 20°C, detekcija pri 237 nm, hidrodinamičko injektiranje 50 mbar, 4 s. Pri ovim uvjetima omogućeno je razdvajanje analita unutar 2 minute uz prisutnost α-ciklodekstrina ili unutar 6 minuta bez α-ciklodekstrina. Osigurati topljivost amidnog onečišćenja je bilo zahtjevno, a većina ispitanih otapala je značajno utjecala na rezultate analize. Nadalje, ispitan je dodatak različitih aditiva (α-ciklodekstrin (10 – 15 mM) i 1% Triton, neionski surfaktant) s ciljem postizanja simetričnog pika etilnog onečišćenja. Modifikacija uvjeta analize pomoću drugih aditiva pri kojima bi se postigao bolji oblik i simetričan pik etilnog onečišćenja bit će predmet daljnjeg istraživanja. Budući da je analiza onečišćenja sastavni dio svakodnevne industrijske proizvodnje lijekova, nužno je razviti što više pouzdanih, točnih i preciznih metoda koje će omogućiti kratkotrajne analize koje su ujedno i ekološki prihvatljive.Impurity analysis is an integral part of the quality control of medicines. Therefore, development of simple and selective methods for monitoring impurities is highly required. Febuxostat is a new drug for gout. In clinical research, it was shown that febuxsostat reduces amount of blood uric acid levels much better than standard doses of allopurinol. Micellar electrokinetic chromatographic approach was chosen to develop a method able to separate a febuxostat and its impurities (DEE, ethyl, amide) with pravastatine as internal standard because of the short time analysis and environmental safety. During the development of MECK method, the most usual parameters were optimized (pH and buffer type, voltage and temperature, detector wavelength, the type and concentration of surfactant) to ensure separation of all analytes in the shortest analysis time. The optimum conditions of analysis were 20 mM borate buffer pH 9.3, 50 mM SDS, applied voltage is 30 kV, 20°C, detection at 237 nm, hydrodynamic injection of 50 mbar, 4 s. Under these conditions the separation was successfull within 2 minutes in the presence of α-cyclodextrin or within 6 minutes without α-cyclodextrin. To ensure solubility of amide was demanding, a majority of tested solvents significantly affected the results of the analysis. Furthermore, the addition of various additives was examined (α-cyclodextrin (10 - 15 mM) and 1% Triton, non-ionic surfactant) in order to achieve a symmetrical ethyl peak. Modification of the conditions of analysis with other additives to achieve better and symmetrical shape will be the subject of further research. Since the analysis of impurities is integral part of industrial production of drugs, it is necessary to develop a more reliable, accurate and precise method that would allow short-term analysis witch are also environmentally friendly