Title Analiza ribocikliba i anastrozola HPLC-DAD metodom u krvnoj plazmi nakon taloženja proteina Title (english) Analysis of ribociclib and anastrozole using HPLC-DAD method in blood plasma after protein precipitation Author Denis Pleše Mentor Miranda Sertić (mentor)Committee member Miranda Sertić (predsjednik povjerenstva)Committee member Petra Turčić (član povjerenstva)Committee member Nina Kočevar Glavaš (član povjerenstva) Granter University of Zagreb Defense date and country 2023-09-19, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Abstract Anastrozol i ribociklib koriste se u kombinaciji kao inicijalna endokrina terapija ER pozitivnog tumora dojke u
postmenopauzalnih žena. Anastrozol djeluje na smanjenje raspoloživog estrogena u tijelu koji je potreban za proliferaciju tumora,
dok ribociklib usporava stanični ciklus tumorskih stanica na taj način smanjujući mogućnost nastanka rezistencije na terapiju.
Cilj rada bio je razviti selektivnu analitičku metodu za istovremenu analizu ribocikliba i anastrozola u ljudskoj plazmi kao
temelj daljnjem razvoju osjetljive metode za određivanje ovih lijekova u plazmi pacijenata.
U tekućinskoj kromatografiji visoke djelotvornosti korištena je stacionarna faza XBridge (Waters) Phenyl (4,6 x 150 mm,
3,5 μm). Temperatura kolone podešena je na 25 °C. Ispitane su mobilne faze različitih omjera acetonitrila i vode s 0,1 %-tnom
mravljom kiselinom. Ispitana je izokratna eluacija i gradijentno ispiranje, kao i različite brzine protoka mobilne faze. Najpovoljnijom se pokazala gradijentna eluacija u kojoj se omjer vode i acetonitrila mijenjao sa 75:25 na 15:85 (V/V) u prvih 6 minuta analize pri brzini protoka od 0,5 mL/min, pri čemu su vremena zadržavanja ribocikliba i anastrozola iznosila 6,4 min i 8,7 min. Za detekciju je korišten detektor niza dioda. Određeni su UV-spektri oba analita te odabrane valne duljine detekcije od 220 nm za anatrozol i 270 nm za ribociklib. Radi izbjegavanja solvent effecta prilikom primjene acetonitrila, kao optimalno otapalo za analizu korištena je smjesa metanola i vode u omjeru 50:50 (V/V). Fenazon je odabran kao unutarnji standard zbog sličnog ponašanja analitima prilikom obrade složenog biološkog uzorka, pogodnog vremena zadržavanja (6,16 min) i svojstava (polarnost, molekulska masa) bliskih analitima. Plazma je po standardnom je postupku uz EDTA kao antikoagulans centrifugiranjem ekstrahirana iz pune krvi. Za uklanjanje matriksa, odnosno taloženje proteina plazme isprobane su metode s različitim omjerima metanola i acetonitrila uz i bez dodatka fosfatne kiseline. Fosfatna kiselina u dodatku organskim otapalima nije pokazala značajno smanjenje interferencija,dok je za taloženje proteina s metanolom potreban vrlo velik voluemn otapala. Optimalnim se pokazao postupak taloženja proteina acetonitrilomm u volumnom omjeru acetonitril:plazma 4:1, uz uparavanje otapala do suha i resuspendiranjem u 50 %-tnom metanolu. Prema ICH smjernicama za validaciju bioanalitičkih metoda na šest slijepih uzoraka plazme šestero različitih donora ispitana je selektivnost razvijene metode.
Abstract (english) Anastrozole and ribociclib are used in combination as initial endocrine therapy for ER-positive breast tumors in
postmenopausal women. Anastrozole works to reduce the available estrogen in the body, which is necessary for tumor proliferation, while ribociclib slows down the cell cycle of tumor cells, thereby reducing the possibility of resistance to therapy. The objective of this work was to develop a selective analytical method for the simultaneous analysis of ribociclib and anastrozole in human plasma as a basis for further development of a sensitive method for determining these drugsin patients' plasma. XBridge (Waters) Phenyl (4.6 x 150 mm, 3.5 μm) stationary phase was used in high-performance liquid chromatography. The column temperature was set at 25 °C. Various mobile phase combinations of acetonitrile and water with 0.1% formic acid were tested, including isocratic elution, gradient washing, and different mobile phase flow rates. The most favorable method was gradient elution, where the ratio of water to acetonitrile changed from 75:25 to 15:85 (V/V) during the first 6 minutes of the analysis, with a flow rate of 0.5 mL/min. The retention times for ribociclib and anastrozole were 6.4 min and 8.7 min, respectively. A diode array detector was employed for detection. The UV spectra of both analytes and the selected detection wavelengths of 220 nm for anastrozole and 270 nm for ribociclib were determined. To mitigate the solvent effect when using acetonitrile, a mixture of methanol and water in a ratio of 50:50 (V/V) was utilized as the optimal solvent for the analysis. Phenazone was chosen as an internal standard due to its similar behavior to the analytes when processing complex biological samples, its convenient retention time (6.16 min), and properties (polarity, molecular weight) close to those of the analytes. The plasma is extracted from whole blood by centrifugation using EDTA as an anticoagulant in accordance with the standard procedure. Various methods involving different ratios of methanol and acetonitrile, with and without the addition of phosphoric acid, were tested for matrix removal, which refers to the deposition of plasma proteins. The addition of phosphoric acid to organic solvents did not significantly reduce interferences, while protein precipitation with methanol required a large volume of solvent. The protein precipitation method with acetonitrile in a volume ratio of acetonitrile:plasma of 4:1, followed by solvent evaporation to dryness and resuspension in 50% methanol, proved to be optimal. In accordance with the ICH guidelines for the validation of bioanalytical methods, the selectivity of the developed method was tested using six blank plasma samples from six different donors.
Keywords
anastrozol
ribociklib
HPLC-DAD
krvna plazma
Keywords (english)
anastrozole
ribociclib
HPLC-DAD
blood plasma
Language croatian URN:NBN urn:nbn:hr:163:572582 Study programme Title: Pharmacy Type of resource Text File origin Born digital Access conditions Embargoed access Terms of use Created on 2024-01-31 11:42:41
