| Abstract | Anastrozol i ribociklib se u kombiniranoj primjeni koriste za liječenje ER pozitivnih tumora dojke kod žena
u postmenopauzi. Cilj rada bio je validirati analitičku metodu za istovremenu analizu ribocikliba i anastrozola u ljudskoj
plazmi.
Kromatografska separacija analita provodila se na XBridge Phenyl koloni pri temperaturi 35 °C. Primijenjena
je gradijentna eluacija kombinacijom mobilnih faza (A: voda i mravlja kiselina; B: metanol i mravlja kiselina), uz brzinu
protoka 0,6 mL/min. Detekcija je vršena pomoću spektrometra masa (fragmentacija ribociklib m/z 435.2→ m/z
322.1(25eV) + m/z 252.1(30eV), anastrozol m/z 294.1→ m/z 225.4 (25eV) + m/z 115.2 (70eV)).
Linearnost metode ispitana je na rasponu koncentracija od 700 do 3500 ng/mL za ribociklib te od 20 do
100ng/ml za anastrozol. Preciznost i točnost metode ispitavani su na 3 koncentracijske razine (LLOQ, do 3xLLOQ,
80%ULOQ). Dobivene su zadovoljavajuće RSD vrijednosti ponovljivosti (ribociklib → 4,71%, 1,33%, 1,51%;
anastrozol → 9,23%, 6,43%, 4,31%) i srednje preciznosti (ribociklib → 6,23%, 3,97%, 2,28%; anastrozol → 11,35%,
11,40%, 14,78%). Izračunati analitički prinosi (ribociklib: unutar dana → 11,22%, 8,94%, −3,95%; između dana →
−1,85%, 8,21%, −3,89%; anastrozol: unutar dana → −10,92%, −6,12%, −4,55%; između dana → −8,98%, −4,66%,
−1,55%) zadovoljili su validacijski kriterij točnosti. Ispitani su i parametri injekcijske reproducibilnosti (RSD<7%) i
carry-over efekta (<2%) te su dobivene vrijednosti oba parametra zadovoljile validacijske kriterije. Prema ICH
smjernicama za validaciju bioanalitičkih metoda na šest slijepih uzoraka plazme (različitih donora) na visokim i niskim
koncentracijama ispitan je utjecaj matriks efekta na rezultate. Prilikom određivanja ribocikliba došlo je do ionskog
pojačanja u svim uzorcima, osim u hemolitičkom i lipemičnom. Utjecaj matriksa kod anastrozola uzrokovao je ionsku
supresiju reproducibilnu kroz sve uzorke i ispitane koncentracijske razine. Po svim ispitanim validacijskim parametrima
ova je metoda zadovoljavajuća za istovremeno određivanje koncentracije ribocikliba i anastrozola u plazmi. |
| Abstract (english) | Anastrozole and ribociclib are used in combination for the treatment of ER-positive breast tumors
in postmenopausal women. The aim of this study was to validate an analytical method for the simultaneous
analysis of ribociclib and anastrozole in human plasma.
Chromatographic separation of the analytes was performed on an XBridge Phenyl column at a
temperature of 35 °C. Gradient elution was applied using mobile phases (A: water and formic acid; B:
methanol and formic acid) at a flow rate of 0.6 mL/min. Detection was carried out using a mass spectrometer
(ribociclib fragmentation m/z 435.2→ m/z 322.1 (25eV) + m/z 252.1 (30eV), anastrozole m/z 294.1→ m/z
225.4 (25eV) + m/z 115.2 (70eV)).
The linearity of the method was tested in the concentration range of 700 to 3500 ng/mL for ribociclib
and 20 to 100 ng/mL for anastrozole. Precision and accuracy of the method were tested at three concentration
levels (LLOQ, up to 3xLLOQ, 80% ULOQ). Satisfactory RSD values for repeatability (ribociclib → 4.71%,
1.33%, 1.51%; anastrozole → 9.23%, 6.43%, 4.31%) and intermediate precision (ribociclib → 6.23%, 3.97%,
2.28%; anastrozole → 11.35%, 11.40%, 14.78%) were obtained. The calculated analytical recoveries
(ribociclib: within-day → 11.22%, 8.94%, −3.95%; between-day → −1.85%, 8.21%, −3.89%; anastrozole:
within-day → −10.92%, −6.12%, −4.55%; between-day → −8.98%, −4.66%, −1.55%) met the accuracy
validation criteria. Injection reproducibility parameters (RSD<7%) and carry-over effect (<2%) were also
tested, and the obtained values for both parameters met the validation criteria. According to ICH guidelines
for the validation of bioanalytical methods, the matrix effect on the results was tested on six blank plasma
samples (from different donors) at high and low concentrations. During the determination of ribociclib, ion
enhancement occurred in all samples except the hemolytic and lipemic ones. The matrix effect for anastrozole
caused ion suppression, which was reproducible across all samples and tested concentration levels. Based on
all the tested validation parameters, this method is satisfactory for the simultaneous determination of
ribociclib and anastrozole concentrations in plasma. |
