| Abstract | Zbog povećanog razvoja rezistencije na ET, ista se često kombinira s inhibitorima CDK4/6 kinaze u liječenju HR-pozitivnog / HER2-negativnog karcinoma dojke. Jedna od takvih kombinacija je kombinacija ABE i LET. Iako je ABE lijek ciljanog djelovanja, postoje velike inter- i intraindividualne razlike u njegovom učinku i toksičnosti zbog čega ga je potrebno terapijski pratiti.
Cilj ovog rada jest validirati bioanalitičku metodu za određivanje ABE i LET u plazmi pacijenata. Ispitani validacijski parametri jesu: selektivnost, linearnost, točnost, preciznost, ekstrakcijski prinos i utjecaj matrice.
Uzorak za analizu sačinjen je od plazme obogaćene standardima ABE i LET, a kao unutarnji standardi korišteni su d4-PAL i d12-ANA. Proteini iz uzorka istaloženi su acetonitrilom te je provedena filtracija na Agilent Captiva EMR-Lipid kolonici za uklanjanje fosfolipida. Nakon filtracije sorbens je ispran otopinom amonijaka u metanolu kako ne bi došlo do gubitka lipofilnijih analita. Filtrat je uparen i suhi ostatak otopljen u 65%-tnom metanolu. Kromatografska analiza provedena je na Agilent 1260 sustavu, na Kinetex biphenyl koloni (150×4,6 mm, 2,6 μm). Mobilna faza sastojala se od faze A (voda i 0,1 %-tna mravlja kiselina) i faze B (acetonitril i 0,1 %-tna mravlja kiselina) uz brzinu protoka 0,5 mL/min. Za detekciju je korišten Ultivo Triple Quadrupole spektrometar masa.
Selektivnost metode potvrđena je usporedbom kromatograma slijepe probe i standarda analita pri LLOQ. Na kromatogramu slijepe probe uočen je neznatan signal (0,56% odgovora analita na LLOQ) na tr LET do kojeg dolazi zbog carry-over-a analita iz prethodećeg uzorka.Metoda je linearna u rasponu od 50 – 500 ng/mL za ABE i 30 – 300 ng/mL za LET. RSD vrijednosti za LET iznosile su 1,66 – 2,7 %, a za ABE 2,79 – 4,81 %. Odstupanje od teorijske koncentracije jest -3,91 - 3,49 % za LET te 1,64 – 3,92% za ABE. ER LET iznosio je 96,58 – 102,52 %, a ABE 71,11 – 73,01 %. Svi su opisani rezultati unutar propisanih vrijednosti što je dokaz uspješne validacije. Relativni utjecaj matrice vrlo je sličan pri različitim koncentracijama LET (razlika < 2%) čime je potvrđeno da je d12-ANA dobar izbor unutarnjeg standarda. D4-PAL nije korigirao utjecaj matrice na ABE jer se značajno razlikuje pri dvije različite koncentracije ABE (94,04% pri 125 ng/mL, 24,11% pri 375 ng/mL). Primjenjivost metode dokazana je određivanjem koncentracije analita u plazmi pacijentica. |
| Abstract (english) | Due to the increased resistance to ET, it is often combined with CDK4/6 kinase inhibitors in treating HR+/HER2- breast cancer. One such combination is ABE and LET. Despite ABE being a targeted therapy, there are significant inter- and intra-individual differences in its effectiveness and toxicity, necessitating therapeutic monitoring.
The aim of this study is to validate a bioanalytical method for determining ABE and LET in patient plasma. Validation parameters examined include selectivity, linearity, accuracy, precision, extraction yield, and matrix effect.
Samples were prepared from plasma enriched with ABE and LET standards, using d4-PAL and d12-ANA as internal standards. Proteins were precipitated with acetonitrile and filtered using an Agilent Captiva EMR-Lipid column. After filtration, the sorbent was washed with an ammonia solution in methanol to prevent loss of more lipophilic analytes. The filtrate was evaporated, and the residue dissolved in 65% methanol. Chromatographic analysis was performed on an Agilent 1260 system with a Kinetex biphenyl column (150×4,6 mm, 2.6 μm), using a mobile phase consisted of phase A (water and 0,1% formic acid) and phase B (acetonitrile and 0,1% formic acid) at a flow rate of 0,5 mL/min. Detection was done using an Ultivo Triple Quadrupole mass spectrometer.
Selectivity was confirmed by comparing blank sample chromatograms with analyte standards at LLOQ. On the chromatogram of the blank sample, an insignificant signal (0.56% of the analyte response at LLOQ) was observed at the tr due to the carry-over of the analyte from the previous sample.The method is linear between 50-500 ng/mL for ABE and 30-300 ng/mL for LET. RSD values for LET were 1,66-2,7% and for ABE 2,79-4,81%. Deviation from theoretical concentration was -3,91 to 3,49% for LET and 1,64 to 3,92% for ABE. Extraction recovery was 96,58-102,52% for LET and 71,11-73,01% for ABE. All validation results were within reference values, confirming successful validation. The relative matrix effect for LET was consistent (difference <2%), confirming d12-ANA as a suitable internal standard. However, d4-PAL did not adequately compensate for the matrix effect on ABE, as it varied greatly at two different concentrations of ABE (94,04% at 125 ng/mL, 24,11% at 375 ng/mL). Introducing a new internal standard for ABE is recommended. The method's applicability was demonstrated by determining analyte concentrations in patient plasma. |
