Razvoj in validacija metode za merjenje empagliflozina v krvni plazmi in prilagoditev za mikrovzorčenje

Luštek, Patricia (author), Trontelj, Jurij (mentor), Vovk, Tomaž (comentor)

Empagliflozin je peroralno zdravilo, ki se uporablja za zniževanje glukoze v krvi pri odraslih bolnikih z neustrezno urejeno sladkorno boleznijo tipa 2 in za zdravljenje bolnikov s srčnim popuščanjem. Načrtuje se farmakokinetična raziskava različnih odmerkov empagliflozina na bolnikih s srčnim popuščanjem. V okviru magistrske naloge smo zato nameravali razviti občutljivo, selektivno, točno in natančno metodo za merjenje koncentracij empagliflozina v krvni plazmi ter postaviti izhodišče za prilagoditev metode na mikrovzorčenje krvi. Preizkusili smo tekočinsko ekstrakcijo ter njeno izboljšano in novejšo različico, podprto tekočinsko ekstrakcijo. V okviru tekočinske ekstrakcije smo preizkusili različnie vrste in volumne organskih topil, pri podprti tekočinski ekstrakciji pa smo preizkusili različne kartuše. Končna metoda, ki smo jo uspešno validirali, temelji na tekočinski ekstrakciji z etil acetatom kot ekstrakcijskim topilom, ki ji sledi detekcija s tekočinsko kromatografijo, sklopljena s tandemsko masno spektrometrijo (LC-MS/MS). Razvito metodo smo ovrednotili v skladu s smernicami ICH M10. Delovno območje analizne metode smo potrdili na koncentracijskem območju med 1 ng/mL in 1000 ng/mL. Izkoristek priprave vzorca je pri nizkem in visokem kontrolnem vzorcu znašal nad 70 %. Večji vpliv ozadja in slabšo ponovljivost smo zaznali le pri najnižjem testiranem kalibracijskem vzorcu pri 0,5 ng/mL. Preverili smo relativni vpliv ozadja, pri čemer je vrednost RSD pri QCL znašala 6,98 % in pri QCH 4,17 %. Potrdili smo ustrezno točnost vseh treh nivojev kontrolnih vzorcev in LLOQ, ki je znašala med 89,2 % in 108,7 %. Prav tako smo pri vseh treh nivojih kontrolnih vzorcev in LLOQ potrdili ustrezno ponovljivost, saj so se vrednosti RSD gibale v območju med 0,45 % ter 12,6 %. Dokazali smo ustrezno dolgoročno stabilnost (vsebnost glede na t = 0 h je po 90 dneh na -20 °C na nivoju QCL znašala 88,3 %, na nivoju QCM 104,7 % in na nivoju QCH 104,6 %) in ustrezno stabilnost v času rokovanja z vzorcem za empagliflozin. Na podlagi rezultatov validacije metode lahko potrdimo, da je metoda ustrezna za določitev koncentracij empagliflozina v plazemskih vzorcih bolnikov na terapiji z empagliflozinom za npr. namen terapevtskega spremljanja koncentracij ali izvedbe farmakokinetičnih študij. Metoda je dobro izhodišče za prilagoditev na mikrovzorčenje, kar smo tudi preverili na manjši seriji posušenih krvnih madežev v območju 5-250 ng/mL.

• Language: Slovenian
• Keywords: empagliflozin, validacija, tekočinska ekstrakcija, LC-MS/MS, DBS
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2025
• PID: 20.500.12556/RUL-167520
• Publication date in RUL: 26.02.2025

Secondary language

• Language: English
• Title: Development and validation of a method for determination of empagliflozin in blood plasma and its adjustment for microsampling

Abstract

Empagliflozin is an oral medicine used to lower blood glucose in adult patients with inadequately controlled type 2 diabetes. It is also used to treat patients with heart failure. A pharmacokinetic study of different empagliflozin doses in patients with heart failure is planned. As part of the master's thesis, we therefore wanted to develop a sensitive, selective, accurate and precise method for measuring empagliflozin concentrations in blood plasma, and set a starting point for adapting the method to blood microsampling. We tested two different methods of plasma sample preparation, specifically liquid–liquid extraction and its improved and newer version, supported liquid extraction. As part of the liquid–liquid extraction, we focused on testing different types and volumes of organic solvents, and in supported liquid extraction, we tested different cartridges. The final method, which we successfully validated, is based on liquid–liquid extraction with ethyl acetate as the extraction solvent, followed by detection by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The developed method was evaluated according to ICH M10 guidelines. The operating range of the analytical method was confirmed in the concentration range between 1 ng/mL and 1000 ng/mL. The sample preparation recovery was above 70% for the low and high control samples. A greater matrix effect and lower reproducibility were detected only for the lowest calibration sample tested at 0.5 ng/mL. We checked the relative matrix effect, with the RSD values being 6.98% and 4.17% for QCL and QCH, respectively. We confirmed the appropriate accuracy of all three levels of control samples and the LLOQ, which ranged between 89.2% and 108.7%. We also confirmed adequate reproducibility at all three levels of control samples and LLOQ, as RSD values ranged between 0.45% and 12.6%. Furthermore, we demonstrated adequate long-term stability and bench-top stability during sample handling for empagliflozin. Based on the method validation results, we can confirm that the method is suitable for determining empagliflozin concentrations in plasma samples of patients on empagliflozin therapy for, e.g., the purpose of therapeutic concentration monitoring or for the purpose of conducting pharmacokinetic studies. The method is a good starting point for adaptation to microsampling, which we also verified on a smaller series of dried blood spots in the range of 5–250 ng/mL.

• Keywords: empagliflozin, validation, liquid–liquid extraction, LC-MS/MS, DBS