Validacija metode za določanje koncentracije imunosupresivov v venski krvi z uporabo tekočinske kromatografije, sklopljene s tandemsko masno spektrometrijo

Žibert, Patricia (author), Černe, Darko (mentor), Lojk, Jasna (comentor)

Imunosupresivi so skupina zdravil, ki zavirajo ali zmanjšujejo intenziteto imunskega odziva in se uporabljajo za preprečevanje zavrnitve presajenega organa. Po podatkih Nacionalnega inštituta za javno zdravje iz leta 2022 je bil izmed imunosupresivov največkrat predpisan takrolimus, sledita mu metotreksat in mikofenolna kislina. Med pogosto predpisanimi sta tudi ciklosporin A in sirolimus, medtem ko everolimus spada med redkeje predpisane imunosupresive. Zaradi ozkega terapevtskega okna in velikih razlik v farmakokinetiki med posamezniki je izrednega pomena terapevtsko spremljanje koncentracije imunosupresivov. Terapevtsko spremljanje koncentracije zdravil je proces določanja koncentracije zdravilne učinkovine v bioloških vzorcih in je v zadnjih desetletjih kot del personalizirane medicine vedno bolj pomembno. Prenizke koncentracije imunosupresivov zmanjšujejo njihove učinke, medtem ko previsoke koncentracije delujejo toksično (povzročajo anemijo, levkopenijo, diarejo, osteoporozo). Zaradi naštetih razlogov je nujna uporaba občutljive in selektivne metode, kot je tekočinska kromatografija, sklopljena s tandemsko masno spektrometrijo, ki je v terapevtskem spremljanju koncentracij imunosupresivov zlati standard. V okviru magistrske naloge smo validirali metodo tekočinske kromatografije, sklopljene s tandemsko masno spektrometrijo. Preverjali smo linearnost, selektivnost, točnost, natančnost metode in stabilnost vzorcev, določili absolutni učinek matriksa, spodnjo mejo določljivosti in ionska razmerja ter preverili prenos med vzorci. Kot kriterije za ustreznost preverjenih validacijskih parametrov smo uporabili priporočila in smernice Inštituta za klinične in laboratorijske standarde (CLSI) in Mednarodne konference za harmonizacijo tehničnih zahtev za zdravila za uporabo v humani medicini (ICH). Dokazali smo, da je metoda linearna, točna, selektivna, natančna in ponovljiva. Dokazali smo enako stabilnost in s tem primernost shranjevanja vzorca polne krvi na sobni temperaturi in v hladilniku, stabilnost pripravljenih vzorcev v hladilniku in v zamrzovalniku ter razpad in nestabilnost internih standardov brez matriksa. Z določitvijo absolutnega učinka matriksa smo preverjali tudi izkoristek in učinkovitost procesa, ki sta bila zadovoljiva. Delo smo zaključili z uspešno validacijo metode, s čimer smo ovrednotili primernost metode za rutinsko uporabo.

• Language: Slovenian
• Keywords: imunosupresivi, terapevtsko spremljanje koncentracij, validacija, tekočinska kromatografija, tandemska masna spektrometrija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2025
• PID: 20.500.12556/RUL-168471
• Publication date in RUL: 15.04.2025

Secondary language

• Language: English
• Title: Validation of the method for determining the concentration of immunosuppressants in venous blood using liquid chromatography coupled with tandem mass spectrometry

Abstract

Immunosuppressants are a group of drugs that suppress or reduce the intensity of the immune response and are used to prevent rejection of a transplanted organ. Based on data from The National Institute for Public Health in 2022, tacrolimus was the most frequently prescribed immunosuppressant, followed by methotrexate and mycophenolic acid. Cyclosporine and sirolimus are also frequently prescribed, while everolimus is among the less commonly prescribed immunosuppressants. Due to narrow therapeutic window and wide inter-individual variability in pharmacokinetics therapeutic drug monitoring of immunosuppressants is of paramount importance. Therapeutic drug monitoring is the process of determining the concentration of the active substance in biological samples and has become increasingly important in recent decades as part of personalised medicine. Suboptimal concentrations of immunosuppressants reduce their effects, while excessive levels lead to toxic effects (causing anaemia, leukopenia, diarrhoea, osteoporosis). Therefore, it is necessary to employ a precise and specific method, such as liquid chromatography tandem mass spectrometry, a gold standard in therapeutic drug monitoring of immunosupressants. We validated the method of liquid chromatography coupled to tandem mass spectrometry. We validated the linearity, selectivity, accuracy, precision of the method and sample stability, determined the absolute matrix effect, the lower limit of quantification and the ion ratios, we also evaluated the carry-over. We followed the guidelines of The Clinical & Laboratory Standards Institute (CLSI) and The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). We proved that the method is linear, accurate, selective, precise and repeatable. We have demonstrated that storing whole blood samples at room temperature and in the refrigerator is equal and therefore suitable, stability of prepared samples in the refrigerator and in the freezer. We also proved the decomposition and instability of internal standards without matrix. The determination of the matrix effect was also used to check the extraction recovery and process efficiency, which were satisfactory. We successfully validated the method, thus evaluating the suitability of the method for use in routine.

• Keywords: immunosuppressants, therapeutic drug monitoring, validation, liquid chromatography, tandem mass spectrometry