20.500.12556/RUL-184420 Razvoj in validacija analizne metode za določanje koncentracije malondialdehida na osnovi mikrovzorčenja krvi Development and validation of an analytical method for determining the concentration of malondialdehyde using blood microsamples Mikrovzorčenje je tehnika odvzema vzorcev v zelo majhnih količinah, ki se najpogosteje uporablja za odvzem biološkega materiala. Ker predstavlja za pacienta manj invaziven in bolj prijazen način odvzema krvi, se vse pogosteje uvaja v različne klinične postopke. Malondialdehid je pomemben kazalnik oksidativnega stresa oz. lipidne peroksidacije, zato lahko služi kot dober kazalnik aktivnosti bolezni, pri katerih je lipidna peroksidacija del patoloških procesov. Metoda za določanje malondialdehida v mikrovzorcih krvi bi omogočila manj invazivno in učinkovitejše klinično spremljanje bolezni. V okviru magistrske naloge smo poskušali validirati in optimizirati dve metodi določanja malondialdehida: metodo posušenih krvnih madežev in metodo volumetričnega absorptivnega mikrovzorčenja ter ju med seboj primerjali. Temelj obeh pristopov je bila že vzpostavljena metoda za določanje malondialdehida v plazemskih in vzorcih posušenih krvnih madežev, ki smo jo poskušali prilagoditi in validirati tudi za vzorce, pridobljene z volumetričnim absorptivnim mikrovzorčenjem. Razvito metodo smo dodatno optimizirali z uporabo različnih antioksidantov. Validacijo obeh metod smo izvajali v koncentracijskem območju 10–90 μM. Validacija metode s posušenimi krvnimi madeži ni bila uspešna, saj noben izmed validacijskih parametrov ni ustrezal zahtevanim kriterijem. Validacija metode volumetričnega absorptivnega mikrovzorčenja je bila delno uspešna, kjer smo potrdili ponovljivost metode (koeficient variacije = 3,5–6,2 %), odsotnost vpliva hematokrita ter stabilnost vzorcev ob uporabi 5 % butiliranega hidroksitoluena pri sobni temperaturi in –20 ⁰C do 14 dni in do 7 dni pri 40 ⁰C, pri čemer so relativne spremembe koncentracije znašale od –15,3 % do 14,9 %. Dodatek butiliranega hidroksitoluena je upočasnil nastajanje malondialdehida in njegovo koncentracijo zmanjšal za približno 20 % ter se izkazal kot najučinkovitejši antioksidant. Linearnost in točnost metode nista bili ustrezni. Določali smo tudi učinkovitost ekstrakcije, učinkovitost celotnega procesa in vpliv matrice, vendar je bila večina izračunanih vrednosti višja od 100 %, kar je nakazovalo na neustreznost obravnavne metode. Pri metodi volumetričnega absoprtivnega mikrovzorčenja bi bilo potrebno prilagoditi koncentracijsko območje in uporabiti 5 % butiliran hidroksitoluen kot antioksidant ter izvesti ponovno validacijo. Microsampling is a technique for collecting samples in very small volumes and is most used for the collection of biological material. As it represents a less invasive and more patient friendly method of blood sampling, it is increasingly being implemented in various clinical procedures. Malondialdehyde is an important biomarker of oxidative stress and an indicator of lipid peroxidation and may serve as a useful marker of disease or the physiological state of a patient. A developed method for malondialdehyde determination in blood microsamples would enable less invasive and more efficient clinical monitoring of diseases. In this study, we attempted to validate and optimize two methods for malondialdehyde determination: the dried blood spot method and volumetric absorptive microsampling, and to compare them with each other. Both approaches were based on an already established method for determining malondialdehyde in plasma and dried blood samples, which we attempted to adapt and validate for samples obtained by volumetric absorptive microsampling. In the final stage, the method was further optimized using different antioxidants. Validation of both methods was performed in the concentration range 10–90 μM. Validation of the dried blood spot method was unsuccessful, as none of the validation parameters met the required criteria. Validation of the volumetric absorptive microsampling method was partially successful, as we confirmed satisfactory repeatability (coefficient of variation = 3,5–6,2 %), absence of hematocrite effect and sample stability when using 5 % butylated hydroxytoluen at room temperature and –20 ⁰C for up to 14 days and for up to 7 days at 40 ⁰C, with relative concentrations ranging from –15,3 % to 14,9 %. The addition of butylated hydroxytoluen reduced malondialdehyde formation, resulting in approximately 20 % lower concentrations, and proved to be the most effective antioxidant. Linearity and accuracy of the method were not satisfactory. Extraction recovery, process efficiency, and matrix effect were also assessed; however, most of the calculated values were higher than 100%, which indicated the inadequacy of the investigated method. For the volumetric absorptive microsampling method, adjustment of the method's concentration range and the use of 5% butylated hydroxytoluene as an antioxidant would be required, followed by revalidation of the method. malondialdehid volumetrično absorptivno mikrovzorčenje posušeni krvni madeži mikrovzorčenje microsampling malondialdehyde volumetric absorptive microsampling dried blood spots true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-07-07 08:46:21 2026-07-07 08:46:31 2026-07-08 04:12:16 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 125664 32346.pdf 32346.pdf 1 CAA283C65BE5917B50AD454F3883F423 32b9916ec119f9e4ceb809487ad4e1fb90b6326f64a33a844670a7b1c186d3c7 78105917-79cf-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=237208 Fakulteta za farmacijo 0 0 0