| Abstract | Spektrofluorimetrija predstavlja jednu od osjetljivnijih metoda s obzirom na mogućnost detekcije željenog spoja u smjesi bez prethodnog odjeljivanja. Malondialdehid (MDA) je stabilan produkt lipidne peroksidacije te je uzet za mjerenje razine oksidacijskog stresa u uzorcima humanog urina.
Metoda korištena u ovom radu za određivanje koncentracije MDA u urinu temelji se na reakciji MDA s dvije molekule 2-tiobarbituratne kiseline (2-TBA) pri visokoj temperaturi od 90ºC i kiselim uvjetima koji su postignuti korištenjem 1% otopine o-H3PO4. Prije mjerenja ispitane su interferencije korištenjem kreatinina, uree, 2-TBA i o-H3PO4. Ispitivanja su napravljena na spektrofluorimetru OLIS RSM 1000F s ekscitacijom na λ=515 nm i detekcijom emisije na λ=553 nm. Ispitivanja interferencija nisu pokazala značajan signal ispitanih supstancija pri λ=553 nm. Za validaciju metode korišteni su standardi otopine MDA dobiveni razrjeđivanjem matične komercijalne otopine 1,1,3,3-tetraetoksipropana te su izrađeni standardi u rasponu od 0,5 do 6,07 μmol/L. Validacijom u vodi dobiven je regresijski pravac y=0,1723x+0,0298 s R2=0,9975. Ponovljivost u seriji za koncentraciju standarda 2,02 μmol/L iznosila je 3,02%, dok je ponovljivost iz dana u dan bila 4,27%. Granica detekcije bila je na 0,078 μmol/L, a granica određivanja na 0,237 μmol/L. Kod validacije u urinu dobiven je regresijski pravac y=0,135x+0,0061 s R2=0,9901. Ponovljivost u seriji za koncentraciju standarda 2,02 μmol/L iznosila je 5,18%, dok je ponovljivost iz dana u dan bila 5,14%. Granica detekcije bila je na 0,248 μmol/L, a granica određivanja na 0,746 μmol/L.
Mjerenjem koncentracije MDA u urinu sedmorice zdravih dobrovoljaca i izračunom koncentracije pomoću kalibracijske krivulje dobivene su vrijednosti koncentracije MDA 1,41±0,59 μmol/L. Kako bi se poništio utjecaj matriksa isti su uzorci izmjereni metodom standardne adicije poznate koncentracije MDA te su dobivene vrijednosti 1,01±0,17 μmol/L. Dobivene vrijednosti validacije i rezultata mjerenja uzoraka upućuju na to kako su obje metode pouzdane za mjerenje koncentracije MDA u urinu, ali metoda standardom adicijom daje točnije vrijednosti. |
| Abstract (english) | Spectrofluorometry is one of the more sensitive methods with the ability to detect the desired compound in a mixture without prior separation. Malondialdehyde (MDA) is a stable product of lipid peroxidation and is used to measure oxidative stress levels in human urine samples. The method used in this work to determine the MDA concentration in urine is based on the MDA reaction with two 2-thiobarbituric acid (2-TBA) molecules at high temperature of 90°C and acid conditions achieved using a 1% o-H3PO4 solution. Before measurement, interferences were investigated using creatinine, urea, 2-TBA and o-H3PO4. Tests were made on the spectrofluorimeter OLIS RSM 1000F with excitation at λ = 515 nm and detection of emission at λ = 553 nm. Interference tests did not demonstrate a significant signal of the tested substances at λ = 553 nm. Standard validation of the MDA solution obtained by diluting the commercial solution of 1,1,3,3-tetraethoxypropane was used to validate the method and to make standards ranging from 0,5 to 6,07 μmol/L. Validation in water gives the regression line y = 0.1723x + 0.0298 with R2 = 0.9975. Repeatability for the 2,02 μmol/L standard concentration range was 3,02%, and day-to-day repeatability was 4,27%. The detection limit was 0,078 μmol/L and the limit of quantitaion was at 0,237 μmol/L. After the validation in the urine a regression line was obtained y = 0.135x + 0.0061 with R2 = 0.9901. Repeatability for the 2,02 μmol / L standard concentration range was 5,18%, while the day-to-day repeatability was 5,14%. The detection limit was 0,248 μmol/L and the limit of quantitaion was at 0,746 μmol/L. Measuring the MDA concentration in the urine of seven healthy volunteers and calculating the concentration by means of the calibration curve, were obtained MDA concentrations of 1,41±0,59 μmol/L. In order to overcome the impact of the matrix, the same samples were measured by the standard addition method adding a known concentration of MDA and the obtained values were 1,01±0,17 μmol/L. The obtained values from validation and the measurement results of the samples indicate that both methods are reliable for measuring MDA concentration in urine, but the standard addition method gives more accurate values. |
