Introduction of the method for determination of copper in serum using atomic absorption spectrophotometry

Jedan od esencijalnih mikroelemenata iznimno važnih za razvoj ljudskog organizma je bakar koji sudjeluje u procesima antioksidativne zaštite, imunosti, eritropoeze, sinteze neuropeptida, a ima i važnu ulogu u aktivnosti redoks enzima ceruloplazmina. Promjena koncentracije bakra u organizmu može dovesti do velikog broja patofizioloških poremećaja. Povećana koncentracija bakra u organizmu uzrokovana je razvojem Wilsonove bolesti koja dovodi do smanjene ugradnje bakra u ceruloplazmin i njegovog nakupljanja u organizmu, posebice u jetri i mozgu. Jedna od metoda koja se koristi u dijagnosticiranju Wilsonove bolesti jest mjerenje koncentracije bakra u biološkim uzorcima atomskom apsorpcijskom spektrofotometrijom. U ovom radu razvijena je i validirana metoda za određivanje bakra u serumu. Ispitivanja su provedena na AAS-u Aanalyst 800 (Perkin Elmer Instruments, Norwalk, CT, SAD) s deuterijskim korektom nespecifične površine na valnoj duljini od 324,8 nm. Analitička metoda je validirana uporabom standarda bakra koncentracija u rasponu od 0,125 do 3,0 mg/L, dobivenih razrijeđivanjem komercijalnog standarda bakra koncentracije 1000 mg/L. Dobivena je kalibracijska krivulja s jednadžbom pravca y= 0,0118x – 1x10-5 i koefcijentom korelacije R2= 1. Vrijednost relativne standardne devijacije (RSD) kojom je iskazana ponovljivost iznosi za ST1(0,1mg/L) 7,28 %, za ST4 (1,0 mg/L) 2,34 % i za ST5 (2,0 mg/L) 0,65%. Vrijednost RSD za iskazivanje srednje preciznosti za ST1 (0,1 mg/L) iznosi 14,41 %, za ST4 (1,0 mg/L) 3,35 % i za ST5 (2,0 mg/L) 1,63%. Dobivene vrijednosti pokazuju da su ponovljivost i srednja preciznost metode u granicama prihvatljivosti za RSD izuzev kod vrlo velikih razrijeđenja gdje dolazi do odstupanja. Točnost (R) za ST4 (1,0 mg/L) i ST6 (3,0 mg/L) iznosi 97,43 % i 99,30%. Granica dokazivanja (LOD) iznosi 0,004 mg/L, dok je granica određivanja (LOQ) 0,011 mg/L. Raspon koncentracije bakra mjerenih u četiri uzorka ljudskog seruma je od 0,66 do 1,55 mg/L. Uvedena i validirana analitička metoda se pokazala prikladnom za točno, precizno i brzo određivanje koncentracije bakra u ljudskom serumu.One of the essential microelements which is of upmost importance for the development of the human organism is copper which participates in the processes of antioxidant protection, immunity, erythropoiesis, the synthesis of neuropeptides, it also has an important role in the activity of the redox enzyme ceruloplasmin. A change in the concentration of copper in the organism can lead to a great number of pathophysiological disturbances. A higher concentration of copper in the organism is caused by the development of Wilson’s disease which can hinder the incorporation of copper into ceruloplasmin and lead to its accumulation in the organism, specifically in the liver and the brain. One of the methods used in diagnosing Wilson’s disease is to measure the concentration of copper in biological samples by using the atomic absorption spectrophotometry. This paper demonstrates the development of a validated method for determining the concentration of copper in the serum. The tests were conducted on an AAS-Analyst 800 (Perkin Elmer Instruments, Norwalk, CT, USA) with a deuterium correction of a nonspecific surface at a wavelength of 324.8 nm. The analytic method was validated using a copper standard with a concentration ranging from 0.125 to 3.0 mg/L, which was created by diluting the commercial standard of copper with a concentration of 1000 mg/L. A calibration curve was generated with the linear equation of y= 0,0118x – 1x10-5 and a coefficient of correlation of R2=1. The value of the relative standard deviation (RSD) for the repeatability of the value of ST1 (0.1 mg/L) is 7.28 %, ST4 (1.0 mg/L) is 2.34 % and ST5 (2.0 mg/L) is 0.65 %. The value of RSD for measuring the intermediate precision for ST1 (0.1 mg/L) is 14.41 % for ST4 (1.0 mg/L) is 3.35 % and for ST5 (2.5 mg/L) is 1.63 %. The obtained values show that the repeatability and the intermediate precision of the method are within the boundaries acceptable for RSD with the exception of deviations in the cases of extremely large dilutions. The accuracy (R) for ST4 (1.0 mg/L) and ST6 (3.0 mg/L) is 97.43 % and 99.30 %. The limit of detection (LOD) is 0.004 mg/L, while the limit of quantification (LOQ) is 0.011 mg/L. The range of the concentration of copper measured in the four samples of human serum varies from 0.66 mg/L to 1.55 mg/L. The implemented and validated analytical method proved suitable for an accurate, precise and quick determination of the concentration of copper in the human serum