Measurement of copper concentrations in human plasma samples using UV-VIS spectrophotometry and inductively coupled plasma mass spectrometry (ICP-MS)

Bakar je esencijalni element koji se nalazi u aktivnim mjestima enzima neophodnih za normalno funkcioniranje ljudskog organizma te je stoga važno pratiti njegovu koncentraciju. Analitičke metode kojima se bakar određuje su spektrofotometrija, spektrometrija masa te atomska apsorpcijska spektroskopija (AAS), a upravo je AAS preporučena metoda za njegovo određivanje. Cilj ovog istraživanja bio je usporediti UV-VIS spektrofotometrijsku metodu i spektrometriju masa (ICP-MS) za određivanje koncentracija bakra u biološkom uzorku. Obje su metode uspoređene s obzirom na parametre validacije i postupak pripreme uzorak te su primijenjene za analizu koncentracija bakra u uzorcima humane plazme. Uzorci humane plazme dobiveni su od dobrovoljnih zdravih i bolesnih darivatelja s bolestima štitne žlijezde (n = 28). Svi prikupljeni rezultati statistički su obrađeni, a dobiveni rezultati za koncentracije bakra u humanoj plazmi UV-VIS spektrofotometrijskom i ICP-MS metodom su uspoređeni upotrebom Spearmanove korelacije i Passing-Bablok regresijske analize (p < 0,05). Ispitivanje validacije sa standardima bakra (500 – 5000 μg/L za spektrofotometrijsku metodu i 5,0 – 150,0 μg/L za ICP-MS) pokazalo je da su obje metode linearne i precizne te da imaju zadovoljavajući limit detekcije (LOD) i kvantifikacije (LOQ). Također pokazano je da priprema uzoraka za obje metode nije zahtjevna, iako je priprema uzoraka za spektrofotometrijsko određivanje koncentracija bakra podložnija mogućim interferencijama. Vrijednosti koncentracija bakra izmjerene UV-VIS spektrofotometrijskom i ICP-MS metodom su se razlikovale (raspon vrijednosti: 575,54 – 4352,54 μg/L za spektrofotometrijsku metodu i 165,01 – 1064,02 μg/L za ICP-MS) što je potvrdila i statistička obrada koja je pokazala da nema korelacije u rezultatima dobivenim dvjema metodama. Vrijednosti koncentracija bakra izmjerene spektrofotometrijskom metodom bile su u referentnom intervalu, dok su vrijednosti izmjerene ICP-MS metodom bile niže od referentnog intervala. Mogući razlog nižih vrijednosti koncentracija bakra izmjerenih ICP-MS metodom je nepotpuno razaranje uzorka i njegova ionizacija, a što se treba ispitati u daljnjim istraživanjima.Copper is essential element found in active sites of enzymes necessary for normal function of human organism. Because of low concentrations in organism, it is important to keep track of copper concentrations. Analytical methods used for copper concentration measurement are spectrophotometry, mass spectrometry and atomic absorption spectroscopy (AAS), from which later is recommended method for copper measurement. The aim of this study was to compare UV-Vis spectrophotometry and mass spectrometry (ICP-MS) as methods for measurement of copper in biological samples. Both methods were compared with validation parameters as well as sample preparation process and were used to measure copper concentrations in human plasma samples. Samples were collected from voluntary healthy subjects and subjects with diagnosed thyroid gland diseases (n = 28). All results were statisticaly processed with Spearman correlation and Passing-Bablok regression analysis (p < 0,05). Validation testing with copper standards (500 – 5000 μg/L for spectrophotometric method and 5,0 – 150,0 μg/L for ICP-MS) showed that both methods are linear in given range of concentrations, precise and that they have satisfactory limit of detection (LOD) and limit of quantification (LOQ). Sample preparation process for both methods is not challenging, although sample preparation for spectrophotometry is more susceptible to possible interferences. Copper concentration values were different (value range: 575,54 μg/L – 4352,54 μg/L for spectrophotometry and 165,01 – 1064,02 μg/L for ICP-MS) which was also confirmed by statistical analysis which showed there is no correlation between values measured by both methods. Values obtained by spectrophotometry were in reference range of values of copper in serum, while values measured with ICP-MS were lower than reference range. Most probable cause of lower values of copper concentrations obtained by ICP-MS is unproper sample preparation and its ionisation that should be investigated in further research