Wilson's disease (WD) is a genetic disorder affecting Cu metabolism, which can lead to severe physiological and neurological symptoms, and even death if untreated. Based on the fact that WD patients show low Cu levels in serum, implementation of screening programs for diagnosis of this condition at the moment of birth, when progression of the disease can be still arrested, has been attempted in the past. These attempts, however, have been unsuccessful, as healthy new-borns often show low Cu levels in serum due to liver immaturity. In this work, the potential use of isotopic analysis of Cu in serum samples as an alternative diagnostic parameter for Wilson's disease has been investigated. For this purpose, the Cu isotopic composition of a set of serum samples from different groups showing either low (i.e. WD patients, patients who had undergone bariatric surgery, infants) or normal (supposedly healthy adults) Cu concentration levels was determined by means of multi-collector ICP-mass spectrometry (MC-ICP-MS), after chromatographic isolation of Cu. For this purpose, AG-MP-1 strong anion exchange resin was relied upon, enabling quantitative recovery of Cu in pure form from the serum samples. MC-ICP-MS measuring conditions were optimized to avoid the influence of spectral overlap, and Ni was admixed as an internal standard for correction of instrumental mass discrimination. The use of this optimized method provided δ⁶⁵Cu for the serum samples with a typical analytical uncertainty of ±0.20‰ (k = 2). Our results show that, for the population considered in this study, combination of Cu concentration values and Cu isotopic information allows classification of WD patients, infants and controls into different groups, while the use of concentration values only is not sufficient for this purpose. Although further studies with a larger number of samples are needed, results are encouraging as far as the use of Cu isotopic analysis for early diagnosis of Wilson's disease is concerned.

This work investigates the potential of a 257 nm femtosecond (fs) laser ablation (LA) device operating at a high repetition rate (10 000 Hz) coupled to a multicollector (MC)-ICPMS to develop a method for the direct determination of Cu ratios in dried urine spots, prepared by deposition of urine (300 μL) onto precut clinical filter paper discs (16 mm diameter). The sampling capabilities offered by the fs LA system, permitting ablation of 150 μm thick coronas in the rim area of the filter, together with the use of admixed Ni as an internal standard, the proper optimization of the MC-ICPMS conditions (e.g., use of pseudo high-resolution mode to avoid interferences) and the use of a data processing approach particularly suitable for short transient signals (linear regression fit) enabled analysis of real urine samples with precision values around 500 ppm (RSD) for urinary Cu contents of a few hundred μg L⁻¹. The methodology developed could prove to be useful for implementing screening protocols to detect Wilson's disease (WD), a well-known disorder related to Cu metabolism. In fact, the use of this analytical methodology permitted us to observe significant differences between (i) untreated WD patients and (ii) WD patients that are under treatment and control samples. This work represents the first time that determination of ⁶⁵Cu/⁶³Cu ratios has been used in the context of WD research, and serves as a proof of principle, suggesting that Cu isotope analysis could help in developing earlier and more reliable means to diagnose WD.

This work describes a novel procedure based on the use of a 1030 nm femtosecond (fs) laser ablation (LA) device operating at a high repetition rate (30 000 Hz) coupled to a sector field-inductively coupled plasma-mass spectrometer (ICP-MS), enabling the complete ablation of dried blood spot (DBS) specimens in a reasonable time (200 s for samples of 5 μL). The integration of the complete signal obtained, in combination with the use of Pt as an internal standard (which can be added to the clinical filter paper prior to the blood deposition, ensuring compatibility with unsupervised sample collection schemes), permits obtaining an analytical response that is independent of the particular characteristics of every sample. On the basis of this methodology, an analytical method was developed for the direct determination of several elements (Cd, Co, Cu and Pb) in four blood reference materials as well as in three real samples, providing accurate results in all cases evaluated, at concentration levels ranging from 0.1 to hundreds of μg L⁻¹. Detection limits of 0.043 (Cd), 0.42 (Co), 0.54 (Cu), and 0.040 (Pb) μg L⁻¹ are achieved, and precision values most often range between 3 and 9% RSD. Finally, the potential to couple the LA device simultaneously to a multicollector-ICP-MS and a sector field-ICP-MS unit by split-flow is also demonstrated, thus allowing us to obtain both elemental (Co, Cu, Cd and Pb) and isotopic (Cu isotopic composition) information from every particular DBS, and therefore maximizing the amount of information that can be drawn from a single DBS specimen. Still, the precision of the approach is limited at this point, as RSD values of approx. 1500 ppm and delta variations of almost 4‰ were observed for five DBS specimens created from the same blood sample.