Marta Costas-Rodríguez

Classification of cultivated mussels from Galicia (Northwest Spain) with European Protected Designation of Origin using trace element fingerprint and chemometric analysis

Inductively coupled plasma-mass spectrometry (ICP-MS) in combination with different supervised chemometric approaches has been used to classify cultivated mussels in Galicia (Northwest of Spain) under the European Protected Designation of Origin (PDO). 158 mussel samples, collected in the five rías on the basis of the production, along with minor and trace elements, including high field strength elements (HFSEs) and rare earth elements (REEs), were used with this aim. The classification of samples was achieved according to their origin: Galician vs. other regions (from Tarragona, Spain, and Ethang de Thau, France) and between the Galician Rías. The ability of linear discriminant analysis (LDA), soft independent modelling of class analogy (SIMCA) and artificial neural network (ANN) to classify the samples was investigated. Correct assignations for Galician and non-Galician samples were obtained when LDA and SIMCA were used. ANNs were more effective when a classification according to the ría of origin was to be applied.

Multielemental determination in breast cancerous and non-cancerous biopsies by inductively coupled plasma-mass spectrometry following small volume microwave-assisted digestion

A microwave-assisted digestion method amenable to analysis of small size biological samples (<30 mg dry mass) has been optimized for determining twelve elements (Ag, As, Cd, Co, Cu, Cr, Mn, Mo, Ni, Pb, Se and Zn) by inductively coupled plasma-mass spectrometry (ICP-MS) in breast cancerous and non-cancerous biopsies. The use of three small volume PTFE closed vials (6-mL capacity) placed inside a conventional microwave vessel allows to drastically diminishing the volume of acid needed for digestion. A Plackett-Burman experimental design was used to evaluate the robustness of the digestion procedure. Effects of nitric acid volume, need for predigestion step, microwave power and digestion time were assessed. No significant effects were found, the digestion method being robust enough to be recommended for a routine practice. The method was successfully validated against CRM BCR 185 (bovine liver), CRM NRCC TORT-2 (lobster hepatopancreas), CRM NRCC DORM-2 (dogfish muscle) and CRM NRCC DOLT-2 (dogfish liver). Procedural detection limits ranged from 0.54 to 40 ng g(-1). Within-batch precision values were less than 3%, whereas between-batch precision values were in the range 2-11%. Forty-seven biopsies from thirty-nine women were analyzed: 20 samples corresponding to mammoplasties from healthy women and 27 samples from patients suffering from cancer pathology, 19 of which corresponded to tumour and 8 to adjacent normal tissue. After applying parametric and non-parametric statistical tests, a significant accumulation of Cu, Mn, Zn and Se in cancerous tissues was demonstrated.

Multiple small volume microwave-assisted digestions using conventional equipment for multielemental analysis of human breast biopsies by inductively coupled plasma optical emission spectrometry

A multiple microwave-assisted digestion procedure using small PTFE closed vials (6 mL capacity) inserted into conventional microwave digestion vessels has been developed as a fast, efficient and clean methodology for multielemental analysis of human breast biopsies by inductively coupled plasma optical emission spectrometry. This small volume strategy allows drastically diminishing the volume of acid needed for digestion, and in turn, a decrease in sample dilution and an increase in sample throughput is achieved. A 2(IV)(4-1) fractional factorial design was used for screening optimization of four variables that can influence the digestion efficiency: (A) nitric acid volume, (B) pre-digestion step, (C) microwave power, and (D) digestion time. A validation study included linearity, precision, detection and quantification limits. Validation against different biological certified reference materials (CRMs) was also performed. The digestion method is suitable for the determination of Al, Ca, Cu, Fe, K, Mg, Mn, P, S and Zn in small size biological samples such as breast biopsies (<30 mg dry mass). Forty-seven biopsies from 39 women were analyzed: 20 samples from healthy women corresponding to mammoplasties and 27 samples from patients suffering from cancer pathology (19 corresponded to tumour and 8 to adjacent normal tissue). A significant accumulation of Al, Ca, Cu, K, Mg, Mn, P and Zn was found in tumour as compared to healthy tissues. When this comparison is made for tumour and adjacent tissues, a significant accumulation of Al, Mg, P and Zn in tumour tissues was observed. Finally, only Ca significantly accumulates in the adjacent tissues as compared to healthy tissues.

High-precision Fe isotopic analysis of whole blood for biomedical purposes without prior isolation of the target element

Recently, it has been documented that Fe isotopic analysis of whole blood and serum by means of multi-collector ICP-mass spectrometry (MC-ICP-MS) provides promising results in a biomedical context and, thus, there is a demand for simple, fast and reliable methodologies, providing a high sample throughput. In this work, the possibility of Fe isotopic analysis by MC-ICP-MS directly in acid-digested whole blood and, thus, without prior Fe isolation was evaluated. The influence of the main mineral matrix elements and the effect of potentially remaining organic compounds were first systematically evaluated using synthetic solutions. The Fe isotopic composition was biased low in the presence of matrix elements such as Na and K, while it was biased high for glucose concentrations ≥ 1% (w/v). Nevertheless, after dilution of the whole blood sample digest to 0.75–1.5 mg L−1 of Fe, followed by adequate correction for instrumental mass discrimination using a combination of internal (with admixed Ni) and external correction, MC-ICP-MS isotope ratio measurements provided accurate and precise results. For actual samples, the Fe isotopic data thus obtained agree well with those obtained using the reference procedure, based on prior chromatographic isolation of Fe from acid-digested blood.

Cu isotopic signature in blood serum of liver transplant patients: a follow-up study.

End-stage liver disease (ESLD) is life-threatening and liver transplantation (LTx) is the definitive treatment with good outcomes. Given the essential role of hepatocytes in Cu homeostasis, the potential of the serum Cu isotopic composition for monitoring a patient’s condition post-LTx was evaluated. For this purpose, high-precision Cu isotopic analysis of blood serum of ESLD patients pre- and post-LTx was accomplished via multi-collector ICP-mass spectrometry (MC-ICP-MS). The Cu isotopic composition of the ESLD patients was fractionated in favour of the lighter isotope (by about −0.50‰). Post-LTx, a generalized normalization of the Cu isotopic composition was observed for the patients with normal liver function, while it remained light when this condition was not reached. A strong decrease in the δ65Cu value a longer term post-LTx seems to indicate the recurrence of liver failure or cancer. The observed trend in favour of the heavier Cu isotopic composition post-LTx seems to be related with the restored biosynthetic capacity of the liver, the restored hepatic metabolism and/or the restored biliary secretion pathways. Thus, Cu isotopic analysis could be a valuable tool for the follow-up of liver transplant patients and for establishing the potential recurrence of liver failure.

Iron isotopic analysis of finger-prick and venous blood by multi-collector inductively coupled plasma-mass spectrometry after volumetric absorptive microsampling

High-precision isotopic analysis of Fe in blood is currently assessed as a complementary approach to achieve a better understanding of human Fe metabolism and for diagnosis of diseases. Volumetric absorptive microsampling (VAMS) is a recently introduced technique that allows the straightforward collection of a well-defined volume of blood by dipping an absorbent polymeric tip into it. In this work, the use of VAMS was evaluated for high-precision isotopic analysis of whole blood Fe by multi-collector inductively coupled plasma-mass spectrometry (MC-ICP-MS). Fe concentrations in whole blood sampled using VAMS were determined by single-collector sector-field ICP-MS. A variety of solvents were evaluated for extraction of Fe from the VAMS-device (Mitra™). Fe was quantitatively extracted from the absorbent using 1 mL of Milli-Q water. The extracted material was subsequently subjected to acid digestion and to a miniaturized chromatographic procedure for isolation of Fe from the blood matrix. The Fe isotopic compositions and concentrations of paired finger-prick and venous blood samples collected at the same time from six individuals were compared. No significant differences were found between the two blood types. The proposed methodology significantly increases the sample throughput and facilitates sample acquisition for Fe isotopic analysis in clinical applications.

Evaluation of the use of cold plasma conditions for Fe isotopic analysis via multi-collector ICP-mass spectrometry: effect on spectral interferences and instrumental mass discrimination

The advantages and disadvantages of using cold plasma conditions in combination with both the standard and the high-transmission (‘jet’) plasma interface and under dry and wet plasma conditions were evaluated in the context of high-precision isotopic analysis of Fe via multi-collector inductively coupled plasma-mass spectrometry (MC-ICP-MS). When using the standard interface and wet plasma, cold plasma conditions suppressed the occurrence of the polyatomic ions 40Ar14N+ and 40Ar16O+, but not that of 40Ar16O1H+. Dry plasma conditions efficiently removed the (hydr)oxide ions, but at the cost of a dramatic increase in the prevalence of 40Ar14N+. Thus, interference-free 56Fe/57Fe measurement can be performed at low mass resolution using cold plasma conditions and the standard plasma interface. No alleviation of the spectral interferences due to Ar-based ions was observed with the ‘jet’ interface with cold plasma conditions. With cold plasma conditions, the instrumental mass discrimination affecting the Fe isotope ratios was mitigated compared to that obtained with hot plasma when the standard interface was used (1.4% per amu versus 3.2% per amu, respectively). This mitigation in instrumental mass discrimination with cold plasma conditions was not observed when the high-transmission interface was used.

Laser ablation-inductively coupled plasma-mass spectrometry for quantitative mapping of the copper distribution in liver tissue sections from mice with liver disease induced by common bile duct ligation

Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was deployed for quantitative mapping of the Cu distribution in cryo-sections of liver tissue from mice with cholestatic liver disease induced via common bile duct ligation (CBDL). Cu distribution maps of the liver sections were obtained from the CBDL-operated mice sacrificed at different time points (2, 4 and 6 weeks) after the surgical intervention and compared with those of the corresponding control (sham-operated) mice. Cu quantification was accomplished versus matrix-matched thin sections of spiked liver tissue homogenates and versus spiked gelatin droplet standards. No statistical differences were obtained between the results using the two calibration approaches and thus, both were considered suitable for quantitative Cu bioimaging of liver cryo-sections. On the basis of practical considerations, i.e. simplicity, low cost and availability of the material, spiked gelatin droplet standards are the preferred choice for quantitative determination of the Cu distribution in liver tissue cryo-sections. An inhomogeneous hepatic Cu distribution was observed in the CBDL mice, in contrast to the homogeneous hepatic Cu distribution established for the sham-operated mice. The Cu levels increased with the progression of the disease and a strong accumulation was observed in some necrotic areas. High-resolution LA-ICP-MS bioimaging, using a circular spot size of 2 μm, was suitable for the visualization of the Cu distribution in liver tissue on a (sub-)cellular level. In addition to the quantitative Cu mapping, the spatial distribution of Zn was also monitored in the liver cryo-sections of the control and the 2, 4 and 6 week CBDL mice, but in all cases, Zn was homogeneously distributed across the tissue.

Natural Fe isotope fractionation in an intestinal Caco-2 cell line model

In this work, Fe isotopic analysis of samples obtained from an in vitro intestinal model was performed via multi-collector ICP-mass spectrometry (MC-ICP-MS) to evaluate the isotope fractionation accompanying Fe uptake and transport mechanisms at a cellular level. The Caco-2 cell line has been used, after cell differentiation, as an enterocyte model and a bi-cameral experimental setup has been developed and optimized for stimulating intracellular Fe fluxes. An Fe : ascorbic acid mixture with a molar ratio of 1 : 5 was used as a source of non-heme bioavailable Fe. Good experimental repeatability and reproducibility were attained with low blank contribution levels, allowing precise and reliable Fe isotope ratio results. Both Fe absorption and transport processes were accompanied by Fe isotope fractionation in favor of the lighter isotopes. After 3 hours of exposure, the isotopic composition of the apical solution and the cells did not significantly differ from that of the original solution added to the cells. After 24 hours of exposure, the trend observed was towards a light Fe isotopic composition in the cells, whereas the apical solutions were enriched in the heavier isotopes. These results were in good agreement with previous in vivo and ex vivo findings. An overall increase in delta Fe values of the cell layers exposed to Fe treatment relative to the corresponding values for the untreated cells also seems to support the assumption of a preferential accumulation of heavy isotopes in enterocyte ferritin. The consistency of the results obtained supports the usefulness of in vitro cell culture models as an interesting complementary tool for studying Fe metabolic pathways at the intestinal level.

High-precision isotopic analysis of Cu in blood serum via multi-collector ICP-mass spectrometry for clinical investigation: steps towards improved robustness and higher sample throughput

High-precision isotopic analysis of Cu in blood serum via multi-collector ICP-mass spectrometry (MC-ICP-MS) has recently been shown valuable for diagnosis and prognosis of diseases affecting Cu metabolism. However, as a result of the low Cu concentration (≤2 mg L−1) in blood serum compared to the high contents of concomitant matrix elements (e.g., ∼3000 mg L−1 Na, ∼1000 mg L−1 S, ∼100 mg L−1 Ca and ∼20 mg L−1 Mg) and the measurable effect that the matrix typically exerts on the extent of instrumental mass discrimination in MC-ICP-MS, an exhaustive sample preparation protocol is required. In this work, an alternative procedure for Cu isolation from serum prior to isotope ratio measurement by MC-ICP-MS was evaluated. This procedure involves removal of the alkali and earth alkaline metals using 1 mL of Chelex-100 chelating ion exchange resin, followed by further purification of the Cu-fraction using 250 μL of AG MP1 anion exchange resin. The matrix elements were removed efficiently and Cu recoveries were quantitative in all cases. The alternative Cu isolation procedure showed several advantages compared to the conventional procedure, e.g., (i) a reduction in the amount of acids, cost and time required and (ii) lower blank levels. The effect of (remaining) concomitant matrix elements on the Cu isotope ratio accuracy and precision attainable was evaluated using both the standard interface (combination of standard cones and a large dry interface pump) and the high-transmission ‘jet’ interface (combination of ‘jet’ cones and a large dry interface pump). For standard solutions, the jet interface provided a superior internal precision (improved by a factor of 1.5) for the raw (uncorrected) 65Cu/63Cu ratio and a mitigated extent of instrumental mass discrimination. However, the influence of matrix elements on the Cu isotope ratio results was stronger with the jet interface than with the standard interface. The proposed procedure was validated using standards with known isotopic composition, a serum reference material and human serum samples. No statistical differences were obtained (at a 95% confidence interval) in the δ65Cu values using the proposed and the conventional (two column passes using 1 mL of AG MP1 resin) procedure. Finally, serum samples collected at different time points during a day from five volunteers were analysed in order to study diurnal variations in the serum Cu isotopic composition. No diurnal variations were established.