Elisa Blanco-González

Solid-phase microextraction as a clean-up and preconcentration procedure for organochlorine pesticides determination in fish tissue by gas chromatography with electron capture detection.

The feasibility of developing a single-step clean-up, enrichment procedure for organochlorine pesticides (OCPs) in fish tissue samples based on solid-phase microextraction (SPME) was investigated. The general analytical methodology developed combines conventional solid-liquid extraction of the OCPs from the sample using an organic solvent with SPME over the organic extract followed by gas chromatography-electron-capture detection (GC-ECD) analysis. Experimental SPME conditions such as extraction time, temperature and matrix effects were optimised. Under optimised conditions, precision, linearity range, detection limits and accuracy were evaluated. Detection limits obtained for fish tissue samples were in the range of 0.1-0.7 ng g(-1). Good recoveries (over 70% in all cases) were achieved from samples spiked at a concentration level of 10 ng g(-1). The accuracy of the developed SPME-GC-ECD procedure in real samples has been verified by analysing, using the standard addition method, a certified reference material (CRM 430, OCPs in pork fat) with satisfactory results.

Quantitative Analysis and Simultaneous Activity Measurements of Cu, Zn-Superoxide Dismutase in Red Blood Cells by HPLC-ICPMS

The interest on accurate and precise determination of metalloproteins such as Cu, Zn-superoxide dismutase (Cu, Zn-SOD) involved in the redox balance of living cells is increasing. For this purpose, analytical strategies that provide absolute protein concentration measurements have to be developed. The determination of Cu, Zn-SOD through the measurement of the Cu associated to the protein, which provides its enzymatic activity, by liquid chromatography with online inductively coupled plasma mass spectrometric (ICPMS) detection is described here. Postcolumn isotope dilution analysis (IDA) of Cu has been applied for quantification after evaluation of the column recovery for the total Cu and also Cu-SOD that turned out to be quantitative. When the concentration results obtained via IDA using high-performance liquid chromatography (HPLC)-ICPMS are plotted versus the activity measurements (using the spectrophotometric pyrogallol autoxidation method) a good correlation curve is obtained. Such results permit us, from ICPMS measurements, to obtain simultaneously the Cu, Zn-SOD absolute concentration as well as its enzymatic activity by interpolation in the previously obtained curve. This possibility was explored in real samples (red blood cells of control individuals and patients with metallic total hip arthroplasty) obtaining a good match between direct enzymatic activity measurements and those obtained by interpolation in the correlation curve. The actual protein identification in the red blood cell extract was conducted by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and two matrixes were compared in order to preserve as much as possible the protein-metal interactions during the MALDI process. Interestingly, using a solution containing trihydroxyacetophenone in citrate buffer permitted us to observe some metal-protein interactions in the MS spectrum of the intact Cu, Zn-SOD from red blood cells.

Evaluation of two commercial capillary columns for the enantioselective gas chromatographic separation of organophosphorus pesticides.

The separation of the enantiomers of 13 organophosphorus pesticides (OPPs) has been investigated by gas chromatography (GC) with flame ionisation detection (FID) using two different commercially available chiral columns, Chirasil-Val (l-valine-tert-butylamide) and CP-Chirasil-Dex CB (heptakis (2,3,6-tri-O-metil)-beta-cyclodextrin). Using the Chirasil-Val column no chiral resolution was obtained for the OPPs investigated under any tested experimental condition. The use of the CP-Chirasil-Dex CB stationary phase enabled good individual enantiomeric separation of two OPPs, ruelene and trichlorfon and partial separation of naled, chloretoxyphos, isophenphos and metamidophos. Also, the obtained chromatographic results showed that Chirasil-Dex could resolve enantiomers through the combination of different mechanism (e.g. formation of inclusion complexes and/or interactions outside the cyclodextrin cavity). Under optimised conditions, precision, linearity range and detection limits were evaluated for the enantiomers of ruelene and trichlorfon using CP-Chirasil-Dex CB column and electron capture detection (ECD). By using the GC-ECD method the enantiomers of these OPPs could be satisfactorily detected at very low concentration levels. The detection limits observed were 1.5ngmL(-1) and 11.5ngmL(-1) for the enantiomers of trichlorfon and ruelene, respectively.

Enantioselective determination of thyroxine enantiomers by ligand-exchange CE with UV absorbance and ICP-MS detection

A simple CE method has been developed for the separation and determination of thyroxine (T4) enantiomers in pharmaceutical formulations. The method was based on ligand‐exchange mechanism using a Cu(II)/L‐proline complex as chiral selector. The effects of different parameters affecting separation such as chiral selector concentration, organic additive, buffer pH and temperature were investigated. A baseline separation of the two enantiomers was obtained at a Cu(II)/L‐proline ratio of 1:8 in a borate buffer (15 mmol/L, pH 9.6) containing 10% v/v acetonitrile. Under the optimized conditions, precision linearity range and detection limits of the developed enantioselective CE method were evaluated and compared using two different detection systems: conventional UV detection at 226 nm and iodine (127I)specific detection (“chiral speciation”) with ICP‐MS. Both methodologies show adequate analytical performance characteristics with detection limits around 0.30 μg/mL for each enantiomer of T4. Finally, a levothroid pharmaceutical formulation sample was successfully analyzed using both developed methods CE‐UV and CE‐ICP‐MS.

Determination of organophosphorus pesticides in spiked river water samples using solid phase microextraction coupled to gas chromatography with EI-MS and ICP-MS detection

Solid-phase microextraction (SPME) has been developed for the gas chromatographic (GC) analysis of a wide range of organophosphorus pesticides (OPPs) in river water samples. Experimental conditions, such as type of fiber, extraction mode, extraction time, extraction temperature, salt additives and pH, were optimised. The optimized SPME procedure was evaluated by means of two different detection systems: GC-MS (using electron impact as ionization source) and GC on-line coupled to an inductively coupled plasma mass spectrometer (ICP-MS). The addition of small percentages of nitrogen in the argon carrier gas flow proved to be very suitable for sensitivity enhancement of the element specific detection by ICP-MS. The analytical figures of merit of the two systems have been compared for the detection of OPPs making use, in the case of GC-ICP-MS, of the selective monitoring of phosphorus and other heteroelements (S, Cl, Br) present in the selected pesticides. Precision, linearity range, detection limits and accuracy of the SPME-GC-ICP-MS method were evaluated and compared with those obtained by SPME-GC-MS. Detection limits ranging from 0.8 ng L−1 to 504 ng L−1 and 0.09 ng L−1 to 143 ng L−1 were obtained with SPME-GC-MS and SPME-GC-ICP-MS, respectively. Both methodologies were applied to the analysis of spiked river water samples.

Metal release in patients with total hip arthroplasty by DF-ICP-MS and their association to serum proteins

A study on the level of metals released to body fluids from patients carrying metal-on-metal (based on Co–Cr alloys) total hip prosthesis and titanium alloys dental implants has been conducted. In the first part, total elemental determination of Co, Cr, Ti, Mo and Mn was done in whole blood and urine for both, control individuals and hip arthroplasty patients. Additionally, the work was extended to patients who carried dental implants. The samples, either acid digested (blood) or just diluted (urine) were analyzed using a double focusing inductively coupled plasma mass spectrometer (DF-ICP-MS). Both strategies are validated using the corresponding certified reference materials. The findings revealed an increased concentration of Cr and Ti and, to a lower extent, Co and Mn in the blood and urine of the patients. The second part of the work tries to explore the possible association of the released metals to human serum proteins. For this purpose, speciation of the above mentioned metals is accomplished using liquid chromatography (anion exchange) with ICP-MS detection. Such studies, firstly conducted in incubated standards and then in fresh serum from the patients, showed the elution of Mn associated to transferrin. Co eluted associated to albumin and Cr could not be detected. Spiking experiments showed that Cr(III) is clearly associated to transferrin and supports the theory that Cr is eliminated from the prosthesis as Cr(VI) that shows no interaction with the studied serum proteins.

Species specific isotope dilution versus internal standardization strategies for the determination of Cu, Zn-superoxide dismutase in red blood cells

Many proteins (more than one third) contain metal ions either within their own structures or bound to some of their active sites. These metals are involved in numerous biological processes and therefore, the quantification of metalloproteins that can serve as clinical biomarkers is of great interest. With this aim, the development of analytical strategies that permit individual (targeted) protein quantitative analysis is attempted in this work. In particular, the evaluation of different strategies for the determination of Cu, Zn-superoxide dismutase (Cu, Zn-SOD), a metalloprotein present in the first-line antioxidant defence system of the body, is conducted. The first analytical strategy is based on the use of bovine Cu, Zn-SOD as internal standard for the quantitative analysis of human Cu, Zn-SOD. For this aim, the chromatographic separation between both species (bovine and human) has been optimized according to their respective isoelectric point by anion exchange chromatography. Interestingly, the obtained results revealed a faster specific degradation of the bovine standard with respect to human SOD during sample preparation. The second strategy involves the production and evaluation of an isotopically enriched metalloprotein standard to be used as tracer in the species specific isotope dilution (SS-IDA) method by measuring the Cu associated to the protein. This is done by liquid chromatography with online inductively coupled plasma mass spectrometric (ICP-MS) detection and applied to the quantification in bovine erythrocytes. This finding is a good example to illustrate the power of SS-IDA for targeted protein quantification in respect to the commonly used alternative standards.

Enantioselective determination of the organochlorine pesticide bromocyclen in spiked fish tissue using solid-phase microextraction coupled to gas chromatography with ECD and ICP-MS detection.

A method for enantioselective determination of bromocyclen enantiomers in fish tissue has been developed. The enantiomers were resolved by capillary gas chromatography (GC) using a commercial chiral column (CP-Chirasil-Dex CB) and a temperature program from 50 degrees C (held for 1 min), raised to 140 degrees C at 40 degrees C min(-1) and then raised at 0.2 degrees C min(-1) to 155 degrees C. This enantioselective gas chromatographic separation was combined with a clean-up/enrichment procedure based on solid-phase microextraction (SPME). Under SPME optimized conditions, precision, linearity range and detection limits of the developed SPME-enantioselective GC procedure were evaluated and compared using two different detection systems: a classical electron-capture detection (ECD) and an element specific detection using inductively coupled plasma mass spectrometry (ICP-MS). The SPME-GC-ECD method exhibited an excellent sensitivity, with detection limits of 0.2 ng L(-1) for each enantiomer of bromocyclen. Although ICP-MS offered poorer detection limits (7 ng L(-1) as Br, equivalent to 36 ng L(-1) of each enantiomer) than conventional ECD detector, it proved to be clearly superior in terms of selectivity. The relative potential and performance of the two compared methods for real-life analysis has been illustrated by the determination of enantiomers of bromocyclen in spiked tissue extracts of trout.

Micellar versus reversed phase liquid chromatography for the determination of desferrioxamine and its chelates with aluminium and iron in uremic serum

Micellar liquid chromatography with sodium dodecyl sulphate or Brij-35 as surfactants in the mobile phase was evaluated and compared with reversed phase liquid chromatography using conventional acetonitrile-water eluents for the separation and determination of desferrioxamine (DFO) and its complexes with aluminium (AIDFO) and iron (FeDFO) in uremic serum. Reversed phase liquid chromatography proved to be superior in terms of sensitivity and selectivity. The three solutes investigated were separated with a mobile phase of 13% (v/v) acetonitrile/phosphate buffer (5 mM, pH = 3.5) on a C(18) column and detected by ultraviolet absorption at 210 nm (DFO) and 220 nm (AlDFO and FeDFO). Limits of detection of 0.1 mug ml(-1) and relative standard deviation of 3-4% were obtained. The recovery from serum samples after ultramicrofiltration was around 90%. The method was applied to the determination of DFO, AlDFO and FeDFO in uremic serum.

Chiral speciation of trace elements: approaches to the speciation of selenoaminoacid enantiomers in biological samples

The “state of the art” of chiral trace element speciation, that is, the recognition of the particular enantiomer in which the trace element sought is occurring, is given here. The importance of such studies and speciation information is highlighted. Recent advances and trends in the development and applications of analytical approaches for chiral speciation of trace elements in biological material are also reviewed. A comparative overview is given of existing selenoaminoacids chiral speciation methods, based on hybridization of an adequate chiral separation technique (e.g., high-performance liquid chromatography, gas chromatography or capillary electrophoresis) coupled on-line with selenium specific detection by inductively coupled plasma mass spectrometry (ICP-MS). Applications of these hybrid methodologies, reported so far in the literature, to selenoaminoacids chiral speciation in biological real samples are also reviewed.