An interface based on a total consumption micronebulizer was developed for the introduction of xylene solutions into ICP MS. The increase in the nebulizer capillary diameter and the elimination of the internal connections reduced the problem of clogging, pressure instability and memory effects. The xylene carrier could be introduced for several hours at a rate of 30 ml min À1 . The sample (2.5 ml) was injected into the carrier flow to produce peaks of 5 s at half-height (20 s at the base) which allowed a throughput of ca. 100 h À1 for the simultaneous determination of Mo, Ni and V. Calibration curves with good linearity (R 2 4 0.999) over at least three orders of magnitude and detection limits at the sub-ng ml À1 levels were obtained. The method was validated by the analysis of a sample by an independent (ICP AES) method and by the analysis of a NIST CRM 1085c lubricating oil material. The use of a helium-pressurized collision cell was essential to obtain good accuracy for Ni and V but was not required for Mo.

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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.

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.

Electrolytes in serum are important biomarkers for skeletal and cellular health. The levels of electrolytes are monitored by measuring the Ca, Mg, K, and Na in blood serum. Many reference methods have been developed for the determination of Ca, Mg, and K in clinical measurements; however, isotope dilution thermal ionization mass spectrometry (ID-TIMS) has traditionally been the primary reference method serving as an anchor for traceability and accuracy to these secondary reference methods. The sample matrix must be separated before ID-TIMS measurements, which is a slow and tedious process that hindered the adoption of the technique in routine clinical measurements. We have developed a fast and accurate method for the determination of Ca, Mg, and K in serum by taking advantage of the higher mass resolution capability of the modern sector field inductively coupled plasma mass spectrometry (SF-ICP-MS). Each serum sample was spiked with a mixture containing enriched (44)Ca, (26)Mg, and (41)K, and the (42)Ca(+):(44)Ca(+), (24)Mg(+):(26)Mg(+), and (39)K(+):(41)K(+) ratios were measured. The Ca and Mg ratios were measured in medium resolution mode (m/Δm ≈ 4 500), and the K ratio in high resolution mode (m/Δm ≈ 10 000). Residual (40)Ar(1)H(+) interference was still observed but the deleterious effects of the interference were minimized by measuring the sample at K > 100 ng g(-1). The interferences of Sr(++) at the two Ca isotopes were less than 0.25 % of the analyte signal, and they were corrected with the (88)Sr(+) intensity by using the Sr(++):Sr(+) ratio. The sample preparation involved only simple dilutions, and the measurement using this sample preparation approach is known as dilution-and-shoot (DNS). The DNS approach was validated with samples prepared via the traditional acid digestion approach followed by ID-SF-ICP-MS measurement. DNS and digested samples of SRM 956c were measured with ID-SF-ICP-MS for quality assurance, and the results (mean ± expanded uncertainty in mg dL(-1) unit) for Ca (DNS = 10.14 ± 0.13, digested = 10.11 ± 0.10), Mg (DNS = 2.093 ± 0.008, digested = 2.098 ± 0.007), and K (DNS = 15.48 ± 0.11, digested = 15.50 ± 0.28) were in good agreement with the certified values (Ca = 10.17 ± 0.06, Mg = 2.084 ± 0.023, K = 15.55 ± 0.13). Major sources of uncertainty are sample measurement, spike calibration, and instrument factor including mass discrimination of the spectrometer and the detector deadtime.

Carbohydrate deficient transferrin (CDT) is a biochemical marker for congenital disorders of glycosylation (CDG), chronic alcohol consumption, and forensic medicine diagnosis.

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.

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