Absolute and accurate quantification of protein phosphorylation by using an elemental phosphorus standard and element mass spectrometry.
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Mass Spectrometry↗
Biomedical subjects
A Sanz-Medel
Publications and source records attributed to A Sanz-Medel.
Study of a room temperature phosphorescence phenomenon to allow the detection of aflatoxigenic strains in culture media.
A novel screening method based on room temperature phosphorescence (RTP) for the visual detection of aflatoxigenic strains from Aspergillus genus is described. Strains were cultured on media widely used in food mycology to which methyl-beta-cyclodextrin plus bile salts (0.6% sodium deoxycholate) were added. Aflatoxin production was readily detectable after 3 days of incubation at 28 degrees C by RTP emission from the mycelium of aflatoxigenic strains observed after exposure to UV light. The method was tested on thirty-two Aspergillus sp. strains. The phosphorescence phenomenon was reproduced in vitro by immobilizing aflatoxin B1 on ion exchange resin beads.
Aflatoxins↗
Solid-supported room temperature phosphorescence from aflatoxins for analytical detection of Aspergillus spp. strains.
A simple, direct and rapid analytical methodology for the detection of aflatoxin producing Aspergillus spp. strains based on the measurement of room temperature phosphorescence from aflatoxins is presented here.
Aflatoxins↗
Application of isotope dilution analysis for the evaluation of extraction conditions in the determination of total selenium and selenomethionine in yeast-based nutritional supplements.
Isotope dilution analysis (IDA) has been used to quantify total selenium, total solubilized selenium, and the selenomethionine (SeMet) amount in yeast and yeast-based nutritional supplements after acid microwave digestion and different enzymatic extraction procedures. For this purpose, both a (77)Se-enriched SeMet spike, previously synthesized and characterized in our laboratory, and a (77)Se(VI) spike were used. In the analysis of the nutritional supplements, the SeMet spike was added to the sample and extracted under different conditions, and the (78)Se/(77)Se and (80)Se/(77)Se isotope ratios were measured as peak area ratios after high-performance liquid chromatography (HPLC) separation and inductively coupled plasma mass spectrometry (ICP-MS) detection. The formation of SeH(+) and mass discrimination were corrected using a natural SeMet standard injected every three samples. Similarly, total solubilized selenium was measured in the extracts after enzymatic hydrolysis using the (77)Se-enriched SeMet as a spike by direct nebulization without a chromatographic separation. To establish a mass balance, total selenium was also determined by IDA-ICP-MS on the yeast tablets after microwave digestion using (77)Se(VI) as a spike. Results showed that all enzymatic procedures tested were able to solubilize total selenium quantitatively from the solid. However, the recovery for the species SeMet, the major selenium compound detected, was seriously affected by the enzymatic procedure employed and also by the matrix composition of the supplement evaluated. For the yeast sample, SeMet recovery increased from 68 to 76% by the combined use of driselase and protease. For the nutritional supplements, the two most effective procedures appeared to be protease and driselase/protease, with a SeMet recovery ranging from 49 to 63%, depending upon the supplement evaluated. In the case of in vitro gastrointestinal enzymolysis, the results obtained showed 26-37% SeMet recovery, while the rest of selenium was solubilized as other unknown compounds (probably Se-containing peptides).
Chromatography, High Pressure Liquid↗
Iodinated molecularly imprinted polymer for room temperature phosphorescence optosensing of fluoranthene.
A novel molecularly imprinted polymer (MIP) of high interest for room temperature phosphorescence (RTP) sensing systems is described; the synthesized MIP contains iodine as internal heavy atom in the polymeric structure and its applicability for RTP sensing of fluoranthene at microg L(-1) levels is demonstrated.
Fluorenes↗
Comparison of different chloroformates for the derivatisation of seleno amino acids for gas chromatographic analysis.
Three chloroformate reagents, ethyl chloroformate (ECF), methyl chloroformate (MCF) and menthyl chloroformate (MenCF), have been used for the derivatisation of seleno amino acids and their performance was compared. Chromatographic parameters and the inertness of the different instrumental configurations used (gas chromatography-atomic emission detection (GC-AED), and GC-MS) were shown to have a significant influence on the detection of various seleno amino acids (selenomethione, selenoethione and selenocysteine) and some sulphur-containing amino acids (methionine, cysteine, cystine and methylcysteine) which were included in the experiments for comparison. Methyl chloroformate was the preferred derivatisation reagent, since it generally performed best in terms of derivatisation yield and reproducibility and also showed less significant conditioning effects than ethyl chloroformate. Methyl and ethyl chloroformate derivatives of selenomethionine, selenoethionine, cysteine and methionine were detectable, while the detection of the menthyl chloroformate derivatives of selenocystine and cystine was not reproducible. Overall efficiencies for the determination of selenomethionine and selenoethionine from aqueous extracts ranged from 40 to 100% for methyl chloroformate, over 30-75% for ethyl chloroformate to 15-70% for menthyl chloroformate for different series measured over a period of months. The relative standard deviation of the method for the methyl and menthyl chloroformate derivatisation ranged from 7 to 13% without internal standard and was improved to 2% for the determination of selenomethionine using selenoethionine as internal standard. This indicates that, despite the limited reproducibility of the method, its repeatability is good enough to allow accurate determination of seleno amino acids, which was also demonstrated by the analysis of selenium supplementation tablets for human diet that contained selenomethionine.
Amino Acids↗
Fluorescence optosensors based on different transducers for the determination of polycyclic aromatic hydrocarbons in water.
This paper presents the development of two optosensors for the determination of four polycyclic aromatic hydrocarbons (anthracene, benzo[a]pyrene, fluoranthene and benzo[b]fluoranthene) using a photomultiplier device and an intensified coupled charge device (ICCD) as optical transducers, respectively. These optosensors are based on the on-line immobilization of the analytes onto a non-ionic resin solid support (Amberlite XAD-4) in a continuous flow system, followed by the measurement of their native fluorescence. The determinations were performed using 15 mM H(2)PO(4)(-)/HPO(4)(2-) buffer solution at pH 7 and 25% 1,4-dioxane. Detection limits were 6.4 and 9.3 for ANT, 3.3 and 2.5 for BbF, 1.4 and 13.2 for FLT, and 1.7 and 7.8 for BaP using optosensor 1 or 2, respectively. Relative standard deviations were 7.9 and 6.7 for ANT at 50 ng mL(-1), 3.5 and 7.4 for BbF at 60 ng mL(-1), 3.6 and 8.9 for FLT at 50 ng mL(-1), and 6.7 and 11.6 for BaP at 50 ng mL(-1) using optosensor 1 or 2, respectively. Finally, a critical comparison between the two configurations based on different transducers (photomultiplier and ICCD) for resolving and simultaneously determining mixtures of the polycyclic aromatic hydrocarbons under study in water samples (tap and mineral waters) were carried out.
Journal Article↗
Determination of total homocysteine in human serum by capillary gas chromatography with sulfur-specific detection by double focusing ICP-MS.
A selective and sensitive method for determination of total homocysteine (Hcy) in human serum, by gas chromatography coupled to ICP-MS(HR), has been developed. After reduction of the sample with sodium borohydride the liberated Hcy and other aminothiols, such as cysteine (Cys) and methionine (Met), were converted to their N-trifluoroacetyl (TFA)- O-isopropyl derivatives and these were injected into a gas chromatograph equipped with an HP-5 capillary column. Detection was carried out by means of a double-focusing inductively coupled plasma mass spectrometer (DF-ICP-MS) monitoring (32)S at m/Delta m (resolving power)=3000. The transfer line used to transport the analytes from the GC column to the ICP-MS had previously been developed in our laboratory. The different parameters affecting the derivatisation process were optimised, as were the instrumental operating conditions. This optimised GC-ICP-MS(HR) method was successfully applied to the determination of total homocysteine in human serum-values obtained were in agreement with data reported in the literature. Quantitative recoveries and good precision were obtained for spiked human serum, demonstrating the suitability of the method for quantitative determination of total homocysteine in serum.
Acylation↗
Determination of tributyltin in marine sediment: Comité Consultatif pour la Quantité de Matière (CCQM) pilot study P-18 international intercomparison.
The capabilities of National Metrology Institutes (NMIs-those which are members of the Comité Consultatif pour la Quantité de Matière (CCQM)of the CIPM) and selected outside "expert" laboratories to quantitate (C(4)H(9))(3)Sn(+) (TBT) in a prepared marine sediment were assessed. This exercise was sanctioned by the 7th CCQM meeting, April 4-6, 2001, as an activity of the Inorganic Analysis Working Group and was jointly piloted by the Institute for National Measurement Standards of the National Research Council of Canada (NRC) and the Laboratory of the Government Chemist (LGC), UK. A total of 11 laboratories submitted results (7 NMIs, and 4 external labs). Two external laboratories utilized a standard calibration approach based on a natural abundance TBT standard, whereas all NMIs relied upon isotope dilution mass spectrometry for quantitation. For this purpose, a species specific (117)Sn-enriched TBT standard was supplied by the LGC. No sample preparation methodology was prescribed by the piloting laboratories and, by consequence, a variety of approaches was adopted by the participants, including mechanical shaking, sonication, accelerated solvent extraction, microwave assisted extraction and heating in combination with Grignard derivatization, ethylation and direct sampling. Detection techniques included ICP-MS (with GC and HPLC sample introduction), GC-MS, GC-AED and GC-FPD. Recovery of TBT from a control standard (NRCC CRM PACS-2 marine sediment) averaged 93.5+/-2.4% ( n=14). Results for the pilot material averaged 0.680+/-0.015 micro mol kg(-1) ( n=14; 80.7+/-1.8 micro g kg(-1)) with a median value of 0.676 micro mol kg(-1). Overall, performance was substantially better than state-of-the-art expectations and the satisfactory agreement amongst participants permitted scheduling of a follow-up Key comparison for TBT (K-28), a Pilot intercomparison for DBT (P-43), and certification of the test sediment for TBT content and its release as a new Certified Reference Material (HIPA-1) with a TBT content of 0.679+/-0.089 micro mol kg(-1) (expanded uncertainty, k=2, as Sn) (80.5+/-10.6 micro g kg(-1)).
Journal Article↗
[Effect of strontium on bone metabolism in hemodialysis patients].
The objective of this study was to assess the relationship between the bone strontium content and bone histomorphometric parameters in bone biopsies from patients with chronic renal failure undergoing hemodialysis. The study was carried out in 74 illiac crest bone biopsies from patients with renal osteodystrophy from different worldwide regions (Argentina, Portugal and Spain). They were underwent to histological and histomorphometric evaluation. The bone strontium/calcium ratio was measured by quadrupole inductively coupled plasma-mass spectrometry. The samples were classified into groups according to histological criteria: hyperparathyroidism (HP), mixed (MX), osteomalacia (OM) and adynamic bone disease (ABD). Serum PTH and alkaline phosphatase before biopsy were available in most of the patients. No correlation was found between the different histomorphometric parameters and the Sr/Ca ratio. The one way ANOVA test showed statistical differences in the Sr/Ca ratio of the different histological forms (HP: 0.58 +/- 0.39; MX: 1.16 +/- 0.74; OM: 1.10 +/- 0.46; ABD: 0.91 +/- 0.40 microgram Sr/mg Ca; p < 0.003). The post-Hoc analysis showed differences between HP and MX. The biopsies having greater or equal values than 1.4 micrograms Sr/mg Ca showed higher levels of bone formation histomorphometric parameters and serum alkaline phosphatase (395 +/- 519 vs 1,022 +/- 989 UI/L, p < 0.05). Although it has been found that the biopsies with higher bone strontium had higher levels of osteoid tissue (characteristic of osteomalacia), the hypothesis of strontium-induced osteomalacia could not be demonstrated.
Alkaline Phosphatase↗
Multielement trace-element speciation in metal-biomolecules by chromatography coupled with ICP-MS.
The state-of-the-art of multi-element speciation in biological materials by hybrid techniques with inductively coupled plasma-mass spectrometric (ICP-MS) detection is critically reviewed with special attention to multi-metal speciation in metallothioneins (MT) and metallothionein-like proteins (MLP). The most common separation techniques used for the purpose include conventional size-exclusion chromatography (SEC) (with off-line detection) and anion-exchange (AE) or reversed-phase (RP) HPLC coupled on-line to ICP-MS. Advantages and limitations of the different commercial element-selective ICP-MS detectors, including the quadrupole (Q), double focusing (DF), and time-of-flight (TOF) mass analysers, are discussed. Procedures reported for speciation analysis, by use of hybrid techniques, for multi-metal speciation in mussel MLP and rabbit liver MT are illustrated with special emphasis on work performed in the authors' laboratory.
Biochemistry↗
Simultaneous determination of mono-, di-, and tributyltin in sediments by isotope dilution analysis using gas chromatography--ICPMS.
A mixed spike containing 119Sn-enriched monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) was prepared by direct butylation of 119Sn-enriched tin metal using a 1:3 molar excess of butyl chloride with iodide and triethylamine as catalysts. The isotopic composition of the different tin species in the spike solution was determined by gas chromatography- ICPMS after aqueous ethylation using sodium tetraethylborate. Reverse isotope dilution analysis was used for the characterization of the spike by means of natural MBT, DBT, and TBT standards. No species transformation was evident during derivatization from the reverse isotope dilution experiments based on the measured isotope ratios both before and after spiking. The mixed spike was applied to the simultaneous analysis of MBT, DBT, and TBT in certified reference materials, PACS-2 and CRM 646, with satisfactory results.
Journal Article↗
Hybridation of different chiral separation techniques with ICP-MS detection for the separation and determination of selenomethionine enantiomers: chiral speciation of selenized yeast.
Enantioseparation and determination of selenomethionine enantiomers in selenized yeast was investigated using chiral separation techniques based on different principles, coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) for selenium-specific detection. High performance liquid chromatography (HPLC) on a beta-cyclodestrin (beta-CD) column, cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC), gas chromatography (GC) on a Chirasil-L-Val column, and HPLC on a Chirobiotic T column have been investigated as the chiral separation techniques. For HPLC separation on the beta-CD column, and also for CD-MEKC, selenomethionine enantiomers were derivatized with NDA/CN(-). For chiral separation by GC, selenomethionine enantiomers were converted into their N-trifluoroacetyl (TFA)-O-alkyl esters. The developed hybridation methodologies are compared with respect to enantioselectivity, sensitivity and analysis time. The usefulness of the best-suited method [HPLC (Chirobiotic T)-ICP-MS] was demonstrated by its application to the successful chiral speciation of selenium and D-and L-selenomethionine content determination in selenized yeast.
Chromatography, Gas↗
Mercury speciation by HPLC--cold-vapour radiofrequency glow-discharge optical-emission spectrometry with on-line microwave oxidation.
Hollow-cathode (HC) radiofrequency glow-discharge (rf-GD) optical-emission spectrometry (OES) has been used as detector for the determination of inorganic mercury by cold-vapour (CV) generation in a flow-injection (FI) system. Both NaBH4 and SnCl2 were evaluated as reducing reagents for production of mercury CV. The conditions governing the discharge (pressure, He flow rate, and delivered power) and Hg CV generation (NaBH4 or SnCl2 concentration and reagent flow rate) were optimized using both reducing agents. The analytical performance characteristics of FI-CV-rf-GD-OES for mercury detection were evaluated at the 253.6 nm emission mercury line. Detection limits (DL) of 0.2 ng mL(-1) using SnCl2 and 1.8 ng mL(-1) using NaBH4 were obtained (100 microliter sample injections were used). When the optimized experimental conditions using SnCl2 had been determined, the analytical potential of this CV-rf-GD-OES method was investigated as on-line detector for high-performance liquid chromatographic (HPLC) speciation of mercury (Hg(II) and methylmercury). The HPLC-CV-rf-GD-OES detection limits for 100 microliter sample injections were found to be 1.2 and 1.8 ng mL(-1) (as mercury) of inorganic mercury and methylmercury, respectively. The reproducibility observed was below +/- 8% for both species. Finally, the HPLC-CV-rf-GD-OES system developed was successfully applied to the determination of methylmercury (speciation) in two certified reference materials, Dorm-2 and Dolt-2.
Borohydrides↗
Reference values for trace and ultratrace elements in human serum determined by double-focusing ICP-MS.
Reference values for trace and ultratrace elements concentrations in healthy human serum, measured by double-focusing inductively coupled plasma-mass spectrometry (ICP-MS), are presented. Blood donors from Asturias (Spain) were selected as the reference population (n=59). Blood samples were collected, after donation, taking the necessary precautions to avoid contamination. All subjects analyzed had normal renal function and nutritional status, as shown from their creatinine and albumin levels. A total number of 14 elements (Al, Ca, Cr, Mn, Fe, Co, Cu, Zn, Rb, Sr, Mo, Cd, Pb, and U) were monitored almost simultaneously. Serum samples were diluted 1+4 with ultrapure water and matrix interferences were corrected using Sc, Ga, Y, and Tl as internal standards. Fe, Cu, and Zn were also determined by isotope dilution analysis (IDA). Reference trace element concentrations intervals observed containing 95% of the reference distribution after excluding outliers are presented. Fourteen serum samples from hemodialysis patients were also analyzed for comparison. High levels of Al, Cr, Sr, Mo, Mn, Pb, U, Co, and Cu and low levels of Fe, Zn, and Rb were found in the serum samples from hemodialysis patients compared to the corresponding reference values observed in this work.
Adult↗
Speciation of metallothionein-like proteins of the mussel Mytilus edulis at basal levels by chromatographic separations coupled to quadrupole and double-focusing magnetic sector ICPMS.
Characterization and partial purification of metallothionein-like proteins (MLPs) of the mussel Mytilus edulis from natural populations of three coastal regions in Spain were performed. Size exclusion chromatography (SEC) with quadrupole (Q-ICPMS) or double-focusing inductively coupled plasma mass spectrometry (DF-ICPMS) detection was used first for speciation of cadmium in such natural samples and those of mussels exposed to 500 mg x L(-1) Cd in an aquarium tank. SEC results showed always a single Cd-MLP peak (MLP fraction). The contents in Cd, Cu, and Zn of this MLP fraction, of the high molecular weight protein pool (HMW), and of the whole cytosol were then measured by DF-ICPMS. Then, a given aliquot (50 microL) of MLPs with the highest values for UV molecular absorption at 254 nm (also the maximum sulfur and Cd, Cu, or Zn contents) was used to further fractionation. Fast protein liquid chromatography "on line" with Q-ICPMS was used for the purpose. Two Cd-MLP isoforms (MLP-1, MLP-2), with retention times (tR) of 15.7 and 16.0 min, were then detected in cytosols of the mussel samples of aquarium tank and also of the industrial area and Galicia coast. Conversely, wild coast mussels did not show any Cd-MLP signals at all. Analysis of essential elements copper and zinc in such cytosols by FPLC-Q-ICPMS revealed that these two metals were associated just to MLP-1. These results tend to indicate a different role for the two MLP isoforms detected in mussels (i.e., essential metals' homeostasis role seems to be tied to the MLP-1 isoform only). They illustrate the fact that trace metal speciation of unknown species in biological materials is becoming a challenge and points to the use of several complementary analytical techniques to obtain the required speciation information.
Animals↗
Development of a stable isotope approach for the inductively coupled plasma-mass spectrometry determination of oxidized metallothionein in biological materials.
The use of isotope dilution analysis (IDA) with inductively coupled plasma-mass spectrometry (ICP-MS) for the determination of oxidized metallothionein (MT) by a Cd-saturation method is investigated. The method developed here is a modification of an earlier methodology which used a radioactive Cd isotope ((109)Cd). While retaining the many advantages of this previous approach, the procedure presented here uses stable isotope ratio measurements ((114)Cd/(111)Cd) for the determination of MT. Experimental parameters governing the instrumental precision and accuracy for isotope ratio measurements of Cd by ICP-MS were characterized. Systematic errors, including mass bias, detector dead time, and spectroscopic interferences, could be easily corrected. The isotope dilution ICP-MS method was validated by the determination of very low levels of cadmium in biological certified reference materials (NIST SRM 2670 freeze-dried urine, IAEA H-8 horse kidney, and BCR TP-25 lichens). Finally, the IDA procedure was evaluated for the determination of oxidized MT by a Cd-saturation method previously developed using radioactive (109)Cd. The final procedure was applied to the quantification of MT in Long-Evans Cinnamon rat liver cytosol samples and the results were compared with data obtained for the same samples using the reference (109)Cd methodology. A good agreement between the analytical values obtained by both methods was observed.
Acetonitriles↗
Comparison of different derivatization approaches for mercury speciation in biological tissues by gas chromatography/inductively coupled plasma mass spectrometry.
A novel interface design for coupling gas chromatography and inductively coupled plasma mass spectrometry (GC/ICP-MS) was used to perform mercury speciation in biological tissues. Three derivatization approaches were optimized and compared for this purpose: anhydrous butylation using a Grignard reagent, aqueous ethylation by means of NaEt(4)B and aqueous propylation with NaPr(4)B. The last reagent was synthesized in the laboratory as it is not commercially available. Detection limits obtained by GC/ICP-MS ranged between 100 and 200 fg (as absolute mass) for methylmercury and between 500 and 600 fg for inorganic mercury using a 1 microl injection. Quantification of methyl- and inorganic mercury was carried out by resorting to aqueous calibration, using ethylmercury as internal standard for both propylation and butylation derivatization techniques. For ethylation procedures, a methylpropylmercury solution was used as internal standard. The absence of transmethylation during sample preparation was checked using a 97% enriched (202)Hg inorganic standard. The accuracy of the three derivatization approaches was evaluated by the analysis of the certified reference material DOLT-2 (dogfish liver) from the National Research Council of Canada and certified for methylmercury, with satisfactory results.
Animals↗