Evaluation of uncertainty sources in the determination of testosterone in urine by calibration-based and isotope dilution quantification using ultra high performance liquid chromatography tandem mass spectrometry.

Three quantification methodologies, namely calibration with internal standard (Cal-IS, non-weighted), weighted calibration with internal standard (wCal-IS) and isotope pattern deconvolution (IPD) have been used for the determination of testosterone in urine by LC-MS/MS. Uncertainty has been calculated and compared for the three methodologies through intra- and inter-laboratory reproducibility assays. IPD showed the best performance for the intra-laboratory reproducibility, with RSD and combined uncertainty values below 4% and 9% respectively. wCal-IS showed similar performance, while Cal-IS where not constant and clearly worse at the lowest concentration assayed (2ng/mL) reaching RSD values up to 16%. The inter-laboratory assay indicated similar results although wCal-IS RSD (20%) was higher than IPD (10%) and Cal-IS get worse with RSD higher than 40% for the lowest concentration level. Uncertainty budgets calculated for the three procedures revealed that intercept and slope were the most important factors contributing to uncertainty for Cal-IS. The main factors for wCal-IS and IPD were the volumes of sample and/or standard measured.

Cd-induced phytochelatin synthesis in Dittrichia viscosa (L.) Greuter is determined by the dilution of the culture medium.

In this paper, we examined Cd accumulation and PC synthesis in two clones of Dittrichia viscosa, one with a metallicolous (DV-A) and the other with a non-metallicolous origin (DV-W). The clones were cultured in vitro with 0 and 10 mg Cd L(-1) in both short-term treatments (up to 72 h) and over 10 days. We also examined the influence of the culture medium dilution and the PC-synthesis inhibitor, L-buthionine-sulfoximine (BSO), on these parameters. Similar Cd accumulation values were found in the two clones. No synthesis of new thiolic compounds was observed in Cd-treated plants cultured in vitro in Murashige and Skoog medium up to 72 h when compared to controls. Dilution of the culture medium affected PC production, increasing it in 1/2 MS and especially in 1/4 MS. Cd uptake did not increase in the same way, but still hyperaccumulation levels were exceeded in all Cd treatments. BSO addition increased the sensitivity of D. viscosa to Cd and diminished Cd accumulation. Nevertheless, a poor correlation between PCs and Cd accumulation capacity was observed since the highest Cd content did not correspond to the highest PC levels. All these results obtained suggest that PCs are important in Cd accumulation and detoxification in D. viscosa and also that other mechanisms might be involved in these traits.

Sulphur tracer experiments in laboratory animals using 34S-labelled yeast.

We have evaluated the use of (34)S-labelled yeast to perform sulphur metabolic tracer experiments in laboratory animals. The proof of principle work included the selection of the culture conditions for the preparation of sulphur labelled yeast, the study of the suitability of this labelled yeast as sulphur source for tracer studies using in vitro gastrointestinal digestion and the administration of the (34)S-labelled yeast to laboratory animals to follow the fate and distribution of (34)S in the organism. For in vitro gastrointestinal digestion, the combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and high-performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS) showed that labelled methionine, cysteine and other low molecular weight sulphur-containing biomolecules were the major components in the digested extracts of the labelled yeast. Next, in vivo kinetic experiments were performed in healthy Wistar rats after the oral administration of (34)S-labelled yeast. The isotopic composition of total sulphur in tissues, urine and faeces was measured by double-focusing inductively coupled plasma mass spectrometry after microwave digestion. It was observed that measurable isotopic enrichments were detected in all samples. Finally, initial investigations on sulphur isotopic composition of serum and urine samples by HPLC-ICP-MS have been carried out. For serum samples, no conclusive data were obtained. Interestingly, chromatographic analysis of urine samples showed differential isotope enrichment for several sulphur-containing biomolecules.

Development of a direct procedure for the measurement of sulfur isotope variability in beers by MC-ICP-MS.

In this work, a multi-collector inductively coupled plasma mass spectrometer (MC-ICP-MS) was evaluated for the direct measurement of sulfur stable isotope ratios in beers as a first step toward a general study of the natural isotope variability of sulfur in foods and beverages. Sample preparation consisted of a simple dilution of the beers with 1% (v/v) HNO(3). It was observed that different sulfur isotope ratios were obtained for different dilutions of the same sample indicating that matrix effects affected differently the transmission of the sulfur ions at masses 32, 33, and 34 in the mass spectrometer. Correction for mass bias related matrix effects was evaluated using silicon internal standardization. For that purpose, silicon isotopes at masses 29 and 30 were included in the sulfur cup configuration and the natural silicon content in beers used for internal mass bias correction. It was observed that matrix effects on differential ion transmission could be corrected adequately using silicon internal standardization. The natural isotope variability of sulfur has been evaluated by measuring 26 different beer brands. Measured delta(34)S values ranged from -0.2 to 13.8 per thousand. Typical combined standard uncertainties of the measured delta(34)S values were < or = 2 per thousand. The method has therefore great potential to study sulfur isotope variability in foods and beverages.

Use of enriched 74Se and 77Se in combination with isotope pattern deconvolution to differentiate and determine endogenous and supplemented selenium in lactating rats.

A quantitative methodology has been developed to differentiate between endogenous and supplemented selenium in lactating rats using two enriched selenium isotopes. Lactating rats were fed for 2 weeks with formula milk containing one enriched Se isotope, (77)Se, as the metabolic tracer. The isotopic composition of selenium in serum and urine samples was then measured by collision cell ICP-MS after the addition of a solution containing another enriched isotope, (74)Se, as quantitation tracer, before analysis. Isotope pattern deconvolution allowed the transformation of measured Se isotopic abundances into concentrations of natural abundance (endogenous) selenium and enriched (77)Se (supplemented) present in the samples. The proposed methodology was validated using serum and urine reference materials spiked with both (77)Se and (74)Se. The obtained results are discussed in terms of selenium exchange and half-life in lactating rats (11-12 days) and selenium levels in serum in comparison with non-supplemented rats and control rats after maternal feeding.

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).

[Pulmonary edema related to administration of 15-methyl-prostaglandin F2alpha during a cesarean section]. / Edema pulmonar en una cesárea relacionado con la administración de 15-metil prostaglandina F2alpha.

An 18-year-old woman pregnant at 37 weeks gestation and with a history of recurrent urinary tract infection was admitted with a clinical picture of pyelonephritis that responded favorably to antibiotic treatment. After 2 days, cervical ripening was induced with prostaglandin E2 gel and labor was induced with oxytocin. The patient requested epidural analgesia. Six hours after induction, cesarean delivery was indicated owing to risk of fetal distress. The operation was carried out under epidural anesthesia with 10 mL of 0.5% bupivacaine without a vasoconstrictor. After delivery, uterine atony was treated unsuccessfully with oxytocin and methylergometrine maleate; the obstetrician then gave an intramyometrial injection of 0.25 mg of 15-methyl-prostaglandin F2alpha (PGF2alpha). After 5 minutes, SpO2 fell to 89%, accompanied by dyspnea and sinus tachycardia of 130-140 beats/min, with normal cardiorespiratory sounds. The patient was transferred to the postoperative recovery unit, where a chest radiograph led to a diagnosis of acute pulmonary edema. Treatment to reduce edema was successful. PGF2alpha and its analogs are useful for treating uterine atony that does not respond to other drugs, but side effects are not unknown. Caution in prescribing PGF2alpha and care in monitoring the patient's reaction are therefore recommended during and after anesthesia. Unnecessary overhydration should be avoided.

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)).

[Effect of strontium on bone metabolism in hemodialysis patients]. / Efecto del estroncio sobre el metabolismo óseo en pacientes en hemodiálisis.

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.

Tos crónica / Chronic cough

No disponible

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.

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.

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.

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.

Determination of n-alkanes and polycyclic aromatic hydrocarbons in atmospheric particulate and vapour phases in Oviedo, Spain, by GC-MS.

A GC-MS procedure for the determination of hydrocarbons in air samples from Oviedo, Spain, was developed. Air hydrocarbons were sampled with a high volume sampler equipped with a holder containing a glass fiber filter, to trap the particulate phase, and two polyurethane foams to capture hydrocarbons of the vapour phase. Compounds were extracted with CH2Cl2 by Soxhlet extraction and then fractionated using column chromatography with alumina silica. Analyses of the fractions were performed by GC-MS in the electron ionization mode. PAHs and n-alkanes were the compounds examined in this work. Samples collected in the vicinity of the Faculty of Chemistry (a semi-urban area) were analysed. The total concentration of PAHs in the air samples analysed ranged from 28 to 76 ng m(-3). The total concentration of n-alkanes and PAHs in the vapour phase exceeded the concentration in the particulate phase in the samples analysed.

Indirect determination of trace amounts of fluoride in natural waters by ion chromatography: a comparison of on-line post-column fluorimetry and ICP-MS detectors.

An alternative method for the determination of trace levels of fluoride in drinking and sea-water samples is presented. It is based on the formation of the aluminium monofluoride complex in excess of Al3+ and separation of the two species formed (AlF2+ and Al3+) in a small (5 cm long, CG2) ion exchange guard column. The final determination is accomplished by both ICP-MS specific detection and post column derivatisation with fluorimetric detection. Fundamental studies on the formation kinetics of the complex, ion chromatographic separation and optimum aluminium concentration were carried out using spectrofluorimetric detection by post-column reaction of the species with 8-hydroxyquinoline-5-sulfonic acid in a micellar medium of cetyltrimethylammonium bromide. Fluorimetric detection showed good detection limits, but interferences from cations such as Mg2+ and Zn2+ required the use of the longer CS2 ion exchange column. Iron interfered in relatively large amounts but adding EDTA to the sample solution eliminated the interference. A similar separation methodology was applied using ICP-MS detection for the indirect determination of fluoride, by monitoring aluminium at mass 27. In this case, a detection limit of 0.1 ng ml-1 was obtained using 0.45 M HNO3 as eluent and no interference caused by high concentrations of iron was observed. The proposed method was applied to the determination of very low levels of fluoride in natural waters.

Speciation of basal aluminium in human serum by fast protein liquid chromatography with inductively coupled plasma mass spectrometric detection.

The coupling of fast protein liquid chromatography (FPLC) with inductively couples plasma mass spectrometry (ICP-MS) was evaluated as a technique for studying aluminium bound to proteins present in human serum. Separation of human serum proteins was achieved on a MonQ (HR5/5) anion-exchange column using an ammonium acetate gradient (0-0.25 mol I-1) at the physiological pH of 7.4 (0.05 mol l-1 TRIS-HC1 buffer). Aluminium contamination was avoided with an on-line Al-chelating scavenger column. Proteins were detected spectrophotometrically at 295 nm and the Al detection was carried out on-line using both quadrupole ICP-MS and double-focusing ICP-MS systems. At metal basal levels in serum the latter detector proved to be adequate for this detection. Results obtained with the procedure developed confirmed clearly that transferrin is the only significant A1-binding proteins in unspiked uraemic serum. In addition, a high-resolution ICP-MS instrument was applied successfully as an A1-specific detector allowing for the first time A1 speciation studies in unspiked normal serum. The technique can also be used for studying the protein binding of elements other than A1.

Different quantification approaches for the analysis of biological and environmental samples using inductively coupled plasma mass spectrometry.

For the analysis of biological and environmental materials by inductively coupled plasma mass spectrometry (ICP-MS), several quantification procedures can be used depending on the precision and accuracy required. Semi-quantitative methods based on the molar response curve were compared with conventional external calibration and standard additions for the analysis of waters and sewage sludges. For the analysis of biological materials, where higher quality data were required, isotope dilution analysis using enriched isotopes was applied. It was observed that the molar sensitivity for different elements in ICP-MS was a simple function of the mass of the isotopes measured after normalization for ionization efficiency which could be fitted to a third-order polynomial equation. Element ionization adjustments for the third-order polynomial, using the Saha equation, allowed the calculation of the plasma ionization temperature and electron density. For the determination of trace metals in waters and sewage sludges, the samples were spiked with different internal standards, ionization corrections were performed and the results obtained agreed with those obtained by external calibration and standard addition within a factor of 2 but, on average, the agreement was within 20%. The determination of molybdenum in biological reference materials was performed by isotope dilution analysis taking into account possible sources of error in the measurements by ICP-MS such as mass discrimination, detector dead time, isobaric interferences and random error propagation.