Cold Vapor Generation Atomic Fluorescence Research Articles

Estimated intake levels of methylmercury in children, childbearing age and pregnant women in a Mediterranean region, Murcia, Spain

Methylmercury (MeHg) is a bioaccumulable toxin in the trophic chain and a powerful neurotoxin during fetal and child development. Consumption of contaminated fish and shellfish is a principal environmental source of MeHg exposure. This study was designed to assess the Hg and estimated MeHg intake in vulnerable groups of the Murcia region, a Mediterranean part of Spain, compared with international regulations. A validated food frequency questionnaire was used to assess seafood consumptions in 320 children younger than 10 years, 301 women of childbearing age, and 537 pregnant women. Hg concentrations were measured in the most consumed fish products by cold vapor generation-atomic fluorescence spectrometry. The weekly intake of MeHg (microg/kg bw/week) was 2.60 (95% CI = 2.10-3.10) in children 1-5 years, 2.65 (95% CI = 2.26-3.03) in children 6-10 years, 0.98 (95% CI = 0.89-1.07) in women of childbearing age, and 0.88 (95% CI = 0.81-0.95) in pregnant women. The main exposure to MeHg, especially in young children, is related to intake of bluefin tuna and swordfish. Fifty-four percent of children aged 1-10 years, 10% of pregnant women, and 15% of women of childbearing age exceed the Joint Expert Committee on Food Additives provisional tolerable weekly intake of MeHg. In the Murcia region, where fish is a central component of the diet, the focus should be on educating vulnerable populations to reorient fish consumption in order to lower the amount of Hg incorporated with the diet as well as to reduce Hg emissions into the environment.

Determination of S-nitrosoglutathione and other nitrosothiols by p-hydroxymercurybenzoate derivatization and reverse phase chromatography coupled with chemical vapor generation atomic fluorescence detection

S-Nitrosoglutathione (GSNO) reacts with the organic mercurial probe, p-hydroxymercury benzoate (PHMB, HO–Hg–(C 6H 4)–COO −Na +) giving the complex GS–Hg(C 6H 4)COOH (GS–PHMB). This reaction has been studied by UV measurements at 334 nm also in the presence of ascorbic acid and the product of reaction, the GS–PHMB complex, characterized by Electrospray Ionization Mass Spectrometry (ESI-MS) and by Reversed Phase Chromatography (RPLC) coupled on-line and sequentially with a UV–visible diode array detector (DAD) followed by a cold vapor generation atomic fluorescence spectrometer (CVGAFS). The simultaneous presence of PHMB and ascorbate produced a synergistic effect on GSNO decomposition rate that can be observed only above a given concentration threshold of ascorbate (ascorbate/GSNO molar ratio ≥ 180). The results indicated that the formation of GS–PHMB, both in the presence and the absence of ascorbic acid, does not involve the formation of free thiolic species but it takes place through a more complex mechanism. The PHMB derivatives of GSH and GSNO obtained by the present method were found to be identical by ESI-MS. GSSG did not interfere because it was not reduced and derivatized to GS–PHMB. Once complexed by the alkylating agent N-ethylmaleimide (NEM), GSH did not interfere with the derivatization reaction. This ensured a good selectivity of the developed PHMB derivatization system for RSNO determination. Thus, we have optimized the operating conditions for the selective reaction of PHMB with GSNO and other nitrosothiols (RSNOs) in order to determinate RSNOs in human plasma. LODc for RSNOs in plasma ultrafiltrate was 30 nM (injected concentration, 50 μL loop), the DLR ranged between 0.08 and 50 μM and the CV% was 6.5% at 300 nM concentration level. Reduced and oxidized thiols spiked to plasma did not interfere with the measurement of RSNOs. We found that the sampling procedure was critical for the recovery of endogenous and spiked RSNOs. The ultrafiltrate samples of plasma of 8 healthy humans contained 1460 ± 310 CysNO, 1000 ± 330 nM HCysNO and 320 ± 60 nM GSNO if blood was sampled in a mixture NEM/ethylendiaminotetracetic acid (EDTA)/serine–borate complex (SBC), where serine–borate complex is a potent inhibitor of γ-glutamyltransferase, an enzyme involved in the conversion of GSNO into CysGlyNO. In the absence of SBC during the sampling of blood GSNO concentration found in the ultrafiltrate was lower (at level of the determination limit in plasma ultrafiltrate, i.e. 75 nM) and the peak of CysGlyNO appeared, which corresponded to a concentration of 200 ± 60 nM ( N = 4 blood samples).

On-line pressurized electromagnetic induction heating digestion: a promising sample preparation technique for trace mercury analysis

A novel, automated on-line pressurized sample digestion system based on electromagnetic induction heating technique was developed to perform solid sample decomposition in acid medium. The efficiency of acid digestion was increased with 0.36 MPa pressure built up in-line by a 0.5 mm id × 25 m length flow restrictor and up to approximately 135 °C reaction temperature. The system's performance was evaluated for on-line digestion of edible seaweeds and subsequent determination of mercury by cold vapor generation atomic fluorescence spectrometry (CVAFS).

Determination and characterization of phytochelatins by liquid chromatography coupled with on line chemical vapour generation and atomic fluorescence spectrometric detection

Liquid chromatography (LC) coupled on line with UV/visible diode array detector (DAD) and cold vapour generation atomic fluorescence spectrometry (CVGAFS) has been developed for the speciation, determination and characterization of phytochelatins (PCs). The method is based on a bidimensional approach, e.g. on the analysis of synthetic PC solutions (apo-PCs and Cd 2+-complexed PCs) (i) by size exclusion chromatography coupled to UV diode array detector (SEC–DAD); (ii) by the derivatization of PC –SH groups in SEC fractions by p-hydroxymercurybenzoate (PHMB) and the indirect detection of PC-PHMB complexes by reversed phase liquid chromatography coupled to atomic fluorescence detector (RPLC–CVGAFS). MALDI-TOF/MS (matrix assisted laser desorption ionization time of flight mass spectrometry) analysis of underivatized synthetic PC samples was performed in order have a qualitative information of their composition. Quantitative analysis of synthetic PC solutions has been performed on the basis of peak area of PC-PHMB complexes of the mercury specific chromatogram and calibration curve of standard solution of glutathione (GSH) complexed to PHMB (GS-PHMB). The limit of quantitation (LOQ) in terms of GS-PHMB complex was 90 nM (CV 5%) with an injection volume of 35 μL, corresponding to 3.2 pmol (0.97 ng) of GSH. The method has been applied to analysis of extracts of cell cultures from Phaeodactylum tricornutum grown in Cd-containing nutrient solutions, analysed by SEC–DAD–CVGAFS and RPLC–DAD–CVGAFS.

Comparison of three strategies to evaluate uncertainty from in-house validation data. A case study: mercury determination in sediments

In the present paper, three approaches are compared for the evaluation of the combined uncertainty in the determination of mercury in aquatic sediments by an aqua regia extraction procedure. For this, the data obtained in validation studies from five certified reference materials (CRMs), covering a range of concentrations from 0.8 to 130 mg kg(-1) of mercury and analysed by three atomic spectroscopic techniques (cold vapour generation atomic fluorescence spectrometry, CV-AFS, cold vapour generation atomic absorption spectroscopy, and inductively coupled plasma mass spectroscopy), were considered. The combined uncertainty was firstly assessed by considering separately the data obtained for each CRM analysed (approach A). Moreover, this assessment was also performed with two other calculation approaches (B and C) based on the pooled data obtained from the validation step. The comparison of the results obtained for the different techniques showed a clear bias effect when using CV-AFS with nitric acid as a diluent. In relation to the strategies tested for the combined uncertainty assessment, approach C proved to be the easiest and friendliest method for uncertainty assessment.

Cloud Point Extraction Preconcentration Prior to High-Performance Liquid Chromatography Coupled with Cold Vapor Generation Atomic Fluorescence Spectrometry for Speciation Analysis of Mercury in Fish Samples

A cloud point extraction methodology was developed for simultaneous preconcentration of Hg(II), methylmercury (MeHg), ethylmercury (EtHg), and phenylmercury (PhHg) prior to reversed-phase high-performance liquid chromatography (HPLC) on-line coupled with cold vapor atomic fluorescence spectrometry for speciation analysis of mercury in fish. The four mercury species were taken into complexes with ammonium pyrrolidine dithiocarbamate (APDC) in aqueous nonionic surfactant Triton X-114 medium and concentrated in the surfactant-rich phase by bringing the solution to the temperature of 40 degrees C. Baseline separation of the enriched complexes was achieved on an RP-C(18) column with a mixture of methanol, acetonitrile, and water (65:15:20, v/v) containing 200 mmol L(-1) HAc (pH 3.5) as the mobile phase. An on-line postcolumn oxidation of the effluent from HPLC, in the presence of K2S2O8 in HCl, was applied in the system followed by an optimal cold vapor generation of mercury species. The variables affecting the complexation and extraction steps were examined. The preconcentration of 10 mL of solution with 0.08% w/v Triton X-114 and 0.04% w/v APDC at pH 3.5 gave enrichment factors of 29, 43, 80, and 98 for MeHg, EtHg, PhHg, and Hg(II), respectively. Low detection limits (S/N = 3) were obtained, ranging from 2 to 9 ng L(-1) (as Hg) for all species. The developed method was successfully applied to the speciation of mercury in real fish samples.

Speciation and Quantification of Thiols by Reversed-Phase Chromatography Coupled with On-Line Chemical Vapor Generation and Atomic Fluorescence Spectrometric Detection: Method Validation and Preliminary Application for Glutathione Measurements in Human Whole Blood

We developed a sensitive, specific method for the low-molecular-mass thiols cysteine, cysteinylglycine, glutathione, and homocysteine and validated the method for measurement of glutathione in blood. The technique was based on reversed-phase chromatography (RPC) coupled on line with cold vapor generation atomic fluorescence spectrometry (CVGAFS). Thiols were derivatized before introduction on the column by use of a p-hydroxymercuribenzoate (PHMB) mercurial probe and separated as thiol-PHMB complexes on a Vydac C4 column. Postcolumn on-line reaction of derivatized thiols with bromine allowed rapid conversion of the thiol-PHMB complexes to inorganic mercury with recovery of 100 (2)% of the sample. HgII was selectively detected by atomic fluorescence spectrometry in an Ar/H2 miniaturized flame after sodium borohydride reduction to Hg0. The relationship between thiol-PHMB complex concentration and peak area (CVGAFS signal) was linear over the concentration range 0.01-1400 micromol/L (injected). The detection limits were 1, 1, 0.6, and 0.8 nmol/L for cysteine, cysteinylglycine, homocysteine, and glutathione in the injected sample, respectively. The CVs for thiols were 1.5%-2.2% for calibrator solutions and 2.1% and 3.0% for real samples. The RPC-CVGAFS method allowed speciation of glutathione (reduced and oxidized) in human whole blood from healthy donors and from the coronary sinus of patients with idiopathic dilated cardiomyopathy during and after chronotropic stress. The RPC-CVGAFS method could be used to measure reduced and oxidized glutathione in human whole blood as disease biomarkers.

Mercury speciation by liquid chromatography coupled with on-line chemical vapour generation and atomic fluorescence spectrometric detection (LC–CVGAFS)

Reverse phase chromatography (RPC) coupled on-line with UV–vis diode array detector (DAD) and cold vapour generation atomic fluorescence spectrometry (CVGAFS) is proposed for the speciation and determination of inorganic and organic mercury (methylmercury, ethylmercury and phenylmercury) in the form of cysteine, penicillamine and glutathione complexes. The mercury–thiol complexes are separated on a C 18 Reverse Phase column and oxidized on-line with bromine, generated in situ by KBr/KBrO 3 in HCl medium, in order to fast convert organic mercury species to inorganic mercury in less than 2.5 s, at room temperature, in a 30 cm knitted coil. Hg(II) is selectively detected by AFS in a Ar/H 2 miniaturized flame after sodium borohydride reduction to Hg 0. Under optimized conditions, on-line bromine treatment gives recoveries of thiol-complexed methylmercury, ethylmercury and phenylmercury with respect to inorganic mercury ranging between 79 and 85%, 80 and 85%, 63 and 76%, respectively, depending on the complexing thiol employed. Optimized elution conditions were provided in the three complexing agents. The detection limits (LODc) for inorganic mercury, methylmercury, ethylmercury and phenylmercury, in the optimized conditions complexed with thiols were about 16, 18, 18 and 20 pg (as mercury), respectively, a relative standard deviation (R.S.D) ranging between 1.5 and 2.0%, and a linear dynamic range between 0.1 and 100 ng injected. LC–DAD–CVGAFS method has been validated by analysing two certificate reference material, DORM-2 and NIES CRM 13, obtaining 98 ± 6 and 97 ± 5% of methylmercury recovered, respectively.

Characterization of denatured metallothioneins by reversed phase coupled with on-line chemical vapour generation and atomic fluorescence spectrometric detection

A new analytical hyphenated technique is proposed for determination and characterization of thiolic proteins, based on reverse phase chromatography (RPC) coupled on-line with cold vapour generation atomic fluorescence spectrometry (CVGAFS). Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0 mol l −1 urea and p-hydroxymercurybenzoate (PHMB). The derivatized proteins are separated on a C4 Vydac Reverse Phase column. Post-column on-line reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO 3 in HCl medium, allowed the fast conversion of both the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury, also in the presence of methanol in the RPC eluent phase. Hg(II) is selectively detected by AFS in a Ar/H 2 miniaturized flame after sodium borohydride reduction to Hg°. Under optimized conditions, on-line bromine treatment gives a 98 ± 2% recovery of both free and protein-complexed PHMB. The effect of methanol on the sensitivity of Hg(II) detection was studied and controlled. RPC-CVGAFS system has been applied to the analysis of metallothioneins from rabbit liver (MT RL) standard solutions, and their commercial isoforms MT-1 and MT-2. The analysis of denatured, PHMB-complexed MTs allowed the determination of the number of thiolic groups complexed by PHMB. It was found that MTs from rabbit liver have 10.0 ± 0.3 (MT-1) and 6.7 ± 0.3 (MT-2 and MT RL) –SH groups complexed by PHMB. The detection limit (LODc) for PHMB in 95% methanol in the optimized conditions was about 9.3 × 10 −9 mol l −1 and for the denatured MTs LODc was about 8.6 × 10 −10 mol l −1, taking into account an approximate complexating ratio PHMB:MTs of 7:1.

Simultaneous determination of trace mercury and cadmium in tobacco samples by cold vapor generation-atomic fluorescence spectrometry

A highly sensitive and simple method was developed for the simultaneous determination of trace cadmium and mercury in tobacco samples by cold vapor generation-atomic fluorescence spectrometry equipped with an intermittent flow reactor. The reaction was accomplished with potassium tetrahydroborate(III) in aqueous solutions. A newly modified atomizer was presented on the basis of the present atomic fluorescence system, which effectively improved the stability of the signals. Special accent was put on the study of the conditions for the generation of volatile cadmium and mercury species were investigated in detail. Some transition metal ions that could be used as sensitivity enhancers, including iron(III), cobalt(II), nickel(II), zinc(II) and molybdenum(VI) in the presence of thiourea. The tolerance to the interference of coexisting ions was carefully examined in this system. Under optimal conditions, the detection limits (3σ) were evaluated to be 2.8 ng l−1 for cadmium and 4.3 ng l−1 for mercury, respectively. The relative standard deviations for 10 replicate determinations of 0.8 µg l−1 mercury and 1 µg l−1 cadmium were 3.2% and 2.9%, respectively. The proposed method was successfully applied to the simultaneous determination of mercury and cadmium in tobacco leaf samples.

Determination of mercury in cosmetics by flow injection-cold generation-atomic fluorescence spectrometry with on-line preconcentration

A method for the determination of trace amounts of mercury in cosmetics is reported. The method involves the acid treatment of the sample in a focused-microwave digester, on-line preconcentration in a C 18 column and cold vapour generation-atomic fluorescence detection. The method can be applied to the determination of the target analyte at the picogram level in different eye cosmetics (eye liner, eye shadow and eye pencil), with recoveries ranging between 89 and 104% and with a precision, expressed as relative standard deviation, of 5.9%.