Heidi Goenaga-Infante

Improved sample preparation and quality control for the characterisation of titanium dioxide nanoparticles in sunscreens using flow field flow fractionation on-line with inductively coupled plasma mass spectrometry

Titanium dioxide in nanoparticulate form is used in large scale in a variety of consumer products including sunscreens. There is an increasing need for methodology for the reliable characterisation of the particle size and size dependent elemental composition in these complex matrices. Such measurement capability is essential for underpinning safety assessments, for quality control of existing products and for correlation of nanoparticle characteristics with biological effects observed in toxicity tests. This work describes the first systematic comparison and optimisation of extraction methods for titanium dioxide nanoparticles in sunscreen samples. Sunscreens were selected because of their wide use, high fat content and matrix of high complexity. Defatting of the sample with hexane followed by bath sonication with an aqueous extractant was found to provide stable suspensions of secondary titanium dioxide particles for their size characterisation by flow field flow fractionation on-line with element selective detection by inductively coupled plasma mass spectrometry. Further addition of a small amount of hexane to the aqueous extractant resulted in particle disaggregation and thus allowed for characterisation of the primary particle size. A novel approach based on sample spiking with aluminium-labelled titanium dioxide reference particles of known size was used to study the effect of extraction and separation conditions on particle size distribution in the presence of the real sample matrix. The developed methodology was applied to analysis of commercial sunscreens with various sun protection factors. Titanium extraction efficiency, particle size distribution and titanium dioxide recovery from the FFF channel were determined for each product.

Dynamic monitoring of metal oxide nanoparticle toxicity by label free impedance sensing.

The increased use of nanoparticles in industrial and medical products is driving the need for accurate, high throughput in vitro testing procedures to screen new particles for potential toxicity. While approaches using standard viability assays have been widely used, there have been increased reports of the interactions of nanoparticles with their soluble labels or optical readouts which raise concerns over the potential generation of false positive results. Here, we describe the use of an impedance spectroscopy approach to provide real-time reagent free detection of toxicity for a panel of metal oxide nanoparticles (ZnO, CuO, and TiO(2)). Using this approach, we show how impedance measurements can be used to track nanoparticle toxicity over time with comparable IC(50) values to those of standard assays (ZnO-55 μg/mL, CuO-28 μg/mL) as well as being used to identify a critical 6 h period following exposure during which the nanoparticles trigger rapid cellular responses. Through targeted analysis during this response period and the use of a novel image analysis approach, we show how the ZnO and CuO nanoparticles trigger the active export of intracellular glutathione via an increase in the activity of the ATP dependent MRP/1 efflux pumps. The loss of glutathione leads to increased production of reactive oxygen species which after 2.5 h triggers the cells to enter apoptosis resulting in a dose dependent cytotoxic response. This targeted testing strategy provides comprehensive information beyond that achieved with standard toxicity assays and indicates the potential for cell-nanoparticle interactions that could occur following in vivo exposure.

Two-dimensional HPLC coupled to ICP-MS and electrospray ionisation (ESI)-MS/MS for investigating the bioavailability in vitro of arsenic species from edible seaweed

Edible seaweed consumption is a route of exposure to arsenic. However, little attention has been paid to estimate the bioaccessibility and/or bioavailability of arsenosugars in edible seaweed and their possible degradation products during gastrointestinal digestion. This work presents first use of combined inductively coupled plasma mass spectroscopy (ICP-MS) with electrospray ionization tandem mass spectrometry (ESI-MS/MS) with two-dimensional HPLC (size exclusion followed by anion exchange) to compare the qualitative and quantitative arsenosugars speciation of different edible seaweed with that of their bioavailable fraction as obtained using an in vitro gastrointestinal digestion procedure. Optimal extraction conditions for As species from four seaweed namely kombu, wakame, nori and sea lettuce were selected as a compromise between As extraction efficiency and preservation of compound identity. For most investigated samples, the use of ammonium acetate buffer as extractant and 1xa0h sonication in a water bath followed by HPLC-ICP-MS resulted in 40–61% of the total As to be found in the buffered aqueous extract, of which 86–110% was present as arsenosugars (glycerol sugar, phosphate sugar and sulfonate sugar for wakame and kombu and glycerol sugar and phosphate sugar for nori). The exception was sea lettuce, for which the arsenosugar fraction (glycerol sugar, phosphate sugar) only comprised 44% of the total extracted As. Interestingly, the ratio of arsenobetaine and dimethylarsinic acid to arsenosugars in sea lettuce extracts seemed higher than that for the rest of investigated samples. After in vitro gastrointestinal digestion, approximately 11–16% of the total As in the solid sample was found in the dialyzates with arsenosugars comprising 93–120% and 41% of the dialyzable As fraction for kombu, wakame, nori and sea lettuce, respectively. Moreover, the relative As species distribution in seaweed-buffered extracts and dialyzates was found to be very similar. Collection of specific fractions from the size-exclusion column to be analysed using anion-exchange HPLC-ESI-MS/MS enabled improved chromatographic selectivity, particularly for the less retained arsenosugar (glycerol sugar), facilitating confirmation of the presence of arsenosugars in seaweed extracts and dialyzates. Using this approach, the presence of arsenobetaine in sea lettuce samples was also confirmed.

Determination of total arsenic and arsenic speciation in tobacco products: from tobacco leaf and cigarette smoke

The efficiency of a sequential extraction procedure (leaching with water followed by extraction with driselase and sodium dodecylsulfate) was investigated for quantitative As speciation in cut tobacco by HPLC-ICP-MS. The total As concentration and size-exclusion HPLC-ICP-MS profiles obtained for the extracts were used to select optimal extraction conditions as a trade-off between As extraction efficiency and preservation of compound identity. Leaching the cut tobacco (taken from 3R4F Kentucky research reference cigarettes) with water by either sonication in a water bath for 2 h or microwave-assisted extraction (MAE) at 50 W (50 °C) for only 10 min enabled approximately 42% of the total As in the solid to be extracted. Using anion-exchange HPLC with ICP-MS detection, 89% of the total water-soluble As was, for the first time, reported to be present as inorganic As. The instrumental limits of detection (3σ criterion) obtained for arsenite and arsenate by anion-exchange HPLC-ICP-MS were 10 and 50 ng kg−1 As, respectively. Using this hyphenated method, the presence of DMA and MMA as minor As species in the water-soluble fraction could also be detected. Complementary cation-exchange HPLC-ICP-MS analysis of unspiked extracts and extracts spiked with As standards pointed to the presence of arsenate (as a major As species) and DMA and MMA (as a minor As species). Interestingly, recent XANES results revealed that the cut tobacco contained almost exclusively As(V). Successive extractions with driselase and sodium dodecylsulfate (SDS) on the solid residue led to further extraction of approximately 13% and 8%, respectively, of As from the solid cut tobacco. The total As concentration in a homogenate of cut tobacco (from 3R4F Kentucky reference cigarettes) was found to be 318 ± 9 ng g−1 As (precision as SD, n = 3), as determined by microwave acid digestion followed by collision reaction cell ICP-MS (He mode) with standard addition calibration. The developed hyphenated MS methodology was used to study the speciation of As in water-soluble extracts of 3R4F mainstream smoke. These extracts were, for the first time, found to contain arsenite (As(III)), arsenate (As(V)) and an unidentified As-containing species. XANES analysis also reported a mixture of As(V) and As(III) in mainstream cigarette smoke condensate. The reduction of As(V) to As(III) during dynamic cigarette smoke formation can be explained by the overall smoke redox properties associated with the cigarette combustion process.

Analysis of mono-phosphate nucleotides as a potential method for quantification of DNA using high performance liquid chromatography–inductively coupled plasma-mass spectrometry

The determination of total deoxyribonucleic acid (DNA) concentration is of great importance in many biological and bio-medical analyses. The quantification of DNA is traditionally performed by UV spectroscopy; however the results can be affected greatly by the sample matrix. The proposed method quantifies phosphorus in digested calf thymus DNA and human DNA by high performance liquid chromatography (HPLC) combined with inductively coupled plasma mass spectrometry (ICP-MS). The method presented showed excellent baseline separation between all four DNA mono-nucleotides and 5′UMP. The ability of LC-ICP-MS to provide an internal check that only DNA derived phosphorus was counted in the assay was demonstrated by establishing a mass balance between the total phosphorous signal from undigested DNA and that from the speciated DNA. Column recoveries ranging from 95% to 99% for phosphorus resulted in a mass balance of 95%u2009±u20090.5% for standard nucleotides, determined by LC-ICP-MS, compared to total DNA determined by flow injection coupled to ICP-MS (FI-ICP-MS). The method for quantification was validated by analysis of NIST SRM 2,372; a total speciated DNA recovery of 52.1xa0ng/μL, compared with an expected value of 53.6xa0ng/μL, was determined by external calibration. From repeat measurements, a mass balance of 97%u2009±u20090.5% for NIST DNA was achieved. The method limits of detection for individual nucleotides were determined between 0.8 and 1.7xa0μgxa0L−1 (31P) for individual nucleotides by LC-ICP-MS, and 360xa0ngxa0L−1 for 5′AMP by direct nebulisation.

Final report of the key comparison CCQM-K98: Pb isotope amount ratios in bronze

Isotope amount ratios are proving useful in an ever increasing array of applications that range from studies unravelling transport processes, to pinpointing the provenance of specific samples as well as trace element quantification by using isotope dilution mass spectrometry (IDMS). These expanding applications encompass fields as diverse as archaeology, food chemistry, forensic science, geochemistry, medicine and metrology. However, to be effective tools, the isotope ratio data must be reliable and traceable to enable the comparability of measurement results. The importance of traceability and comparability in isotope ratio analysis has already been recognized by the Inorganic Analysis Working Group (IAWG) within the CCQM. While the requirements for isotope ratio accuracy and precision in the case of IDMS are generally quite modest, absolute Pb isotope ratio measurements for geochemical applications as well as forensic provenance studies require Pb isotope ratio measurements of the highest quality. To support present and future CMCs on isotope ratio determinations, a key comparison was urgently needed and therefore initiated at the IAWG meeting in Paris in April 2011. The analytical task within such a comparison was decided to be the measurement of Pb isotope amount ratios in water and bronze. Measuring Pb isotope amount ratios in an aqueous Pb solution tested the ability of analysts to correct for any instrumental effects on the measured ratios, while the measurement of Pb isotope amount ratios in a metal matrix sample provided a real world test of the whole chemical and instrumental procedure. A suitable bronze material with a Pb mass fraction between 10 and 100 mg•kg-1 and a high purity solution of Pb with a mass fraction of approximately 100 mg•kg-1 was available at the pilot laboratory (BAM), both offering a natural-like Pb isotopic composition. The mandatory measurands, the isotope amount ratios n(206Pb)/n(204Pb), n(207Pb)/n(204Pb) and n(208Pb)/n(204Pb) were selected such that they correspond with those commonly reported in Pb isotopic studies and fully describe the isotopic composition of Pb in the sample. Additionally, the isotope amount ratio n(208Pb)/n(206Pb) was added, as this isotope ratio is typically measured when performing Pb quantitation by IDMS involving a 206Pb spike. Each participant was free to use any method they deemed suitable for measuring the individual isotope ratios. However, the majority of the results were obtained by using muIti-collector ICPMS or TIMS. The key requirements for all analytical procedures were a traceability statement for all results and the establishment of an uncertainty budget meeting a target uncertainty for all ratios of 0.2 %, relative (k=1). Additionally, the use of a Pb-matrix separation procedure was encouraged. The obtained overall result was excellent, demonstrating that the individual results reported by the NMIs/DIs were comparable and compatible for the determination of Pb isotope ratios. MC-ICPMS and MC-TIMS data were consistent with each other and agree to within 0.05 %. The corresponding uncertainties can be considered as realistic uncertainties and mainly range from 0.02 % to 0.08 % (k=1). As stated above isotope ratios are being increasingly used in different fields. Despite the availability and ease of use of new mass spectrometers, the metrology of unbiased isotope ratio measurements remains very challenging. Therefore, further comparisons are urgently needed, and should be designed to also engage scientists outside the NMI/DI community. Possible follow-up studies should focus on isotope ratio and delta measurements important for environmental and technical applications (e.g. B), food traceability and forensics (e.g. H, C, N, O, S and 87Sr/86Sr) or climate change issues (e.g. Li, B, Mg, Ca, Si). Main text. To reach the main text of this paper, click on Final Report. The final report has been peer-reviewed and approved for publication by the CCQM.