Evaluation of two-phase sample transport upon ablation of gelatin as a proxy for soft biological matrices using nanosecond laser ablation - inductively coupled plasma - mass spectrometry.

BACKGROUND: Recent papers on LA-ICP-MS have reported that certain elements are transported in particulate form, others in gaseous form and still others in a combination of both upon ablation of C-based materials. These two phases display different transport behaviour characteristics, potentially causing smearing in elemental maps, and could be processed differently in the ICP which raises concerns as to accuracy of quantification and emphasizes the need for matrix-matching of external standards. This work aims at a better understanding of two-phase sample transport by evaluating the peak profile changes observed upon varying parameters such as laser energy density and wavelength. RESULTS: It is demonstrated that upon ablation of gelatin, elements are transported predominantly in particulate phase, but already at low laser energy density, a significant fraction of some elements is transported in the gaseous phase, which is even more expressed at higher energy density. This behaviour is element-specific since the ratio of the signal intensity for the analyte element transported in gas phase to the total signal intensity was 0 % for 23Na, 43 % for 66Zn and as high as 95 % for 13C using a 193 nm laser. The results also suggest an effect of the laser wavelength, as all elements show either the same or higher amount of gas phase formation upon ablating with 213 nm versus 193 nm. It was even established that elements that fully occur in particulate form upon ablation using 193 nm laser radiation are partly converted into gaseous phase when using 213 nm. SIGNIFICANCE: This work provides a thorough investigation of the underexposed phenomenon of two-phase sample transport upon ablation of biological samples upon via LA-ICP-MS. It is shown that for some elements a fraction of the ablated material is converted and transported in the gas phase, which can lead to significant smearing effects. As such, it is important to evaluate element-specific peak profiles on beforehand and, if necessary, adapt instrument settings and slow down data acquisition.

Semiquantitative Analysis for High-Speed Mapping Applications of Biological Samples Using LA-ICP-TOFMS.

Laser ablation (LA) in combination with inductively coupled plasma time-of-flight mass spectrometry (ICP-TOFMS) enables monitoring of elements from the entire mass range for every pixel, regardless of the isotopes of interest for a certain application. This provides nontargeted multi-element (bio-)imaging capabilities and the unique possibility to screen for elements that were initially not expected in the sample. Quantification of a large range of elements is limited as the preparation of highly multiplexed calibration standards for bioimaging applications by LA-ICP-(TOF)MS is challenging. In this study, we have developed a workflow for semiquantitative analysis by LA-ICP-TOFMS based on multi-element gelatin micro-droplet standards. The presented approach is intended for the mapping of biological samples due to the requirement of matrix-matched standards for accurate quantification in LA-ICPMS, a prerequisite that is given by the use of gelatin-based standards. A library of response factors was constructed based on 72 elements for the semiquantitative calculations. The presented method was evaluated in two stages: (i) on gelatin samples with known elemental concentrations and (ii) on real-world samples that included prime examples of bioimaging (mouse spleen and tumor tissue). The developed semiquantification approach was based on 10 elements as calibration standards and provided the determination of 136 nuclides of 63 elements, with errors below 25%, and for half of the nuclides, below 10%. A web application for quantification and semiquantification of LA-ICP(-TOF)MS data was developed, and a detailed description is presented to easily allow others to use the presented method.

Comparison of single pulse, multiple dosage, and 2D oversampling / deconvolution LA-ICPMS strategies for mapping of (ultra)low-concentration samples.

Elemental LA-ICPMS mapping in continuous scanning mode gathers the counts generated upon laser ablation in line scanning mode. Acquisition of counts can be performed for each single laser pulse separately or by summing the counts of multiple laser pulses. Conventionally, pixels in an LA-ICPMS map are associated with spot-resolved single laser pulses (zero-dimensional, 0D), but also sub-pixel convolution strategies are in use, associated with one-dimensional (1D) or two-dimensional (2D) overlapping laser shots, and where possible followed by deblurring. The imaging quality of several 0D, 1D, and 2D LA-ICPMS strategies were compared for mapping of (ultra)low-concentration samples, both via computational and experimental approaches. The data presented will be helpful to make the right decision about the best possible LA-ICPMS mapping strategy for the highest image quality.

Nanoparticle Analysis in Biomaterials Using Laser Ablation-Single Particle-Inductively Coupled Plasma Mass Spectrometry.

In the past decade, the development of single particle-inductively coupled plasma mass spectrometry (SP-ICPMS) has revolutionized the field of nanometallomics. Besides differentiation between dissolved and particulate metal signals, SP-ICPMS can quantify the nanoparticle (NP) number concentration and size. Because SP-ICPMS is limited to characterization of NPs in solution, we show how solid sampling by laser ablation (LA) adds spatial-resolution characteristics for localized NP analysis in biomaterials. Using custom-made gelatin standards doped with dissolved gold and commercial or synthesized gold nanoparticles, LA-SP-ICPMS conditions such as laser fluence, beam size, and dwell time were optimized for NP analysis to minimize NP degradation, peak overlap, and interferences from dissolved gold. A data-processing algorithm to retrieve the NP number concentration and size was developed for this purpose. As a proof-of-concept, a sunflower-root-sample cross-section, originating from a sunflower plant exposed to gold NPs, was successfully imaged using the optimized LA-SP-ICPMS conditions for localized NP characterization.

Perceptual Image Quality Metrics Concept in Continuous Scanning 2D Laser Ablation-Inductively Coupled Plasma Mass Spectrometry Bioimaging.

This work focuses on the structural similarity (SSIM) index as a tool for optimization of the perceived visual image quality obtainable by continuous scanning 2D LA-ICPMS bioimaging, but also other mass spec imaging techniques may benefit from this approach. This index quantifies the differences between a distorted image and a reference image based on parameters associated with luminance, contrast, and noise. Since reference images are not normally available, a protocol was developed to virtually apply distortion-related information introduced by the LA-ICPMS imaging system to a reference image of one's choice. Distortion-related information in the form of blur and noise was experimentally retrieved from line scans across a laser milled knife edge on custom-prepared gelatin standards (mimicking proteinaceous biomatrixes). Distorted images were generated via computational procedures developed earlier, warranting objective image quality assessment via the SSIM indices. We illustrate the potential of this approach for image quality optimization for a suite of LA-ICPMS imaging conditions.

Differential cadmium and zinc distribution in relation to their physiological impact in the leaves of the accumulating Zygophyllum fabago L.

Cadmium and zinc share many similar physiochemical properties, but their compartmentation, complexation and impact on other mineral element distribution in plant tissues may drastically differ. In this study, we address the impact of 10 µm Cd or 50 µm Zn treatments on ion distribution in leaves of a metallicolous population of the non-hyperaccumulating species Zygophyllum fabago at tissue and cell level, and the consequences on the plant response through a combined physiological, proteomic and metabolite approach. Micro-proton-induced X-ray emission and laser ablation inductively coupled mass spectrometry analyses indicated hot spots of Cd concentrations in the vicinity of vascular bundles in response to Cd treatment, essentially bound to S-containing compounds as revealed by extended X-ray absorption fine structure and non-protein thiol compounds analyses. A preferential accumulation of Zn occurred in vascular bundle and spongy mesophyll in response to Zn treatment, and was mainly bound to O/N-ligands. Leaf proteomics and physiological status evidenced a protection of photosynthetically active tissues and the maintenance of cell turgor through specific distribution and complexation of toxic ions, reallocation of some essential elements, synthesis of proteins involved in photosynthetic apparatus or C-metabolism, and metabolite synthesis with some specificities regarding the considered heavy metal treatment.

Development of a 2D laser ablation inductively coupled plasma mass spectrometry mapping procedure for mercury in maize (Zea mays L.) root cross-sections.

A LA-ICP-MS method based on a 213 nm Nd:YAG laser and a quadrupole ICP-MS has been developed for mapping of mercury in root cross-sections of maize (Zea mays L.) to investigate the mechanism of mercury uptake from soil and its potential translocation to the edible parts. Conventional rastering was found to be unusable due to sorption of mercury onto the internal parts of the LA device, giving rising to memory effects resulting in serious loss of resolution and inaccurate quantification. Spot analysis on a virtual grid on the surface of the root sections using washout times of 10s in between spots greatly alleviated problems related to these memory effects. By ablating straight through the root sections on a poly(methyl methacrylate) support the calibration process was simplified as internal standardization and matrix-matching could be circumvented. Mercury-spiked freeze-drying embedding medium, sectioned similarly to the root sections, was used for the preparation of the standards. Standards and root sections were subjected to spot analysis using the following operational parameters: beam diameter, 15 µm; laser fluence, 2.5 J cm(-2); repetition rate, 20 Hz; dwell time, 1s; acquisition time, 0.1s. The mercury peaks for standards and roots sections could be consistently integrated for quantification and construction of the 2D mercury maps for the root sections. This approach was successfully used to investigate the mercury distribution in root sections of maize grown in soil spiked to a level of 50 mg kg(-1) DW HgCl2. It was found that at given Hg concentrations in the substrate Hg ions practically do not cross root plasma membranes of the endodermal barrier, but are entirely retained in the root apoplastic space. This suggests that maize plants grown in Hg-contaminated areas translocate Hg to the upper edible parts of the plant only to a small extent.

As(2)O (3) oxidation by vitamin C: cell culture studies.

The ability of As(2)O(3) to induce apoptosis in various malignant cell lines has made it a potential treatment agent for several malignancies. In this study the chemical stability of As(2)O(3) (As(III)) in cell-free growth media with various compositions was studied (MEM with different amount of amino acids and DMEM). Special attention was given to evaluate the influence of serum (FBS; fetal bovine serum) absence and vitamin C addition on the oxidation of As(III) to As(V) in cell-free growth media. FBS is an important source of antioxidants and vitamin C (ascorbic acid) is acting as a prooxidant in millimolar concentrations. Media were incubated with As(III) (0.6, 2 and 7 µmol l(-1)) up to 72 h. Experiments were performed at 37°C in light or/and in the dark, with or without added serum (10%) or vitamin C (1.4, 0.14 mM). Metabolites were followed with high-performance liquid chromatography directly coupled to a hydride generation-atomic fluorescence spectrometry system. After 72 h up to 30% of As(III) was transformed into As(V) in MEMs and up to 35% in DMEM when exposed in dark. Light had no influence on transformations in MEMs, but changed the situation dramatically in DMEM where almost all As(III) was oxidized to As(V) after 72 h when exposed to light. Except for some faster oxidation rate the absence of FBS had little effect on the transformation rate in all media. The most visible impact on As(III) oxidation was observed by addition of vitamin C. Addition of vitamin C (1.4 mM) transformed almost all As(III) to As(V) within 72 h. In lower concentrations (0.14 mM) a pro-oxidative effect was still observed reaching approximately 60% oxidation of As(III) during 72 h. All oxidation processes could be explained by pseudo first order reaction kinetics, yielding reaction rates increasing with initial As(III) concentration and vitamin C concentration whereas the FBS content additionally increased the As(III) oxidation rate in the DMEM (light). The temporal oxidation of As(III) to As(V) in various cell-free growth media necessitates routine checking of the valence state of arsenic during cell culture experiments and the results of biological effects attributed to As(III) should be interpreted with caution. Special attention is needed particularly in cases with vitamin C which was acting pro-oxidatively in all conditions examined.

Arsenic trioxide versus tetraarsenic oxide in biomedical research: misunderstandings and misinterpretations.

This work presents an analytical chemist's view on the sometimes unconscious use of arsenic trioxide in (bio)medical research. Arsenic trioxide is a frequently used chemical in cancer treatment research and its action to various malignant cells has been extensively studied and published. Unfortunately some research articles show trivial errors with regards to background knowledge of the chemical, handling the chemical, experimental design and interpretation of results like e.g. in a range of articles comparing advantages of tetraarsenic oxide over arsenic trioxide (dimeric/monomeric) although the dissolution of both yields the same active compound (HAsO(2)). To fully understand the implications of these errors we will highlight some of them with the intent to harmonize future work in this field.

Analytical artefacts in the speciation of arsenic in clinical samples.

Urine and blood samples of cancer patients, treated with high doses of arsenic trioxide were analysed for arsenic species using HPLC-HGAFS and, in some cases, HPLC-ICPMS. Total arsenic was determined with either flow injection-HGAFS in urine or radiochemical neutron activation analysis in blood fractions (in serum/plasma, blood cells). The total arsenic concentrations (during prolonged, daily/weekly arsenic trioxide therapy) were in the microg mL(-1) range for urine and in the ng g(-1) range for blood fractions. The main arsenic species found in urine were As(III), MA and DMA and in blood As(V), MA and DMA. With proper sample preparation and storage of urine (no preservation agents/storage in liquid nitrogen) no analytical artefacts were observed and absence of significant amounts of alleged trivalent metabolites was proven. On the contrary, in blood samples a certain amount of arsenic can get lost in the speciation procedure what was especially noticeable for the blood cells although also plasma/serum gave rise to some disappearance of arsenic. The latter losses may be attributed to precipitation of As(III)-containing proteins/peptides during the methanol/water extraction procedure whereas the former losses were due to loss of specific As(III)-complexing proteins/peptides (e.g. cysteine, metallothionein, reduced GSH, ferritin) on the column (Hamilton PRP-X100) during the separation procedure. Contemporary analytical protocols are not able to completely avoid artefacts due to losses from the sampling to the detection stage so that it is recommended to be careful with the explanation of results, particularly regarding metabolic and pharmacokinetic interpretations, and always aim to compare the sum of species with the total arsenic concentration determined independently.

Arsenosugars and other arsenic compounds in littoral zone algae from the Adriatic Sea.

In 10 different marine algae from the littoral zone (found between the highest and lowest tide marks on the seashore) arsenic compounds were determined by means of a high-performance liquid chromatography (anion and cation exchange)-UV photochemical digestion-hydride generation-atomic fluorescence spectrometry (HPLC-UV-HGAFS) system. Samples (Ceramium sp., Cystoseira barbata, Enteromorpha sp., Fucus virsoides, two different species of Gelidium, Padina pavonica, Polisyphonia sp. and Ulva rigida) were collected along the Adriatic Sea coast of Slovenia. The total arsenic content of the algal samples, as determined by ICP-MS, ranged from 1.35 to 28.1 microg g(-1) (fresh weight). In all algae but two, the most abundant arsenic species found were arsenosugars with minor amounts of other arsenic compounds. Cystoseira barbata and Ceramium sp. contained high amounts of mainly inorganic arsenic. A small quantity of arsenobetaine was detected in most of the investigated Adriatic algae, which probably originates from mesofauna attached to the algae in their natural habitat.

Validation of bismuth film electrode for determination of cobalt and cadmium in soil extracts using ICP-MS.

A study is presented on the use of the bismuth film electrode (BiFE) operated in the anodic stripping and the cathodic adsorptive stripping voltammetry (ASV, CAdSV) modes, for the determination of two trace heavy metals (Cd and Co, respectively), in soil extract samples. Two types of BiFE were examined in this study: the in situ prepared BiFE, which was employed in ASV determination of Cd, and the ex situ prepared BiFE, which was used in CAdSV of Co with dimethylglyoxime (DMG) as complexing agent. A series of unpretreated soil extracts with varying Cd and Co concentrations were analyzed, and the results obtained compared to those determined using inductively coupled plasma-mass spectrometry (ICP-MS). The results revealed the suitability of stripping analysis at the BiFE for determination of mugl(-1) levels of heavy metals in soil extracts. The promising results obtained here, coupled with the non-toxic nature of bismuth (in comparison to commonly used mercury electrodes employed in stripping analysis), offer great promise in centralized and decentralized analysis of trace heavy metals in complex environmental matrices.