Validation of Analytical Method for Determination of Thallium in Rodent Plasma and Tissues by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).

Thallium (Tl) can be released as a byproduct of smelting, mining, and other industries, causing human exposure. There are knowledge gaps on the toxicity of thallium compounds, so the National Toxicology Program is investigating the toxicity of thallium (I) sulfate in rodents. We developed and validated a method to quantitate Tl in rodent plasma and secondary matrices. Primary matrix standards and validation samples were digested with nitric acid and analyzed for Tl by inductively-coupled plasma - mass spectrometry (ICP-MS). Method performance was validated for linearity, accuracy, precision, and other criteria. Calibration was linear from 1.25 to 500 ng Tl/mL plasma; accuracy (RE) was -5.9 to 2.6% for all calibration standards. The lower limit of quantitation (LLOQ) was 1.25 ng Tl/mL plasma, and the limit of detection was 0.0370 ng Tl/mL plasma. Intra- and interday RE and precision (RSD) were -5.6 to -1.7% and ≤0.8% (intraday) and -4.8 to -1.3% and ≤4.3% (interday), respectively, at three sample concentration levels. Standards up to 10.0 × 103 ng/mL could be analyzed by dilution with digested blank matrix, with -6.4% RE and 5.4% RSD. Method was also evaluated in post-natal day 4 (PND4) Hsd:Sprague Dawley SD (HSD) dam and pup plasma, gestation day 18 (GD 18) HSD rat fetal homogenate, HSD rat urine, female HSD rat brain homogenate, female B6C3F1 mouse plasma. Background Tl was detected in control fetal and brain homogenates and urine at < 30% of LLOQ response. Results demonstrate that the method is suitable for determination of Tl in rodent matrices for toxicology studies.

Systemic exposure and urinary excretion of vanadium following perinatal subchronic exposure to vanadyl sulfate and sodium metavanadate via drinking water.

Vanadium is a ubiquitous environmental contaminant although there are limited data to assess potential adverse human health impact following oral exposure. In support of studies investigating the subchronic toxicity of vanadyl sulfate (V4+) and sodium metavanadate (V5+) following perinatal exposure via drinking water in male and female rats, we have determined the internal exposure and urinary excretion of total vanadium at the end of study. Water consumption decreased with increasing exposure concentration following exposure to both compounds. Plasma and urine vanadium concentration normalized to total vanadium consumed per day increased with the exposure concentration of vanadyl sulfate and sodium metavanadate suggesting absorption increased as the exposure concentration increased. Additionally, females had higher concentrations than males (in plasma only for vanadyl sulfate exposure). Animals exposed to sodium metavanadate had up to 3-fold higher vanadium concentration in plasma and urine compared to vanadyl sulfate exposed animals, when normalized to total vanadium consumed per day, demonstrating differential absorption, distribution, metabolism, and excretion properties between V5+ and V4+ compounds. These data will aid in the interpretation of animal toxicity data of V4+ and V5+ compounds and determine the relevance of animal toxicity findings to human exposures.

Quantitation of Total Vanadium in Rodent Plasma and Urine by Inductively Coupled Plasma - Mass Spectrometry (ICP-MS).

Human exposure to vanadium (V) is anticipated because it is a drinking water contaminant. Due to limited data on soluble V salts, the National Toxicology Program is investigating the toxicity in rodents following drinking water exposure. Measurement of internal V dose allows for interpretation of toxicology data. The objective of this study was to develop and validate an inductively coupled plasma-mass spectrometric method to quantitate total V in rat plasma. The method was linear (r ≥ 0.99) from 5.00 - 1,000 ng V/mL. Intra- and inter-day relative error (% RE) and relative standard deviation (% RSD) of spiked plasma samples were 8.5% - 15.6% RE and ≤ 1.8% RSD and 7.3% - 11.7% RE and ≤ 3.1% RSD, respectively. The limit of detection was 0.268 ng V/mL plasma and absolute percent recovery was 113%. Standards up to 7,500 ng V/mL plasma were diluted into the validated range (5.6% RE, 0.9% RSD). V in extracted plasma samples over 15 days at ambient and refrigerated conditions was from 97.7 - 126% of day 0. Determined plasma V concentrations after three freeze-thaw cycles and after frozen storage for up to 63 days ranged from 100 - 106% and 100 - 122% of day 0, respectively. The method was extended to rat urine (accuracy and precision -2.0 - 0.3% RE and <0.6% RSD, respectively for same linear range). These data demonstrate that the method is suitable to quantitate V in rat plasma and urine.

Simulated Gastric Digestion and In Vivo Intestinal Uptake of Orally Administered CuO Nanoparticles and TiO2 E171 in Male and Female Rat Pups.

Oral exposure to nanoparticles (NPs) during early life is an understudied area. The goals of this study were to evaluate the effect of pre-weaned rat gastric fluids on 50 nm CuO NPs and TiO2 E171 in vitro, and to evaluate uptake in vivo. The NP uptake was studied in vivo in male and female Sprague-Dawley rat pups following oral administration of four consecutive daily doses of 10 mg/kg CuO NPs, TiO2 E171, or vehicle control (water) between postnatal day (PND) 7-10. Rat pups were sacrificed on either PND10 or PND21. Simulated digestion led to dissolution of CuO NPs at the later ages tested (PND14 and PND21, but not PND7). In vivo intestinal uptake of CuO NPs and TiO2 E171 was observed by hyperspectral imaging of intestinal cross sections. Brightfield microscopy showed that the number of immune cells increased in the intestinal tissue following NP administration. Orally administered NPs led to low intestinal uptake of NPs and an increase in immune cells in the small and large intestine, suggesting that oral exposure to NPs during early life may lead to irritation or a low-grade inflammation. The long-term impact of increased immune cells in the intestinal tract during early life is unknown.

Air-Quality Assessment of On-Site Brick-Kiln Worker Housing in Bhaktapur, Nepal: Chemical Speciation of Indoor and Outdoor PM2.5 Pollution.

Brick workers and their families in Nepal generally live in poorly ventilated on-site housing at the brick kiln, and may be at higher risk for non-occupational exposure to fine particulate matter air pollution and subsequent respiratory diseases due to indoor and outdoor sources. This study characterized non-occupational exposure to PM2.5 by comparing overall concentrations and specific chemical components of PM2.5 inside and outside of brick workers' on-site housing. For all samples, the geometric mean PM2.5 concentration was 184.65 µg/m3 (95% confidence interval: 134.70, 253.12 µg/m3). PM2.5 concentrations differed by kiln number (p = 0.009). Kiln number was significantly associated with 16 of 29 (55%) air pollutant, temperature, or relative humidity variables. There was not a significant interaction between kiln number and location of sample for PM2.5 (p = 0.16), but there was for relative humidity (p = 0.02) and temperature (p = 0.01). Results were qualitatively similar when we repeated analyses using indoor samples only. There was no difference in the chemical makeup of indoor and outdoor PM2.5 in this study, suggesting that outdoor PM2.5 air pollution easily infiltrates into on-site brick worker housing. Outdoor and indoor PM2.5 concentrations found in this study far exceed recommended levels. These findings warrant future interventions targeted to this vulnerable population.

Elemental analysis of infant airborne particulate exposures.

Air pollution is hypothesized to have negative impacts on infant pulmonary health because of infants' increased rates of respiration and ongoing lung development. The severity and type of impact may differ depending on elemental concentrations. We conducted a study of 21 infants <6 months old whose parents carried a small personal particulate monitoring device (RTI MicroPEM) and GPS unit with the infant for 7 days in January and February 2015. The study area was Utah County, UT, USA. Real-time particulate exposure levels, as well as optical density and elemental analysis of the particulate matter (PM), were compared with levels from an outdoor stationary monitor. Infants spent an average of 87.4% of their time indoors. PM levels varied widely by infant and time of day (average=19.07 µg/m3, range=0.63-170.25 µg/m3). Infant particulate exposures were not well approximated by the outdoor monitor. Infants had lower exposures to Sb, Mn, Pb, W and Fe than the outdoor monitor and higher exposures to Cd, Ni and Na. Differences were most pronounced for Na. Brown carbon was only detected by personal monitors and not by the outdoor monitor. Further research is needed to understand the potential implications of indoor elemental exposures on early respiratory development.

Validation of a Metallomics Analysis of Placenta Tissue by Inductively-Coupled Plasma Mass Spectrometry.

Trace elements can play an important role in maternal health and fetal development, and deficiencies in some essential minerals including zinc and copper have been correlated in some individuals to the development of birth defects and adverse health outcomes later in life. The exact etiology of conditions like preeclampsia and the effects of fetal exposure to toxic metals has not been determined, making the assessment of trace element levels crucial to the elucidation of the causes of conditions like preeclampsia. Previous studies analyzing serum and placenta tissue have produced conflicting findings, suggesting the need for a robust, validated sample preparation and analysis method for the determination of trace elements in placenta. In this report, an acid digestion method and analysis by ICP-MS for a broad metallomics/mineralomics panel of trace elements is developed and validated over three experimental days for inter- and intraday precision and accuracy, linear range, matrix impact, and dilution verification. Spike recovery experiments were performed for the essential elements chromium (Cr), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), and zinc (Zn), and the toxic elements arsenic (As), cadmium (Cd), and lead (Pb) at levels equal to and in excess of native concentrations in control placenta tissue. The validated method will be essential for the development of scientific studies of maternal health and toxic metal exposure effects in childhood.

Development, validation, and application of an ultra-performance liquid chromatography-sector field inductively coupled plasma mass spectrometry method for simultaneous determination of six organotin compounds in human serum.

Organotin compounds (OTCs) are heavily employed by industry for a wide variety of applications, including the production of plastics and as biocides. Reports of environmental prevalence, differential toxicity between OTCs, and poorly characterized human exposure have fueled the demand for sensitive, selective speciation methods. The objective of this investigation was to develop and validate a rapid, sensitive, and selective analytical method for the simultaneous determination of a suite of organotin compounds, including butyl (mono-, di-, and tri-substituted) and phenyl (mono-, di-, and tri-substituted) species in human serum. The analytical method utilized ultra-performance liquid chromatography (UPLC) coupled with sector field inductively coupled plasma mass spectrometry (SF-ICP-MS). The small (sub-2 µm) particle size of the UPLC column stationary phase and the sensitivity of the SF-ICP-MS enabled separation and sensitive determination of the analyte suite with a runtime of approximately 3 min. Validation activities included demonstration of method linearity over the concentration range of approximately 0.250-13.661 ng mL(-1), depending on the species; intraday precision of less than 21%, interday precision of less than 18%, intraday accuracy of -5.3% to 19%, and interday accuracy of -14% to 15% for all species; specificity, and matrix impact. In addition, sensitivity, and analyte stability under different storage scenarios were evaluated. Analyte stability was found to be limited for most species in freezer, refrigerator, and freeze-thaw conditions. The validated method was then applied for the determination of the OTCs in human serum samples from women participating in the Snart-Foraeldre/MiljØ (Soon-Parents/Environment) Study. The concentration of each OTC ranged from below the experimental limit of quantitation to 10.929 ng tin (Sn) mL(-1) serum. Speciation values were confirmed by a total Sn analysis.

Evaluation of methods for analysis of lead in air particulates: an intra-laboratory and inter-laboratory comparison.

In 2008, the United States Environmental Protection Agency (USEPA) set a new National Ambient Air Quality Standard (NAAQS) for lead in total suspended particulate matter (Pb-TSP) which called for significant decreases in the allowable limits. The Federal Reference Method (FRM) for Pb-TSP promulgated in 1978 prescribes analysis of Pb by flame atomic absorption spectroscopy (FAAS), but the new limits approach the limits of quantitation of FAAS. On August 2, 2013, the USEPA finalized a new FRM for Pb-TSP. This new FRM describes two extraction methods and analysis by inductively coupled plasma-mass spectrometry (ICP-MS). The study described here was performed to evaluate the use of ICP-MS in the analysis of Pb-TSP for implementation of this new FRM. A multi-laboratory study of the new FRM demonstrated acceptable intra- and inter-laboratory precision and comparability for glass fiber, quartz, and PTFE filters, and acceptable accuracy for the analysis of three National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs). A comparison was made between analytical results obtained using the 1978 FRM and those obtained using the new FRM. The results demonstrate that the ICP-MS method performs acceptably for the determination of Pb-TSP with lower limits of quantitation and strong inter- and intra-laboratory precision.

Effects of lead on Na⁺, K⁺-ATPase and hemolymph ion concentrations in the freshwater mussel Elliptio complanata.

Freshwater mussels are an imperiled fauna exposed to a variety of environmental toxicants such as lead (Pb) and studies are urgently needed to assess their health and condition to guide conservation efforts. A 28-day laboratory toxicity test with Pb and adult Eastern elliptio mussels (Elliptio complanata) was conducted to determine uptake kinetics and to assess the toxicological effects of Pb exposure. Test mussels were collected from a relatively uncontaminated reference site and exposed to a water-only control and five concentrations of Pb (as lead nitrate) ranging from 1 to 245 µg/L in a static renewal test with a water hardness of 42 mg/L. Endpoints included tissue Pb concentrations, hemolymph Pb and ion (Na⁺, K⁺, Cl⁻, Ca²âº) concentrations, and Na⁺, K⁺-ATPase enzyme activity in gill tissue. Mussels accumulated Pb rapidly, with tissue concentrations increasing at an exposure-dependent rate for the first 2 weeks, but with no significant increase from 2 to 4 weeks. Mussel tissue Pb concentrations ranged from 0.34 to 898 µg/g dry weight, were strongly related to Pb in test water at every time interval (7, 14, 21, and 28 days), and did not significantly increase after day 14. Hemolymph Pb concentration was variable, dependent on exposure concentration, and showed no appreciable change with time beyond day 7, except for mussels in the greatest exposure concentration (245 µg/L), which showed a significant reduction in Pb by 28 days, suggesting a threshold for Pb binding or elimination in hemolymph at concentrations near 1000 µg/g. The Na⁺, K⁺-ATPase activity in the gill tissue of mussels was significantly reduced by Pb on day 28 and was highly correlated with tissue Pb concentration (R² = 0.92; P = 0.013). The Na⁺, K⁺-ATPase activity was correlated with reduced hemolymph Na⁺ concentration at the greatest Pb exposure when enzyme activity was at 30% of controls. Hemolymph Ca²âº concentration increased significantly in mussels from the greatest Pb exposure and may be due to remobilization from the shell in an attempt to buffer the hemolymph against Pb uptake and toxicity. We conclude that Na⁺, K⁺-ATPase activity in mussels was adversely affected by Pb exposure, however, because the effects on activity were variable at the lower test concentrations, additional research is warranted over this range of exposures.

Aspects of bioanalytical method validation for the quantitative determination of trace elements.

Bioanalytical methods are used to quantitatively determine the concentration of drugs, biotransformation products or other specified substances in biological matrices and are often used to provide critical data to pharmacokinetic or bioequivalence studies in support of regulatory submissions. In order to ensure that bioanalytical methods are capable of generating reliable, reproducible data that meet or exceed current regulatory guidance, they are subjected to a rigorous method validation process. At present, regulatory guidance does not necessarily account for nuances specific to trace element determinations. This paper is intended to provide the reader with guidance related to trace element bioanalytical method validation from the authors' perspective for two prevalent and powerful instrumental techniques: inductively coupled plasma-optical emission spectrometry and inductively coupled plasma-MS.

Validation and application of a method for the determination of total chromium in rat tissues by inductively coupled plasma mass spectrometry.

The validation of a method for the determination of chromium (Cr) in F-344/N rat tissues by inductively coupled plasma-mass spectrometry is described. Samples were analyzed after a rapid, open-vessel microwave digestion procedure. Performance of the method was evaluated using kidney tissue across a concentration range of 0.50-5.00 microg Cr/g tissue. Data for method linearity, accuracy, precision, digest stability, and storage stability are presented along with limits of detection and quantitation data. Data from a method cross-validation for B6C3F1 mouse kidney tissue are also presented. After validation, the method was applied to analyze samples collected in support of two chronic toxicity and carcinogenesis studies conducted by the National Toxicology Program.

Determination of mercury in an assortment of dietary supplements using an inexpensive combustion atomic absorption spectrometry technique.

The concentrations of mercury in forty, commercially available dietary supplements, were determined using a new, inexpensive analysis technique. The method involves thermal decomposition, amalgamation, and detection of mercury by atomic absorption spectrometry with an analysis time of approximately six minutes per sample. The primary cost savings from this approach is that labor-intensive sample digestion is not required prior to analysis, further automating the analytical procedure. As a result, manufacturers and regulatory agencies concerned with monitoring lot-to-lot product quality may find this approach an attractive alternative to the more classical acid-decomposition, cold vapor atomic absorption methodology. Dietary supplement samples analyzed included astragalus, calcium, chromium picolinate, echinacea, ephedra, fish oil, ginger, ginkgo biloba, ginseng, goldenseal, guggul, senna, St John's wort, and yohimbe products. Quality control samples analyzed with the dietary supplements indicated a high level of method accuracy and precision. Ten replicate preparations of a standard reference material (NIST 1573a, tomato leaves) were analyzed, and the average mercury recovery was 109% (2.0% RSD). The method quantitation limit was 0.3 ng, which corresponded to 1.5 ng/g sample. The highest found mercury concentration (123 ng/g) was measured in a concentrated salmon oil sample. When taken as directed by an adult, this product would result in an approximate mercury ingestion of 7 mug per week.