Sean W. Linder

Capillary Electrophoresis/Inductively-Coupled Plasma-Mass Spectrometry: Development and Optimization of a High Resolution Analytical Tool for the Size-Based Characterization of Nanomaterials in Dietary Supplements

We report the development and optimization of a system consisting of capillary electrophoresis (CE) interfaced with inductively coupled plasma mass spectrometry (ICPMS) for rapid and high resolution speciation and characterization of metallic (e.g., gold, platinum, and palladium) nanoparticles in a dietary supplement. Multiple factors, including surfactant type and concentration, pH of running buffer, and applied voltage, were investigated to optimize the separation conditions. It was found that by using the anionic surfactant sodium dodecyl benzenesulfonate (SDBS) in the running buffer the separation resolution was significantly improved, allowing for easy distinction of adjacent size fractions in a gold nanoparticle mixture with very small size differences (e.g., 5, 15, 20, and 30 nm). The type and concentration of the surfactant was found to be critical in obtaining sufficient separation while applied voltage and pH values of the running buffers largely affected the elution times by varying the electroosmotic flow. Quantum dots were used as mobility markers to eliminate the run-to-run variation. The diameters of the nanoparticles followed a linear relationship with their relative electrophoretic mobility, and size information on unknown samples could be extrapolated from a standard curve. The accuracy and precision of this method was confirmed using 10 and 30 nm gold nanoparticle standard reference materials. Furthermore, the method was successfully applied to the analysis of commercially available metallic nanoparticle-based dietary supplements, as evidenced by good agreement between the particle sizes calculated by CE/ICPMS and transmission electron microscopy (TEM).

Asymmetric Flow-Field Flow Fractionation Hyphenated ICP-MS as an Alternative to Cloud Point Extraction for Quantification of Silver Nanoparticles and Silver Speciation: Application for Nanoparticles with a Protein Corona

Production and application of nanoparticles in consumer products is at an all-time high due to the emerging field of nanotechnology. Direct detection and quantification of trace levels of nanoparticles within consumer products is very challenging and problematic. Although multiple methodologies are available for this purpose, each method has its own set of limitations. Herein, we developed an analytical platform consisting of asymmetric flow-field flow fractionation (AF4) coupled with inductively coupled plasma mass spectroscopy (ICP-MS) for the speciation and quantification of silver ions and silver nanoparticles at the ng/kg level (ppt). AF4 is utilized to concentrate the nanoparticles, and ICP-MS acts as the detector. The protein corona that forms upon exposure of nanoparticles to bovine serum albumin was utilized as a nanoparticle stabilization and AF4 recovery enhancement mechanism. Speciation of silver ions and nanoparticles was achieved with the assistance of penicillamine as a complexation ligand. The effect of nanoparticle size, surface coating, and ionization state toward the detection and quantification of the developed methodology was evaluated. The detection limit was found to be 4 ng/kg with the application of a 5 mL sample loop. Further application of this developed methodology on environmentally relevant samples was demonstrated by the analysis of Arkansas River water spiked with silver nanoparticles and nanoparticle spiked into humic acid solution (50 mg/L) at an environmentally relevant level.

Simple Functionalization Strategies for Enhancing Nanoparticle Separation and Recovery with Asymmetric Flow Field Flow Fractionation

Due to the increasing use of engineered nanomaterials in consumer products, regulatory agencies and other research organizations have determined that the development of robust, reliable, and accurate methodologies to characterize nanoparticles in complex matrices is a top priority. Of particular interest are methods that can separate and determine the size of nanomaterials in samples that contain polydisperse and/or multimodal nanoparticle populations. Asymmetric-flow field flow fractionation (AF4) has shown promise for the separation of nanoparticles with wide size range distributions; however, low analyte recoveries and decreased membrane lifetimes, due to membrane fouling, have limited its application. Herein, we report straightforward strategies to minimize membrane fouling and improve nanoparticle recovery by functionalizing the surface of the nanoparticles, as well as that of the AF4 membranes. Gold nanoparticles (AuNP) were stabilized through functionalization with a phosphine molecule, whereas the surface of the membranes was coated with a negatively charged polystyrenesulfonate polymer. Improved nanoparticle separation, recoveries of 99.1 (±0.5) %, and a detection limit of 6 μg/kg were demonstrated by analyzing AuNP reference materials of different sizes (e.g., 10, 30, and 60 nm), obtained from the National Institute of Standards and Technology (NIST). Furthermore, the stability of the polymer coating and its specificity toward minimizing membrane fouling were demonstrated.

Arsenic Speciation in Rice by Capillary Electrophoresis/Inductively Coupled Plasma Mass Spectrometry: Enzyme-Assisted Water-Phase Microwave Digestion

We report an analytical methodology for the quantification of common arsenic species in rice and rice cereal using capillary electrophoresis coupled with inductively coupled plasma mass spectrometry (CE-ICPMS). An enzyme (i.e., α-amylase)-assisted water-phase microwave extraction procedure was used to extract four common arsenic species, including dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), arsenite [As(III)], and arsenate [As(V)] from the rice matrices. The addition of the enzyme α-amylase during the extraction process was necessary to reduce the sample viscosity, which subsequently increased the injection volume and enhanced the signal response. o-Arsanilic acid (o-ASA) was added to the sample solution as a mobility marker and internal standard. The obtained repeatability [i.e., relative standard deviation (RSD %)] of the four arsenic analytes of interest was less than 1.23% for elution time and 2.91% for peak area. The detection limits were determined to be 0.15-0.27 ng g(-1). Rice standard reference materials SRM 1568b and CRM 7503-a were used to validate this method. The quantitative concentrations of each organic arsenic and summed inorganic arsenic were found within 5% difference of the certified values of the two reference materials.

Detection and Characterization of SiO2 and TiO2 Nanostructures in Dietary Supplements

Nanomaterials are beginning to enter our daily lives through various consumer products as the result of technology commercialization. The development of methodologies to detect the presence of nanomaterials in consumer products is an essential element in understanding our exposure. In this study, we have developed methods for the separation and characterization of silicon dioxide (SiO2) and titanium dioxide (TiO2) nanostructures in dietary supplements marketed in products specifically targeted for women. A total of 12 commercial products claiming the inclusion of SiO2 and TiO2, but not making any claims regarding the particle size, were randomly selected for purchase through various retailers. To isolate nanostructures from these products, a simple methodology that combines acid digestion and centrifugation was utilized. Once isolated, the chemical composition, size, morphology, and crystal structure were characterized using mass spectroscopy, light scattering, electron microscopy, and X-ray diffraction techniques. SiO2 and TiO2 nanostructures were detected in 11 of 12 products using these methods. Many of the isolated nanoscale materials showed a high degree of aggregation; however, identified individual structures had at least one dimension below 100 nm. These robust methods can be used for routine monitoring of commercial products for nanoscale oxides of silica and titanium.

Rapid determination of silver in nanobased liquid dietary supplements using a portable X-ray fluorescence analyzer.

This paper reports a rapid and straightforward method for the quantitation of total Ag content in nanobased commercially available liquid dietary supplements using a portable X-ray fluorescence (pXRF) analyzer. Figures of merits were evaluated by analyzing a series of AgNO3 standards. This method was shown to have a detection limit of 3 ppm, a quantitation limit of 10 ppm, and a broad linear range from the detection limit to 10000 ppm (1%). Accurate detection and quantitation of Ag content in well-characterized Ag nanoparticle samples and in nanobased liquid dietary supplements were achieved with good correlation (i.e., percentage difference average values under 15%) between the total Ag concentration obtained by the pXRF analyzer and by inductively coupled plasma mass spectrometry (ICP-MS). Furthermore, accurate quantitation of Ag in the presence of high concentrations of potential spectral interferences was also demonstrated.

In vitro enrofloxacin binding in human fecal slurries.

Most antibiotic inactivation studies have been conducted through in vitro incubations of human use aminoglycosides, beta-lactams, and fluoroquinolones, usually at fecal concentrations expected with therapeutic dose regimens in humans and animals. Less is known about the inactivation of these molecules when ingested at concentrations consistent with residue levels present in animal-derived foods from antibiotic treated animals. In this investigation, we used the fluoroquinolone, enrofloxacin which is specifically marketed for veterinary medicine as test compound. Fecal suspensions at 10%, 25%, and 50% (w/v) were subjected to physicochemical and molecular characterization and used in the drug binding studies. The fecal binding of enrofloxacin added at concentrations of 0.06, 0.1, 1, 5, 15, 50, and 150 mg/L was determined in various fecal slurry suspensions using analytical chemistry and microbiological assay methods. There was consistent correlation between both assay methods. By the analytical chemistry assay, the 10%, 25% and 50% diluted autoclaved fecal samples dosed with enrofloxacin showed binding of 50±4.6%, 54±6.5% and 56±6.8% of the enrofloxacin, respectively. Binding of enrofloxacin to fecal contents occurred rapidly within 10 min and remained constant over the incubation period. Denaturing gradient gel electrophoreses and pyrosequencing analysis showed varied profiles of the bacterial composition of the human intestinal microbiota for fecal samples from different individuals. This study provided information on methodological questions that have concerned regulatory authorities on in vitro testing to determine if concentrations of veterinary antimicrobial agent residues entering the human colon remain microbiologically active.

Capillary electrophoresis coupled with inductively coupled mass spectrometry as an alternative to cloud point extraction based methods for rapid quantification of silver ions and surface coated silver nanoparticles

Speciation and accurate quantification of ionic silver and metallic silver nanoparticles are critical to investigate silver toxicity and to determine the shelf-life of products that contain nano silver under various storage conditions. We developed a rapid method for quantification of silver ions and silver nanoparticles using capillary electrophoresis (CE) interfaced with inductively-coupled plasma mass spectrometry (ICPMS). The addition of 2-mercaptopropionylglycine (tiopronin) to the background electrolyte was used to facilitate the chromatographic separation of ionic silver and maintain the oxidation state of silver. The obtained limits of detection were 0.05 μg kg(-1) of silver nanoparticles and 0.03 μg kg(-1) of ionic silver. Nanoparticles of varied sizes (10-110 nm) with different surface coating, including citrate acid, lipoic acid, polyvinylpyrrolidone and bovine serum albumin (BSA) were successfully analyzed. Particularly good recoveries (>93%) were obtained for both ionic silver and silver nanoparticle in the presence of excess amount of BSA. The method was further tested with six commercially available dietary supplements which varied in concentration and matrix components. The summed values of silver ions and silver nanoparticles correlated well with the total silver concentration determined by ICPMS after acid digestion. This method can serve as an alternative to cloud point extraction technique when the extraction efficiency for protein coated nanoparticles is low.

An improved methodology of asymmetric flow field flow fractionation hyphenated with inductively coupled mass spectrometry for the determination of size distribution of gold nanoparticles in dietary supplements

Engineered nanoparticles are available in large numbers of commercial products claiming various health benefits. Nanoparticle absorption, distribution, metabolism, excretion, and toxicity in a biological system are dependent on particle size, thus the determination of size and size distribution is essential for full characterization. Number based average size and size distribution is a major parameter for full characterization of the nanoparticle. In the case of polydispersed samples, large numbers of particles are needed to obtain accurate size distribution data. Herein, we report a rapid methodology, demonstrating improved nanoparticle recovery and excellent size resolution, for the characterization of gold nanoparticles in dietary supplements using asymmetric flow field flow fractionation coupled with visible absorption spectrometry and inductively coupled plasma mass spectrometry. A linear relationship between gold nanoparticle size and retention times was observed, and used for characterization of unknown samples. The particle size results from unknown samples were compared to results from traditional size analysis by transmission electron microscopy, and found to have less than a 5% deviation in size for unknown product over the size range from 7 to 30 nm.

Surface coating and matrix effect on the electrophoretic mobility of gold nanoparticles: a capillary electrophoresis-inductively coupled plasma mass spectrometry study.

Capillary electrophoresis (CE) is considered as a versatile technique in the size-based separation and speciation of nanomaterials. The electrophoretic mobility is determined by charge and size of an analyte which are affected by the surface composition of nanomaterials. Size-dependent differential electrophoretic mobility is used as a mechanism for size-based separation of nanoparticles. Understanding the effect of surface chemistry on the electrophoretic mobility of nanomaterials in CE is critical in obtaining accurate results in retention-based size calculation. A suite of gold nanoparticles (NPs) varied in sizes with different coatings, including citric acid (CA), lipoic acid (LA), tannic acid (TA), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), branched polyethyleneimine (BPEI), and bovine serum albumin (BSA), were selected to evaluate their impact to the migration pattern of gold NPs. Additionally, surface-coated gold NPs dispersed in Suwannee River humic acid (SRHA) solution and fetal bovine serum (FBS) were used to investigate the matrix effect. It was found that the correlation between NP size and relative electrophoretic mobility is highly dependent on the capping agents. The matrix component in the SRHA solution only exhibited limited influence to the migration of NPs while electrophoretic behaviors were drastically altered in the presence of FBS matrix.