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Analyses of reduced glutathione (GSH), oxidized glutathione (GSSG), and total glutathione (tGSH) in red blood cell samples from 30 children diagnosed with autism and 30 age, gender, and socioeconomic status matched controls were undertaken. The children's ages ranged from 2 to 9. Samples were obtained from subjects residing in Western Pennsylvania, an area of the United States greatly affected by high levels of mercury deposition and airborne PM 2.5 particulates. Liquid chromatography – mass spectrometry was utilized by following EPA Method 6800 for sample analyses. The children with autism had a significantly lower mean red blood cell (RBC) reduced to oxidized glutathione ratio (GSH/GSSG) compared to the control children (p = 0.025). In addition, compared to the controls, the children with autism had significantly higher RBC tGSH values (p = 0.0076) and GSH values (p = 0.022). These results suggest that exposure to toxic elements may prompt compensatory increases in production of GSH in children with autism in environments higher in toxins. The compensation did not fully correct the anti-oxidant properties of exposure to xenobiotics as demonstrated by the significantly lower GSH/GSSG in children with autism compared to controls. Out of a set of glutathione biomarkers, GSH/GSSG may best determine the degree of compensation for oxidative stress in children with autism.

In this work, we describe a method developed to quantify persistent organic pollutants (POPs) including polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCPs) in dietary supplement samples using stir-bar sorptive extraction (SBSE)-GC-MS/MS-isotope dilution mass spectrometry (IDMS). This method enables accurate, precise, and sensitive quantification of POPs in plant-extract based dietary supplement products commercially available in the United States. When compared with calibration curves, IDMS provided more accurate and precise measurements. The mean error of measurements using this method was 7.24% with a mean RSD of 8.26%. The application of GC-MS/MS enabled approximately two-order-of-magnitude lower limit of quantifications compared with GC-MS. 12 commercially available plant-extract based dietary supplement samples were analysed using this method. PAHs including naphthalene, acenaphthene, fluorene, phenanthrene, fluoranthene, pyrene, chrysene, and benzo[a]pyrene were detected in most of the products and had average concentrations over 1 ng/g. OCPs were detected less frequently than PAHs in these products, and none of the OCPs had mean concentrations over 1 ng/g. The mean toxin concentration of each product was calculated, and the highest value was 3.20 ng/g. These results were compared with existing guidelines and none of the analytes in the samples were found to be above the daily allowable limits.

Stir-bar sorptive extraction has been proven an effective and green extraction method for persistent organic pollutants (POPs) in aqueous samples such as drinking water, wastewater, and human serum. In this study, a method was developed to quantify POPs in human whole blood samples using stir bar sorptive extraction-thermal desorption-GC/MS/MS-isotope dilution mass spectrometry (IDMS). This method enables accurate, precise, and sensitive quantification of POPs in human whole blood samples. Compared with calibration curves, IDMS provided measurements with a higher level of accuracy and precision, especially at lower measured concentrations. The use of GC/MS/MS enabled a lower limit of quantification compared with GC/MS methods. A reverse-IDMS method was performed in this study to further eliminate biases from the labelled concentrations of the commercially available standards. Ten human whole blood samples were analyzed using this validated method. On average, 10 POPs were detected in each blood sample. Naphthalene, α-HCH, DDD, DDE were detected in all ten blood samples, whereas benzo[a]anthracene, chrysene, benzo[k]fluoranthene, and chlorpyrifos were detected in none of the ten samples. The mean xenobiotic body-burden was also calculated for each sample and ranged from 0.719 to 1.12 ng/g with a mean value of 0.897 ng/g.

The analysis of hexavalent chromium, Cr(VI), in soil and sediment samples has been predominantly carried out in materials containing elevated levels. Reliable analysis of trace-level of Cr(VI) in sediment samples remains challenging. Cr(VI) analyses with multipoint calibration and speciated isotope dilution (SID) adapted from U.S. EPA method 6800 were used to measure lower-level Cr(VI) on an ion chromatograph coupled with a tandem mass spectrometer (IC-MS/MS). Lake sediment samples were collected from various locations in Northern Ontario and Cr(VI) was extracted using both alkaline digestion and ethylene diaminetetraacetic acid (EDTA) extraction. Certified reference materials were extracted and analyzed by IC-MS/MS and UV-VIS detection. The SID-MS approach allowed for the quantification of Cr(VI) in samples with concentration levels below 0.5 μg.g-1 wet weight.

A biomonitoring method was developed for the determination of inorganic-, methyl-, and ethylmercury (Hg(2+), CH3Hg(+), and C2H5Hg(+), respectively) in whole blood by triple-spiking speciated isotope dilution mass spectrometry (SIDMS) using headspace (HS) solid-phase microextraction (SPME) in combination with gas chromatographic (GC) separation and inductively coupled plasma mass spectrometric (ICP-MS) detection. After spiking the blood sample with isotopically enriched analogues of the analytes ((199)Hg(2+), CH3(200)Hg(+) and C2H5(201)Hg(+)), the endogenous Hg species were solubilized in 2.0 mol L(-1) HNO3 and equilibrated with the spikes using a microwave-enhanced protocol. The microwaved sample was treated with a 1% (w/v) aqueous solution of sodium tetrapropylborate (buffered to pH 5.2), and the propylated Hg species were sampled in the HS using a Carboxen/polydimethylsiloxane-coated SPME fiber. The extracted species were thermally desorbed from the fiber in the GC injection port and determined by GC-ICP-MS. The analytes were quantified, with simultaneous correction for their method-induced transformation, on the basis of the mathematical relationship in triple-spiking SIDMS. The method was validated using a bovine blood standard reference material (SRM 966, Level 2). Analysis of human blood samples demonstrated the accuracy and reproducibility of the method, which can detect the Hg species down to 30 pg g(-1) in blood. The validity of the analytical results found for the blood samples was demonstrated using mass balance by comparing the sum of the concentrations of the individual Hg species with the total Hg in the corresponding samples; the latter was determined by isotope dilution mass spectrometry (IDMS) after decomposing the blood using EPA Method 3052 with single-spiking.

As. • Analysis of widely consumed pre- natal and children's dietary supple- ments. • Validation of analytical data using g r a p h i c a l a b s t r a c t a b s t r a c t A study was conducted to develop a microwave-enhanced extraction method for the determination of arsenic species in prenatal and children's dietary supplements prepared from plant materials. The method was optimized by evaluating the efficiency of various solutions previously used to extract arsenic from the types of plant materials used in the dietary supplement formulations. A multivitamin standard reference material (NIST SRM 3280) and a prenatal supplement sample were analyzed in the method optimization. The identified optimum conditions were 0.25 g of sample, 5 mL of 0.3 mol L −1 orthophos- phoric acid (H3PO4) and microwave heating at 90 ◦C for 30 min. The extracted arsenic was speciated by cation exchange ion chromatography-inductively coupled plasma mass spectrometry (IC-ICP-MS). The method detection limit (MDL) for the arsenic species was in the range 2-8 ng g−1. Ten widely con- sumed prenatal and children's dietary supplements were analyzed using the optimized protocol. The supplements were found to have total arsenic in the concentration range 59-531 ng g −1 . The extraction procedure recovered 61-92% of the arsenic from the supplements. All the supplementary products were found to contain arsenite (As 3+ ) and dimethylarsinic acid (DMA). Arsenate (As 5+ ) was found in two of the supplements, and an unknown specie of arsenic was detected in one product. The results of the analysis were validated using mass balance by comparing the sum of the extracted and non-extracted arsenic with the total concentration of the element in the corresponding samples.

In order to determine the health impact of chromium in dietary supplements, the Cr(III) and Cr(VI) must be independently measured and verified with mass balance (sum of both species equaling independent measurements of total chromium), as both may be present in finished products. Because Cr(III) is stable in acidic conditions and Cr(VI) in alkaline conditions, interconversions between species may occur in complex matrices and during analytical extraction, increasing the difficulty of quantification. A study was conducted to determine Cr(VI) and Cr(III) in dietary supplements. EPA Method 3060A extraction protocol was performed to extract Cr(VI), and EPA Method 3052 was performed on the extracted residue to digest the remaining Cr(III). Speciated isotope dilution mass spectrometry (SIDMS), as described in the EPA Method 6800 (update V), was implemented with ion-exchange chromatography inductively coupled plasma mass spectrometry (IC-ICP-MS). Method 6800 uniquely enables tracking and correcting for the bidirectional chromium interspecies conversions that occur during extraction and sample handling prior to instrumental analysis. Mass balance results indicated that the off-the-shelf dietary supplements analyzed during this study contained hexavalent chromium ranging fromDL (detection limit) to 122.4 ± 13.0 μg/g, which corresponds to concentrations from below detection levels to up to 16% of the total chromium content. This type of variation in the final products raises public health issues and points to a need to use a robust method that can accurately and reliably make species measurements including correcting for species conversions.

The reliable analysis of highly toxic hexavalent chromium, Cr(VI), at ultra-trace levels remains challenging, given its easy conversion to non-toxic trivalent chromium. This work demonstrates a novel analytical method to quantify Cr(VI) at low ngL(-1) concentration levels in environmental water samples by using speciated isotope dilution (SID) analysis and double-spiking with Cr(III) and Cr(VI) enriched for different isotopes. Ion chromatography tandem mass spectrometry (IC-MS/MS) was used for the analysis of Cr(VI) as HCrO4(-) → CrO3(-). Whereas the classical linear multipoint calibration (MPC) curve approach obtained a method detection limit (MDL) of 7ngL(-1) Cr(VI), the modified SID-MS method adapted from U. S. EPA 6800 allowed for the quantification of Cr(VI) with an MDL of 2ngL(-1) and provided results corrected for Cr(VI) loss occurred after sample collection. The adapted SID-MS approach proved to yield more accurate and precise results than the MPC method, allowed for compensation of Cr(VI) reduction during sample transportation and storage while eliminating the need for frequent external calibration. The developed method is a complementary tool to routinely used inductively-coupled plasma (ICP) MS and circumvents typically experienced interferences.

Dietary supplements were analyzed by evaluating the elemental content in six widely consumed products manufactured by four well-known companies. The elements included the neurotoxic and carcinogenic elements cadmium, mercury, aluminum, lead, arsenic, and antimony, as well as the essential elements zinc, selenium, chromium, iron, and copper, which were often not listed as ingredients on the product labels. Contamination from either xenobiotic or essential elements was found in all samples analyzed. The samples were prepared using US Environmental Protection Agency (EPA) Method 3052, microwave-enhanced digestion. The resulting digests were analyzed by Inductively Coupled Plasma-Mass Spectrometry based on EPA Method 6020B. The analytical protocols were validated by analyzing a multivitamin standard reference material, the National Institute of Standards and Technology Standard Reference Material 3280. The application of EPA standard methods demonstrated their utility in making accurate and precise measurements in complex matrices with multiple ingredients and excipients. In the future, the use of these methods could provide a uniform quality assurance protocol that can be implemented along with other industry guidelines to improve the production of dietary supplements.

Novel protocols were developed to accurately quantify reduced (GSH), oxidized (GSSG) and total (tGSH) glutathione in biological samples using molecular speciated isotope dilution mass spectrometry (SIDMS). For GSH and GSSG measurement, the sample was spiked with isotopically enriched analogues of the analytes ((310)GSH and (616)GSSG), along with N-ethylmaleimide (NEM), and treated with acetonitrile to solubilize the endogenous analytes via protein precipitation and equilibrate them with the spikes. The supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the analytes were quantified with simultaneous tracking and correction for auto-oxidation of GSH to GSSG. For tGSH assay, a (310)GSH-spiked sample was treated with dithiothreitol (DTT) to convert disulfide-bonded glutathione to GSH. After removing the protein, the supernatant was analyzed by LC-MS/MS and the analyte was quantified by single-spiking isotope dilution mass spectrometry (IDMS). The mathematical relationships in IDMS and SIDMS quantifications are based on isotopic ratios and do not involve calibration curves. The protocols were validated using spike recovery tests and by analyzing synthetic standard solutions. Red blood cell (RBC) and saliva samples obtained from healthy subjects, and whole blood samples collected and shipped from a remote location were analyzed. The concentrations of tGSH in the RBC and whole blood samples were 2 orders of magnitude higher than those found in saliva. The fractions of GSSG were 0.2-2.2% (RBC and blood) and 15-47% (saliva) of the free glutathione (GSH + 2xGSSG) in the corresponding samples. Up to 3% GSH was auto-oxidized to GSSG during sample workup; the highest oxidations (1%) were in the saliva samples.

An ion chromatography–inductively coupled plasma mass spectrometry (IC-ICP-MS) method was developed for the redox speciation analysis of iron (Fe) based on in-column complexation of Fe 2+ and Fe 3+ by dipicolinic acid (DPA). The effects of column type, mobile phase composition and molecular ion interference were studied in the method optimization. The carryover of the target species in the IC-ICP-MS method was uniquely and effectively evaluated using isotopically enriched analogues of the analytes ( 54 Fe 2+ and 57 Fe 3+ ). Standard solutions of the enriched standards were injected into the system following analysis of a sample, and the ratios of the isotopes of iron in the enriched standards were calculated based on the chromatographic peak areas. The concentrations of the analytes carried over from the sample to the enriched standards were determined using the quantitative relationship in isotope dilution mass spectrometry (IDMS). In contrast to the routine way of evaluating carryover effect by injecting a blank solution after sample analysis, the use of isotopically enriched standards identified significant analyte carryover in the present method. Extensive experiments were carried out to systematically identify the source of the carryover and to eliminate the problem; the separation column was found to be the exclusive source. More than 95% of the analyte carryover was eliminated by reducing the length of the column. The detection limit of the IC-ICP-MS method (MDL) for the iron species was 2 ng g -1 . The method was used to determine Fe 2+ and Fe 3+ in synthetic aqueous standard solutions and a beverage sample.

Strategies were designed to quantify hexavalent (Cr(VI)), soluble trivalent (Cr(III)) and insoluble chromium (Cr) species in soil by integrating existing methods of Cr(VI) and total Cr determination with speciated isotope dilution mass spectrometry (SIDMS, EPA Method 6800). Two different extraction methods that utilize a NaOH–Na2CO3 solution (EPA Method 3060A) and alkaline solution of ethylenediaminetetraacetic acid (EDTA) were used to extract Cr(VI) (along with soluble Cr(III) in the latter case). The extracted Cr was speciated by ion chromatography-inductively coupled plasma mass spectrometry (IC-ICP-MS), and the separated species were quantified using the mathematical relationships in SIDMS with simultaneous correction for their method-induced transformations. The Cr species that fell out as insoluble solid during extraction were determined by isotope dilution mass spectrometry (IDMS) after decomposing the extraction residues in a mixture of mineral acids according to EPA Method 3052. Several certified reference materials and a soil sample were analyzed using the proposed strategies. The measured mass fractions of Cr(VI) and total Cr in the reference materials statistically agreed with the certified values at 95% CL. The insoluble fraction of Cr accounted for 64–107% of the total Cr in the samples. The validity of the strategies was proved using mass balance by comparing the sum of the mass fractions of Cr(VI), soluble Cr(III) and insoluble Cr with the total Cr measured in the corresponding samples; the latter was determined by IDMS after decomposing the soils using EPA Method 3052 with single spiking.