Shaogang Chu

Determination of non-halogenated, chlorinated and brominated organophosphate flame retardants in herring gull eggs based on liquid chromatography–tandem quadrupole mass spectrometry

Numerous triester organophosphate flame retardants (OPFRs) have been used for several decades and continue to be used in a variety of commercial products. We developed a sensitive quantitative method for the analysis of, seven non-halogenated, three chlorinated and two brominated OPFRs of known or possible environmental relevance in herring gull eggs. This method is based on a simple two-step sample extraction followed by liquid chromatography-electrospray ionization(+)-tandem mass spectrometry. Instrumental detection limits and method limits of quantification (MLOQs) among the 12 OPFRs ranged from 0.01 to 0.12 ng/mL and 0.06 to 0.20 ng/g, respectively. The mean OPFR recovery efficiencies of replicate analyses (n=6) were very quantitative and ranged from 89% to 104%, with the two brominated OPFRs being somewhat lower but reproducible, i.e., 67% and 72%, respectively. Essentially negligible matrix effects were indicated by a standard addition approach that revealed mean percent signal recoveries (n=5 replicates) of 89-106% for most OPFRs. In the analysis of n=13 herring gull eggs from the Channel-Shelter Island colony (Lake Huron), tris(2-chloroisopropyl) phosphate (<MLOQ - 4.1 ng/g wet weight, ww), tris(2-chloroethyl) phosphate (<MLOQ - 0.6 ng/g ww) and tris(2-butoxyethyl) phosphate (<MLOQ - 2.2 ng/g ww) were detected and/or quantified.

Linear and Branched Perfluorooctane Sulfonate Isomers in Technical Product and Environmental Samples by In-Port Derivatization-Gas Chromatography-Mass Spectrometry

Perfluorooctane sulfonate (PFOS) is found globally as an environmental contaminant and is highly bioaccumulative in exposed biota including humans. However, there is a dearth of environmental information on the isomeric profile of PFOS, especially in biological samples, which requires suitable analysis methods for the identification and quantification of ultratrace amounts. In the present study, a novel method was developed that incorporates clean up by solid-phase extraction (SPE) WAX cartridges and in-port derivatization-gas chromatography-mass spectrometry (GC/MS) to identify and quantitatively determine linear PFOS (L-PFOS) and branched (monotrifluoromethyl and bistrifluoromethyl) isomers in PFOS technical product and in environmentally relevant biological samples. Tetrabutylammonium hydroxide (TBAH) was used for derivatization via an in situ pyrolytic alkylation reaction that occurred in the GC injector and generated butyl PFOS isomer derivatives. In addition to L-PFOS, ten branched PFOS isomers were identified in the technical product. The environmental relevance of branched PFOS isomers in addition to L-PFOS was evidenced by the presence of six branched and L-PFOS in representative herring gull and double-crested cormorant egg, and polar bear liver and plasma samples from the Great Lakes and Arctic, respectively. For all PFOS isomers in the technical product and biota samples the method demonstrated high sensitivity with the limit of detection (LOD) ranging from 0.05 to 0.25 ng/mL, with exception of L-PFOS where the LOD was 1.46 ng/mL. For the biotic samples, the method detection limits (MDLs) were slightly higher than the LODs and ranged from 0.09 to 0.46 ng/g wet weight (w.w.) with exception of L-PFOS (MDL = 6.87 ng/g w.w.).

High-Sensitivity Method for Determination of Tetrabromobisphenol-S and Tetrabromobisphenol-A Derivative Flame Retardants in Great Lakes Herring Gull Eggs by Liquid Chromatography-Atmospheric Pressure Photoionization-Tandem Mass Spectrometry

Tetrabromobisphenol-A-bis(2,3-dibromopylether) (TBBP-A-dbpe), tetrabromobisphenol-A-bis(allyl ether) (TBBP-A-ae), and tetrabromobisphenol-S-bis(2,3-dibromopropyl ether) (TBBP-S-dbpe) are derivatives of tetrabromobisphenol-A (TBBP-A), and are all used as brominated flame retardants (BFRs). Using high-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry with atmospheric pressure photoionization (APPI) in the negative mode (LC-APPI(-)-Q-TOF-MS) and the novel use of pure acetone as dopant and LC mobile phase, full scan mass spectra showed that for these BFRs the dominant isotopic ion cluster was [M + O₂](-), and with other lesser abundant [M + O₂ - HBr](-), and [M - H](-) fragment ions. Subsequently, highly sensitive quantification of TBBP-A-dbpe, TBBP-A-ae, and TBBP-S-dbpe was accomplished via LC-triple quadrupole mass spectrometry with APPI(-) (LC-APPI(-)-MS/MS) via multiple ion monitoring based on the [M + O₂](-) > [Br](-) transition. Low to sub ng/g (wet weight (w.w.)) method limits of detection (LODs) were achieved, i.e., 0.07, 0.03, and 1.28 ng/g w.w. for TBBP-A-dbpe, TBBP-A-ae, and TBBP-S-dbpe, respectively. A variety of herring gull eggs were screened for these BFRs. The eggs were collected during 2008-2009 from several colony sites in the eastern Laurentian Great Lakes (Ontario) and in the St. Lawrence River (Québec). All egg samples had TBBP-S-dbpe concentrations below the LOD, and TBBP-A-ae and TBBP-A-dbpe were quantifiable in 67%-83% of the samples at concentrations up to 0.56 ng/g wet weight. Thus, TBBP-A-ae and TBBP-A-dbpe are present in herring gull eggs from these populations, bioaccumulate in the herring gull food chain, and are transferred from gull to egg.

Analysis of fluorotelomer alcohols and perfluorinated sulfonamides in biotic samples by liquid chromatography-atmospheric pressure photoionization mass spectrometry.

A quantitative analytical method was developed to simultaneously detect fluorotelomer alcohols (6:2 FTOH, 8:2 FTOH and 10:2 FTOH) and polyfluorinated sulfonamides (perfluoro-1-octanesulfonamide (FOSA) and N-methylperfluoro-1-octanesulfonamide (NMeFOSA)) in biotic samples with liquid chromatography-atmospheric pressure photoionization mass spectrometry (LC-APPI-MS/MS). APPI mass spectra for FOSA and NMeFOSA showed that the major ionization mechanism was not photoionization, whereas for the FTOHs it was photoionization. For FTOHs, a [M+O(2)](-) ion was generated with a similar response as the deprotonated molecular ion [M-H](-). We demonstrated that FTOHs, FOSA and NMeFOSA can be measured in various biota samples using APPI with a minimized matrix effect. Using APPI, the linear response range for the FTOHs was 0-1,000 ng/mL (r(2)>0.9997), and for FOSA and NMeFOSA ranged from 0 to 250 ng/mL (r(2)>0.995). The instrument and method detect limits ranged from 0.16 to 0.63 pg and below 1 ng/g wet weight (w.w.), respectively. For the overall method applied to the test matrices, recovery efficiencies ranged from 73 to 102% for egg homogenate and 89-100% for liver tissue. The present study demonstrates for the first time that a far more response and sensitive approach for the detection and quantification of FTOHs and polyfluorinated sulfonamides is possible using APPI as opposed to electrospray ionization.

Dicationic ion-pairing of phosphoric acid diesters post-liquid chromatography and subsequent determination by electrospray positive ionization-tandem mass spectrometry.

Several organophosphate triesters are widely used as flame retardants and can be metabolized to dibutyl (DBP), diphenyl (DPhP), di(2-ethylhexyl) (DEHP) and di(1,3-dichloro-2-propyl) (or bis(1,3-dichloro-2-propyl); DDCPP) phosphoric acid, respectively. A highly sensitive liquid chromatography-electrospray ionization(+)-triple quadrupole mass spectrometry (LC-ESI(+)-QQQ-MS/MS) based analysis method was presently developed. In this method the target compounds were separated with a C(18)-based reversed phase LC column, and decamethonium hydroxide (dicatonic reagent) was introduced post-LC to form ion-pairs, which were subsequently detected by ESI(+). For the phosphate acid diester ion-pairs, the mass spectra showed the most abundant ion to be [(CH(3))(2)N(CH(2))(10)N(CH(3))(3)](+), with lesser abundances of [[M-H](-)[(CH(3))(3)N(CH(2))(9)CH(2)](2+)](+) and [CH(2)CH(CH(2))(8)N(CH(3))(3)](+). For DDCPP, the fragment ions of [[Cl](-)[(CH(3))(3)N(CH(2))(10)N(CH(3))(3)](2+)](+) and [[Cl](-)[(CH(3))(3)N(CH(2))(9)CH(2)](2+)](+) could also be observed. The limits of quantitation (LOQs) by LC-ESI(+)-MS/MS (based on multiple reaction monitoring) were 0.14, 0.03, 0.14 and 0.02 ng/mL for DPhP, DBP, DDCPP and DEHP, respectively. The response was highly linearly correlated (r>0.995) with concentration over the range of the LOD to 1000 ng/mL. The matrix effect on ESI+ was negligible for the samples in experiment of in vitro metabolism using rat liver microsomes.

Tissue bioaccumulation patterns, xenobiotic biotransformation and steroid hormone levels in Atlantic salmon (Salmo salar) fed a diet containing perfluoroactane sulfonic or perfluorooctane carboxylic acids

In the present study, groups of juvenile Atlantic salmon (Salmo salar) were fed gelatine capsules containing fish-food spiked with PFOA or PFOS (0.2 mg kg(-1) fish) and solvent (methanol). The capsules were given at days 0, 3 and 6. Blood, liver and whole kidney samples were collected prior to exposure (no solvent control), and at days 2, 5, 8 and 14 after exposure (Note: that day 14 after exposure is equal to 7d recovery period). We report on the differences in the tissue bioaccumulation patterns of PFOS and PFOA, in addition to tissue and compound differences in modulation pattern of biotransformation enzyme genes. We observed that the level of PFOS and PFOA increased in the blood, liver and kidney during the exposure period. Different PFOS and PFOA bioaccumulation patterns were observed in the kidney and liver during exposure- and after the recovery periods. Particularly, after the recovery period, PFOA levels in the kidney and liver tissues were almost at the control level. On the contrary, PFOS maintained an increase with tissue-specific differences, showing a higher bioaccumulation potential (also in the blood), compared with PFOA. While PFOS and PFOA produced an apparent time-dependent increase in kidney CYP3A, CYP1A1 and GST expression, similar effects were only temporary in the liver, significantly increasing at sampling day 2. PFOA and PFOS exposure resulted in significant decreases in plasma estrone, testosterone and cortisol levels at sampling day 2, and their effects differed with 17α-methyltestostrerone showing significant decrease by PFOA (also for cholesterol) and increase by PFOS. PFOA significantly increased estrone and testosterone, and no effects were observed for cortisol, 17α-methyltestosterone and cholesterol at sampling day 5. Overall, the changes in plasma steroid hormone levels parallel changes in CYP3A mRNA levels. Given that there are no known studies that have demonstrated such tissue differences in bioaccumulation patterns with associated differences in toxicological responses in any fish species or lower vertebrate, the present findings provide some potential insights and basis for a better understanding of the possible mechanisms of PFCs toxicity that need to be studied in more detail.

Perfluorooctane sulfonate (PFOS) toxicity in domestic chicken (Gallus gallus domesticus) embryos in the absence of effects on peroxisome proliferator activated receptor alpha (PPARα)-regulated genes.

Perfluorooctane sulfonate (PFOS) is a widely distributed industrial compound that has been detected in the eggs of various wild avian species. Laboratory studies have indicated that PFOS is embryotoxic to domestic chickens (Gallus gallus domesticus), but the mechanisms of toxicity in the developing avian embryo remain unknown. We recently demonstrated that PFOS acts as a peroxisome proliferator by causing increased expression of peroxisome proliferator activated receptor alpha (PPARalpha)-regulated genes in cultured primary chicken embryo hepatocytes. The present study examined whether PPARalpha-regulated genes were dose-dependently affected in chicken embryos exposed in ovo to PFOS. White leghorn chicken eggs were injected with 0.1, 5.0 or 100.0 microg PFOS/g egg into the air cell prior to incubation. Embryos were incubated until pipping, after which the expression of PPARalpha-regulated genes was measured in the liver tissue of surviving embryos using real-time reverse transcription polymerase chain reaction. A dose-dependent decrease in embryo pippability was observed with an LD50 of 93 microg/g (3.54 microg/g-672,910 microg/g, 95% confidence interval). Hepatic PFOS concentrations increased concomitantly with dose. The PPARalpha-regulated genes measured were peroxisomal acyl CoA oxidase, bifunctional enzyme, liver fatty acid binding protein and peroxisomal 3-ketoacyl thiolase. PFOS exposure via egg injection prior to incubation did not affect the transcriptional activity of any of the assayed PPARalpha-regulated genes at any of the doses examined in day 21 chicken embryos.

Determination of organophosphate flame retardants and plasticizers in lipid-rich matrices using dispersive solid-phase extraction as a sample cleanup step and ultra-high performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry.

A fast, robust and highly sensitive analysis method for determination of trace levels of organophosphate ester (OPE) flame retardants and plasticizers in lipid-rich samples was presently developed, and based on ultra-high performance liquid chromatography-tandem mass spectrometry coupled to a positive atmospheric pressure chemical ionization source (UHPLC-MS/MS-APCI(+)). The target OPEs in the sample were extracted from the biota samples, such as egg and liver, by ultrasonic extraction, and cleaned up further by dispersive solid phase extraction (d-ESP). As a result, background contamination was largely reduced. Different dispersive ESP sorbents were tested and primary secondary amine (PSA) bonded silica sorbents showed the best recoveries for these target OPEs. The recoveries obtained were in the range 54-113% (RSD<17%), with method limits of quantification (MLOQs) ranging between 0.06 and 0.29ng/g in egg, and 0.05 and 0.50ng/g w.w. in liver sample. The matrix effects (MEs) associated with using APCI(+) and ESI(+) sources were investigated. APCI(+) showed much less ion suppression than ESI(+) for the determination of these OPEs. For egg and liver samples, the APCI(+) ME values ranged from 40% to 94%, while ESI(+) ME values ranged from 0% to 36%. Although APCI(+) was used for the determination of OPEs, the ionization mechanism might mainly be a thermospray ionization process. This UHPLC-MS/MS-APCI(+) method showed good response linearity for calibration (R2>0.99). The proposed method was applied to real environmental bird egg and fish samples, where several OPE were quantifiable and different OPE patterns was observed between samples.

Isomer‐specific accumulation of perfluorooctane sulfonate in the liver of chicken embryos exposed in ovo to a technical mixture

Prior to its recent phaseout, perfluorooctane sulfonate (PFOS) was produced by electrochemical fluorination processes, which yielded technical mixtures composed of linear isomer (∼65-79%) and several branched isomers (∼21-35%). Because PFOS can biomagnify in wildlife, birds that occupy higher trophic levels are at increased risk of exposure. We hypothesized that the pharmacokinetic properties of PFOS are isomer-specific in developing chicken (Gallus gallus domesticus) embryos exposed to technical grade PFOS (T-PFOS). In the present study, T-PFOS was composed of 62.7% linear isomer (L-PFOS), and 37.3% branched isomer, including six mono(trifluoromethyl)-branched isomers and four bis(trifluoromethyl)-branched isomers. Concentrations of 0.1, 5, or 100u2009µg/g of T-PFOS were injected into the air cell of chicken eggs prior to incubation. After pipping, compared with T-PFOS, the PFOS isomer profile in embryonic liver tissue for the 0.1u2009µg/g dose group showed 21% enrichment in the proportion of L-PFOS with a corresponding decrease in the proportion of branched isomers. Not all branched isomers were discriminated against at equal rates. The proportion of two mono(trifluoromethyl)-branched isomers and three bis(trifluoromethyl)-branched isomers decreased to a greater degree than other branched isomers. In contrast, the mono-branched isomer, P6MHpS, was overrepresented in the low-dose group. In the higher dose groups, L-PFOS was still enriched but only by approximately 10%, which indicated a dose-dependent change in isomer composition relative to T-PFOS. These results show that accumulation of PFOS in chicken embryo livers is dependent on the presence and position of branches on the alkyl backbone. This supports the hypothesis that the pharmacokinetics of PFOS are isomer-specific in biota, and may help explain why wildlife PFOS burdens are dominated by L-PFOS relative to T-PFOS mixtures.

Concentrations, patterns and metabolites of organochlorine pesticides in relation to xenobiotic phase I and II enzyme activities in ringed seals (Phoca hispida) from Svalbard and the Baltic Sea

The present study investigates the concentrations and patterns of organochlorine pesticides (OCPs) and their metabolites in liver and plasma of two ringed seal populations (Phoca hispida): lower contaminated Svalbard population and more contaminated Baltic Sea population. Among OCPs, p,p-DDE and sum-chlordanes were the highest in concentration. With increasing hepatic contaminant concentrations and activities of xenobiotic-metabolizing enzymes, the concentrations of 3-methylsulfonyl-p,p-DDE and the concentration ratios of pentachlorophenol/hexachlorobenzene increased, and the toxaphene pattern shifted more towards persistent Parlar-26 and -50 and less towards more biodegradable Parlar-44. Relative concentrations of the chlordane metabolites, oxychlordane and -heptachlorepoxide, to sum-chlordanes were higher in the seals from Svalbard compared to the seals from the Baltic, while the trend was opposite for cis- and trans-nonachlor. The observed differences in the OCP patterns in the seals from the two populations are probably related to the catalytic activity of xenobiotic-metabolizing enzymes, and also to differences in dietary exposure.