Quadrupole ion storage tandem mass spectrometry and high-resolution mass spectrometry: complementary application in the measurement of 2,3,7,8-chlorine substituted dibenzo-p-dioxins and dibenzofurans in US foods.

The US Food and Drug Administration has simultaneously utilized both high-resolution mass spectrometry (HRMS) and quadrupole ion storage tandem mass spectrometry (QISTMS) in the measurement of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) in 147 food samples collected in 1998 and 1999 in the US. In 1998, 20 egg samples, six scallop, 10 blue crab, eight American lobster, 10 pollack, 15 striped bass, five rockfish, 10 crawfish, seven aqua-cultured and 13 wild-caught salmon, along with 19 cream and 18 mozzarella cheese samples were measured for PCDD/Fs. QISTMS provided limits of detection (LODs) close to those produced using HRMS for many congeners in 56 samples analyzed by both techniques in 1998 and three salmon and three striped bass collected in 1999. The I-TEQs of the mean levels for measured congeners in 40 samples of fish and shellfish and 16 cheese and eggs from 1998 analyzed by HRMS and QISTMS were 0.99 and 1.1 ng/kg wet weight, respectively. The I-TEQ for mean congener levels in the 40 fish and shellfish measured by HRMS was 1.4 ng/kg wet weight. A higher sample throughput with greater data quality at a lower cost is achievable by using both QISTMS and HRMS.

Selection of alternative 5' splice sites: role of U1 snRNP and models for the antagonistic effects of SF2/ASF and hnRNP A1.

The first component known to recognize and discriminate among potential 5' splice sites (5'SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5'SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5'SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5'SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5'SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5'SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5'SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.

Elevated TCDD in chicken eggs and farm-raised catfish fed a diet with ball clay from a Southern United States mine.

The U.S. Food and Drug Administration (FDA) terminated the use of ball clay from a mine in Mississippi as an additive in animal feed after discovering nanogram per gram concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). The FDA collected chicken eggs and farm-raised catfish in affected areas and throughout the remaining continental United States to assess levels of 2,3,7,8-TCDD. A new method using quadrupole ion storage tandem-in-time mass spectrometry (QISTMS) measured the 2,3,7,8-TCDD levels in 42 catfish fillet composites, 3 Tilapia fillet composites, 46 chicken egg samples, and 6 chicken feeds. Six catfish composites and 20 egg samples had 2,3,7,8-TCDD concentrations significantly above 1.0 pg/g wet weight of fillet or whole egg. Farm-raised catfish not exposed to feed containing ball clay had a mean 2,3,7,8-TCDD concentration of 0.12 pg/g. The TCDD isomer pattern in ball clay differed from the TCDD isomer pattern in a fly ash sample and from the "chick edema factor" TCDD pattern in a sample of reference toxic fat used as a feed ingredient in the 1950s.

Tandem-in-time mass spectrometry method for the sub-parts-per-trillion determination of 2,3,7,8-chlorine-substituted dibenzo-p-dioxins and -furans in high-fat foods.

Limits of quantitation (LOQs) for a quadrupole ion storage tandem-in-time mass spectrometry (QISTMS) method were evaluated through replicate analysis of unfortified peanut oil, shortening, lamb fat, and butter for all 2,3,7,8-chlorine-substituted polychlorodibenzo-p-dioxins (PCDDs) and polychlorodibenzofurans (PCDFs). Ten congeners were measurable in butter (0.27-2.5 pg/g) and nine congeners were measurable in lamb fat (0.09-2.6 pg/g) with good precision. LOQs for high-fat foods were estimated by triplicate analysis of peanut oil fortified at two levels. Accurate and reproducible results were achieved at 0.5 pg/g for most PCDD/Fs (1.0 pg/g for heptachlorodibenzo-p-dioxin and heptachlorodibenzofuran and 2.0 pg/g for octachlorodibenzofuran) and at 0.2 pg/g for 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). QISTMS distinguished between catfish and chicken eggs with elevated TCDD levels from background samples collected from the most regions of the continental United States. QISTMS determined the extent of TCDD contamination in butter, lamb fat, and cottonseed oil collected from rural villages in Kazakhstan. Replicate analysis of catfish and chicken eggs by the QISTMS method produced comparable results to high-resolution mass spectrometry (HRMS). Lower limits of detection will be needed if QISTMS is to fully complement HRMS in the measurement of TCDD levels in food.

Determination of polychlorinated dibenzo-p-dioxin and dibenzofuran background in milk and cheese by quadrupole ion storage collision induced dissociation MS/MS.

Recent developments in quadrupole ion storage scanning techniques have made possible the acquisition of mass spectrometry/mass spectrometry (MS/MS) data with high sensitivity, selectivity and reproducibility. Retail dairy products were analyzed successfully for bioincurred or background contamination by all 17 of the 2,3,7,8 substituted polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). Analytes were measured by both full scan electron impact low resolution MS (EI-LRMS) and collision induced dissociation MS/MS (CID MS/MS). Results were comparable using either technique. MS/MS, however, provided higher sensitivity and selectivity on many congeners. Results for the MS/MS technique were reproducible, with little reduction in sensitivity or spectral quality during the analyses of all test samples. The MS/MS signal to noise ratio (S/N) was 10 to 100 times greater than low resolution electron impact performed with the same instrument in food matrices. Signal to noise ratios increased on most parent ions as well as for all daughter ions. Further development of this technique may provide a cost effective alternative to traditional HRMS analyses of PCDDs and PCDFs in food.

Biotransfer and bioaccumulation of dioxins and furans from soil: chickens as a model for foraging animals.

Chickens were used as a model for foraging animals to examine the bioavailability of all 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/PCDFs) from soil. Three groups of chickens were exposed through their diet to soil contaminated with PCDD/PCDFs at less than 0.5 pg/g I-TEQ (control group), 42 pg/g I-TEQ (low exposure group), and 460 pg/g I-TEQs (high exposure group). Eggs, tissues, feces and feed were analysed throughout the exposure and depuration period. Daily intake was estimated at 2.5 ng/kg-day for the high and 0.3 ng/kg-day for the low exposure groups. Bioavailability was chlorination-dependent ranging from 80% for tetrachlorinated to less than 10% for octachlorinated congeners. During exposure, tissue distribution was congener-dependent with 5-30% of the intake excreted in the eggs, 7-54% deposited in the adipose and less than 1% present in the liver. On a fat weight basis, the highest concentrations were observed in the liver, implying that mechanisms other than lipid solubility operate in that tissue. Bioconcentration factors and elimination half-lives were also congener- and tissue-dependent. Results from this study indicate that animals foraging on soil contaminated at low ppt PCDD/PCDF levels may bioaccumulate these compounds to unacceptable levels.

World Health Organization international intercalibration study on dioxins and furans in human milk and blood.

Under the sponsorship of the World Health Organization (WHO), an interlaboratory calibration on the analysis of PCDD/PCDFs in human milk and blood was carried out which included 19 laboratories from 14 countries. The study design involved the analysis of three samples of each matrix in triplicate. Selected samples were spiked with native standards of certain 2,3,7,8-substituted congeners at concentrations known only to WHO staff. The study design resulted in approximately 4000 individual pieces of PCDD/PCDF data generated by a variety of analytical methods, at various concentrations, and by laboratories of widely different experience. This was, by considerable margin, the largest study which allowed for the direct comparison of laboratory and method performance. The results of statistical analysis of this data base addresses the effect on data quality of clean up methods, instrumental methods, analyte concentration, laboratory QA programs, and laboratory experience. The study has shown that the laboratory is the single most important determinant of data precision and accuracy. The method of analyte enrichment (sample clean up), analyte measurement [gas chromatography/mass spectrometry (GC/MS) protocol], and analyte concentration have weaker correlations with data quality.