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Rapid Quantitative Detection & Speciation of Contamination in the Urban Watershed

Rapid Quantitative Detection & Speciation of Contamination in the Urban Watershed. Continuation of WQ1 669 524 94. N W R I. David C. White, Cory Lytle, Ying Dong Gan, Aaron M. Peacock, Kimberly Salome, Yevette M. Picenco,

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Rapid Quantitative Detection & Speciation of Contamination in the Urban Watershed

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  1. Rapid Quantitative Detection & Speciation of Contamination in the Urban Watershed Continuation of WQ1 669 524 94 N W R I • David C. White, Cory Lytle, Ying Dong Gan, Aaron M. Peacock, Kimberly Salome, Yevette M. Picenco, • Institute for Applied Microbiology, University of Tennessee, Knoxville, TN • Microbial Insights, Inc., Rockford, TN, Microbial Insights, Inc. -IAM

  2. WQ1 669 524 94 Objectives: 1). Rapid (< 1 hr) Quantitative sensitive method for CONTAMINATION* Detection to monitor multiple sites in the watershed     2). Allows for” regionalized” intensive treatment in an integrated watershed system CONTAMINATION* Drugs, hormones & other pharmaceutically active compounds -ppb Differentiate Pathogenic Bacteria including especially VBNC pathogens ~ Enterics, Legionella, Mycobacteria, indicators for virus Protozoa Cryptosporidium, Giardia , Microsporidium, Algae Indicators pf pathogen infectivity

  3. Sampling Urban Watershed --- Scheme #1 • Strategically placed coupons at multiple sites for biofilm formation • Biofilms are readily invaded by Pathogens and Nurtured • Lipids in biofilms serve as a built in solid phase extractor for hydrophobic drugs, hormones, bioactive agents • 5. Convenient to recover & analyze for biomarkers • Its not in the water but the slime on the coupon

  4. Sampling Urban Watershed --- Scheme # 2 • Coupon sequentially extracted high pressure/temperature • Supercritical Carbon Dioxide  Neutral Lipids • (residue) Chloroform/methanol  Polar lipids • Analysis by HPLC/ES/MS/MS • (residue) Mild Acid , SFE  Lipopolysaccharide OH FA Analysis by GC/MS • Analysis on GIS Basis by automatons neural network ANN • Feedback to purification interdiction systems

  5. In the Drinking Water Biofilm Reproducibly Generate a Drinking Water Biofilm: • 1. Add from continuous culture vessels: • Pseudomonas Spp. • Acetovorax spp. • Bacillus spp. • 2. Seed with trace surrogate/pathogen E. coli (GFP), Mycobacteriumpflei (GFP), Legionellabosmanii , Sphingomonas

  6. Biofilm Test System

  7. Detecting specific pollution:Drugs, hormones bioactive compounds Recover from biofilms: Triculture biofilm + E. coli (GFP) established 3 days in presence of 1000 ppb drug  Extract  EI/MS/MS Beads Beads +Biofilm Waste Caffeine 4.7% 50 ppb 3.7% 37 ppb ~97% Triclosan* 3.7% 37 ppb 15% 160 ppb ~ 84% Monensin** ~ 0 % 0.18 1.8 ppb ~99% Finasterdine*** ~ 0% 0.4 % 4.2 ppb ~98 % Caffeine LOD ~ 2 ppb *disinfectant widely used in toiletries LOD ~25 ppb * **macrolide antibiotic used as antiparastic in cattle & chickens factories LOD~2 ppb * ****(Proscar) inhibitor of testosterone hydroxylase LOD ~ 300 ppt * Biofilms concentrate bioactives compared to sterile surface

  8. ESI (cone voltage) Q-1 CAD Q-3 ESI/MS/MS

  9. Finasterdine Q1 scan 373.3 373.3 Product ion scan 305.4

  10. Coupon + Biofilm Extract with SFECO2  1. Neutral Lipids UQ isoprenologues UQ-8 Enterics, UQ-9 Pseudomonas, UQ-10 Protozoa Derivatize –N-methyl pyridyl Diglycerides (cell lysis) Sterols, Cholesterol (Protozoa), Ergostrerol (Fungi) Extract Residue with Chloroform.methanol 2. Polar Lipids  Lipid Biomarkers Phospholipids, PC, PE, PG, & sn1 sn2 FA Amino Acid PG, 0rnithine lipids, Plasmalogens Acidify, Extract residuewithSFECO2  3. LPS OH FA Transesterify, GC/MS .  30H 10:0, 12:0 – Pseudomonas 30H 14:0 -- pathogens & enterics

  11. Respiratory Ubiquinone (UQ) LOD = 3 ppb (3.7 fmol/uL) ~ 104 Bacteria , UQ-8 E. coli & Enterics, UQ-9 Pseudomonas, UQ-10 Protozoa, Algae, UQ-12,13 Legionella Q7 Q10 Q6 197 m/z

  12. Parent product ion MS/MS of synthetic PG Q-1 1ppm PG scan m/z 110-990 (M –H) - Sn1 16:0, Sn2 18:2 Q-3 product ion scan of m/z 747scanned m/z 110-990 Note 50X > sensitivity SIM additional 5x > sensitivity ~ 250X

  13. Gram-negative Bacteria  lipid-extracted residue,  hydrolize [1% Acetic acid ],  extract = Lipid A • Acid sensitive bond [to KDO]   14* 14* E. Coli Lipid A  3 OH 14:0*

  14. Lipid A from E. coli Fatty acids liberated by acid hydrolysis followed by acid–catalyzed (trans) esterification 3OH 14:0 TMS GC/MS of Methyl esters 3OH 14:0 14:0 phthalate siloxane

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