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Rapid Quantitative Detection & Speciation of Contamination in the Urban Watershed David C. White, Cory Lytle, Ying Dong Gan, Aaron M. Peacock, Kimberly.

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Presentation on theme: "Rapid Quantitative Detection & Speciation of Contamination in the Urban Watershed David C. White, Cory Lytle, Ying Dong Gan, Aaron M. Peacock, Kimberly."— Presentation transcript:

1 Rapid Quantitative Detection & Speciation of Contamination in the Urban Watershed 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, - IAM Continuation of WQ Microbial Insights, Inc. N W R I

2 WQ 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 1.Strategically placed coupons at multiple sites for biofilm formation 2.Biofilms are readily invaded by Pathogens and Nurtured 3.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 Sampling Urban Watershed --- Scheme #1

4 6.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 7.Analysis on GIS Basis by automatons neural network ANN 8.Feedback to purification interdiction systems Sampling Urban Watershed --- Scheme # 2

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

6 Biofilm Test System

7

8

9 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 % 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

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

11 Finasterdine Q 1 scan Product ion scan

12 Coupon + Biofilm Extract with SFECO 2 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 Transesterify, GC/MS.  30H 10:0, 12:0 – Pseudomonas 30H 14:0 -- pathogens & enterics Lipid Biomarkers Phospholipids, PC, PE, PG, & sn1 sn2 FA Amino Acid PG, 0rnithine lipids, Plasmalogens 3. LPS OH FA   Acidify, Extract residue with SFECO 2 

13 Q6Q6 Q7Q7 Q m/z Respiratory Ubiquinone (UQ) LOD = 3 ppb (3.7 fmol/uL) ~ 10 4 Bacteria, UQ-8 E. coli & Enterics, UQ-9 Pseudomonas, UQ-10 Protozoa, Algae, UQ-12,13 Legionella

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

15 14 * Gram-negative Bacteria  lipid-extracted residue,  hydrolize [1% Acetic acid ],  extract = Lipid A    Acid sensitive bond [to KDO] E. Coli Lipid A  3 OH 14:0 *

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


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