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Rapid Quantitative Detection & Speciation of
Contamination in the Urban Watershed
Continuation of WQ1 669 524 94
NW 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
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
Sampling Urban Watershed --- Scheme #1
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 # 2
6. Coupon sequentially extracted high
pressure/temperature
 Neutral Lipids
(residue) Chloroform/methanol  Polar lipids
Supercritical Carbon Dioxide
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
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),
Mycobacterium pflei (GFP), Legionella
bosmanii , Sphingomonas
Biofilm Test System
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
Caffeine
4.7% 50 ppb 3.7% 37 ppb
Triclosan*
3.7% 37 ppb 15% 160 ppb
Monensin**
~0%
0.18 1.8 ppb
Finasterdine*** ~ 0%
0.4 % 4.2 ppb
Waste
~97%
~ 84%
~99%
~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
ESI
(cone voltage)
Q-1
ESI/MS/MS
CAD
Q-3
Finasterdine Q1 scan
+Q1: 0.118 to 0.480 min from Sample 1 (finasterdine) of 0928002.wiff
CH 3
7.4e6
7.0e6
Max. 7.4e6 cps.
373.3
373.7
H3C
6.5e6
O
CH 3
6.0e6
NH
CH 3
5.5e6
In te n s ity , c p s
5.0e6
4.5e6
CH 3
4.0e6
374.7
3.5e6
3.0e6
2.5e6
2.0e6
O
N
H
1.5e6
395.6
C23H36N2O2
Exact Mass: 372.28
Mol. Wt.: 372.54
H
1.0e6
5.0e5
202.4
0.0
200
210
231.2
220
230
365.3
343.4
253.5
240
250
260
270
280
290
300
310
m/z, amu
320
330
340
350
366.5
360
397.7
371.7
370
+Product (373.7): 119 MCA scans from Sample 2 (finasterdine) of 0928002.wiff
380
390
400
Max. 1.6e8 cps.
373.3
373.3
1.6e8
1.5e8
Product ion scan
1.4e8
1.3e8
305.4
305.4
1.2e8
In te n s ity , c p s
1.1e8
1.0e8
9.0e7
8.0e7
7.0e7
6.0e7
5.0e7
374.4
4.0e7
3.0e7
2.0e7
1.0e7
107.2
0.0
100
121.3
120
175.1
147.4 161.3
140
160
317.2
189.3
180
215.4 220.3
200
220
249.3
255.4
240
260
m/z, amu
355.3
272.4
280
300
320
340
360
380
400
Extract with SFECO2
Coupon + Biofilm

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
Phospholipids, PC, PE, PG, & sn1 sn2 FA
Biomarkers Amino Acid PG, 0rnithine lipids, Plasmalogens
Acidify, Extract residue with SFECO2
3. LPS OH FA

Transesterify, GC/MS .  30H 10:0, 12:0 – Pseudomonas
30H 14:0 -- pathogens & enterics
Respiratory Ubiquinone (UQ)
LOD = 3 ppb (3.7 fmol/uL) ~ 104 Bacteria ,
UQ-8 E. coli & Enterics,
UQ-9 Pseudomonas, UQ-10 Protozoa, Algae,
Q7
Q6
UQ-12,13 Legionella
Q10
O
H3OC
CH3
H3OC
O
197 m/z
H
]n
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 747 scanned
m/z 110-990
Note 50X > sensitivity
SIM additional 5x > sensitivity ~ 250X
Gram-negative Bacteria  lipid-extracted residue, 
hydrolize [1% Acetic acid ],  extract = Lipid A
Acid sensitive bond
O
[to KDO]
O
O P O
OH O
O
HO
O
HN
O
O
O
Exact Mass: 1765.19
Mol. Wt.: 1766.32
HN
O
OH
O
2C93H174N2O24P2
O
O
P O
O
OH
O
OH
O
O


14*
14*
E. Coli Lipid A  3 OH 14:0*
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
14:0
3OH 14:0
phthalate
siloxane