1. AXYS ANALYTICAL SERVICES Ltd “Ultra Trace Organic Analysis, POPs, and Emerging Contaminants”
For ASU April 8, 2011
By Richard Grace – Director – Sales, Marketing and Service – AXYS Analytical Services Ltd. Or

2. AGENDA
About AXYS
Ultra- Trace POPs and Emerging Contaminant Analysis Basics
Selecting and Developing Methods
Attributes
Accreditation + Quality Systems
Detection + Reporting Limits
Applicable Levels + Blank Levels
Analyte Lists + Time
Some topical items on CECs

3. About AXYS 100 employees located in Victoria B.C. (Vancouver Island)
ISO Certified, Multiple NELAP accreditations
Focus – Multi Target Ultra Trace Organic Analysis (pg or ng levels)
POPs
Emerging Contaminants
HRMS, LC MS/MS
some GC/MS, GC-ECD options
High barrier to entry
3 Analytical Areas
Environmental (All matrices associated with Bioaccumulation cycle)
Bio-monitoring
Method Development and Validation (i.e. EPA 1668, 1614, 1694, 1698, 1699)
Big “D” , little “R”

4. Where is Victoria?

5. AXYS HRMS Analysis (AXYS Developed EPA Methods in brackets)
Chlorinated and Brominated Dioxins and Furans
PCBs (EPA 1668A and 1668C)
OC, OP, ON Pesticides (EPA 1699)
PBDEs (EPA 1614)
Hormones and Sterols (EPA 1698)
Acid Extractable Herbicides
Chlorinated Naphthalenes
PBBs
Pyrethroids

6. AXYS LC MS/MS Analysis (EPA Reference Methods in Brackets)
Perfluoronated Compounds (pending)
Personal Care and Pharmaceutical Products (EPA 1694)
Hormones (EPA 1645)
Phthalate Esters Metabolites
Parabens and metabolites
Bisphenol A and metabolites
Nonyl Phenols and Nonyl Phenol Ethoxylates
Variety of Pesticide Panels

7. Who do we work for? 70% U.S., 30% Cdn. + International
Government and Regulatory Authorities (50% of work)
National and State Governments
POP and CEC Assessment
Legal issues
Method Development
Private Sector Industry + Consultants (40% of work)
Assessment and Remediation
Regional and Ambient / Baseline Work / TMDLs
POP and CEC Risk Assessment / Litigation
Academic, Legal, NGO – 10%

8. AXYS Analysis Early Days (1980 to 2000)
Occurrence of POPs in all environmental and bioaccumulation matrices
Arctic
Regulatory Framework, Litigation and Settlements
POPs in humans, NRD assessments
Early Reconnaissance Studies and NGOs
Awareness of CECs
Epidemiology Studies and CECs
Explosion of programs
CEC focus
AXYS does not publish studies, our clients do
2-4 year lag vs. time of work

9. AXYS Analytical Process (75 Staff)

10. Expanded AXYS Analytical Process (15 client services staff)

11. AXYS Analysis
All analysis performed with multiple mass or mass fragment detection per target
HRMS (EI or CI)
GC/MS (EI or CI in SIM)
LC MS/MS in MRM mode (multiple transitions where possible)
All analysis use labelled standards added at start of analytical process, recovery corrected vs. recovery standard
All “active” analysis EPA Tier 1 validation (IPR, MDL, MS/MSD by EPA Federal Register 40 CFR Part 136, Appendix B, no iteration

12. GCMS Scan Chromatogram in Urine Matrix Ultra Trace Detection Not Required for Targets

13. GC/MS with Ultra Trace DLs Not Met pp-DDT by GC EI MS SIM

14. Meeting Ultra Trace Requirements 13C12 pp DDT and pp DDT by HRMS

15. LC / MS vs. LC MS/MS TIC (Total Ion Chromatogram) vs. MRM Monitoring
Dehydronefidipine

16. Ultra Trace Analysis Principles Positive Identification and Quantification
Performance of multiple ions monitored and reported to meet response, ratio, and retention criteria
Recovery correction required
Max. chromatographic separation and selectivity achieved through cleanup, columns, instrument type
Selectivity achieves low detection limits, ruggedness
Common to all CDC and EPA 1600 series methods for POPs and CECs
Basis for EU 657 standard for positive identification and quantification of targets with no specified reference method
Many bio-monitoring methods would be considered a “non-positive screen by this standard

17. # of Ions (native plus labeled surrogate)
EU 657 Point Criteria for Positive Identification if Using Isotope Dilution (4 Points Required for a Positive)
Technique
# of Ions (native plus labeled surrogate)
# of Points
GC/MS (EI or CI) N
LC/MS
GC MS/MS
1 precursor, 2 daughter 4
LC MS/MS
2 precursor, each with one daughter 5
HRMS 2N

18. Applying Point System to PBDE Analysis
Method
HRGC/HRMS
(8+ points)
HRGC/LRMS
(1-2 points)
HRGC/ECD
(0 Points)
Details
EI (Electron Impact) / Voltage SIR
NCI (Negative Chemical Ionization) /SIM
Scan
Quantification
13C-labeled standards for isotope dilution
Labeled standards
not possible
Specificity
High: Monitor two to six ions from parent ion cluster, compare to theoretical ratio
Medium: Monitor Br- ions lost from PBDEs. Other brominated compounds e.g. PBBs may interfere if have same GC retention time
Low: Identification based on GC retention time. Other halogenated compounds may interfere e.g. PBBs, PCBs, Cl pesticides if same GC retention time.
Sensitivity
Best (Lowest)
detection limit
Highest
detection limits
Medium

19. PFC Methods That Meet Criteria MRM Transitions and Surrogates Used
Target Analyte
MRM Transition IS
MRM
Surrogate
PFBA (C4 Acid)
213>169
13C4 PFBA
217>172
13C4 PFOA
417>372
PFPeA (C5 Acid)
263>219
PFHxA (C6 Acid)
313>269, 313>119
13C2 PFHxA
315>270
PFHpA (C7 Acid)
363>319, 363>169
PFOA (C8 Acid)
413>369, 413>219, 413>169
13C2 PFOA
415>370
PFNA (C9 Acid)
463>419, 463>219, 463>169
13C5 PFNA
468>423
PFDA (C10 Acid)
513>469, 513>269, 513>219
13C2 PFDA
515>470
PFUnA (C11 Acid)
563>519, 563>269, 563>219
13C2 PFUnA
565>520
PFDoA (C12 Acid)
613>569, 613>319, 613>169
13C2 PFDoA
615>570
PFBS (C4 Sulfonate)
299>80, 299>99
303>84
13 C2 FOUEA 18 O2 PFOS
PFHS (C6 Sulfonate)
399>80, 399>99
18O2 PFHS
403>84
PFOS (C8 Sulfonate)
499>80, 499>99, 499>130
13C2 PFOS
503>80
FOSA (C8 Sulfonamide)
498>70

20. CEC Surprises – Triggers for Change PFOS Pharmacokinetics and Distribution
Well absorbed orally (95% within 24 h)
Distributed mainly in serum and liver
Not metabolized
Fecal and urinary excretion
Perception change in early 2000’s
30 ng/mL serum average in North Americans in 2004
Rat: Plasma t½ = 7.5 days
Monkey: Serum t½ = 200 days
Human: Estimated Serum t½ = ~8.7 years

21. Standard AXYS PFC In Water Method
AXYS PFC Method Basics
Standard AXYS PFC In Water Method
MLA 060
For water, soils, sediments, tissues, serum, air
Applies principles of EPA 1600 series performance based methods
Key AXYS PFC Method Attributes
Recovery Corrected
Matrix Matched Calibration
Pre-Concentration by SPE Cartridge
Low detection limits and blank levels
Big “D”, small “R”
Benchmarked, multiple method validations to insure accuracy
LOQ is LMCL point above MDL or client specific level
Adjust pH to 5.5 ±0.5 With HCOOH
Spike labeled surrogates
SPE extraction with WAX
Condition: 5 mL 0.3% NH4OH in MeOH
and 5mL0.1 M HCOOH
Wash: with 5 mL of H2O 5 mL &
With 1:1 (0.1 M HCOOH: MeOH)
Elute: 4 mL of 0.3% NH4OH in MeOH
Microvial 300uL portion
Analyze by (-ESI) LC-MS/MS

22. POPs and CECs Analyses Accreditation and Benchmarking
POPs and CECs = Environmental Contaminants
ISO 17025:2005 is correct quality standard
ISO 17025: 2005 = ISO 9001: technical requirements (method and statistical QC)
National Bodies
NELAP not ISO 17025
Accreditation by test and environmental matrix
NELAP offers regulatory method programs
Human matrices not offered in past
CALA / SCC has now made available
Proficiency not available, must go to other forums

23. POPs and CECs – Benchmarking Accuracy
No “regulatory” programs or methods
Tools for establishing accuracy
Inter-calibrations / Round Robins / PE Programs
SRMs / CRMs
Inter-calibrations available
AMAP
Dioxin Inter-calibrations
PFC Inter-calibrations
Some academic programs for endocrine disruption (BPA, PEMs)
NIST developed
Available for most legacy (regulated) POPs
Some persistent CECs in development (PFCs, PBDEs)
Not available for most other CECs (BPA, PEM, Parabens etc.)

24. 1st Worldwide Interlaboratory Study on PFCs in Environmental and Human Samples
*Van Leeuwen et al. Environ. Sci. Technol. (2006) Vol. 40, p
*Fish Fillet Results
PFOS
PFOA
Spiked Concentration (ng/g) 37 10
Analytical Results (ng/g)
Minimum
2.8
0.54
Median 40 13
Maximum
295
204
%RSD
125
201
Evaluation of Results
%Satisfactory 17 25
%Questionable - 30
%Unsatisfactory 83 45

25. Limits of Accuracy in early POPs and CEC Analysis
Phthalate Ester metabolites
Standards accuracy issue
i.e. MCPP standard used for most studies appears to be 17% of reported value
PFCs in Eggs
BPA
0.25 to 10 ng/mL gives 90% population coverage in urine (NHANES)
Urine deconjugated BPA 10X blood values
Some studies show much higher blood values (BPA instrument values)

26. LC MS/MS False Positive Example
Analysis – PFCs by LC MS/MS
Matrix – Eggs for human consumption
Analytical Result – 120 mean ng/g PFOS per Egg detected using LC MS/MS in MRM mode (Carbon labelled PFOS surrogate employed, one transition specified retention time, UPLC technology submitted for publication)
Net Result – Paper withdrawn, results were identified as false positive (TDCA, a bile acid in many animals)
Issue Identified – not enough positive identification criteria
Identified need for monitoring multiple transitions
Did not focus on chromatographic separation

27. LC MS/MS Interferences PFOS and Taurodeoxycholate Acid (TDCA) Same MW, Common Transition
Compound MW
Parent
Ion 1
Ion 2
Ion 3 CE CV
Taurochendeoxycholate
499.29
498.2
79.8
106.8
123.8 55 80
Taurodeoxycholate
Tauroursodeoxycholate
PFOS
499.93
498.9
79.9
98.9
 N/A
45/40

28. PFOS / TDCA Interference TIC Chromatogram Separation PFOS – Top, 3 TDCA Isomers

29. Detection and Reporting Limits
Select analytes, then required reporting limits, then determine sample size
Define reporting limits by study objectives
NHANES is an excellent resource
Literature MAY be useful
Be aware of very different languages
Many Laboratories and Publications
Reporting Limit < MDL < Blank Level
ISO Reference Laboratories
Reporting Limit > MDL > Blank Level (ideal)

30. AXYS Reporting Limits Default Sample Reporting Limits (RL)
HRMS and GC/MS – LMCL or 3:1 signal to noise ratio of peaks
LC MS/MS – LMCL or 3:1 signal to noise ratio if above LMCL
IDL not calculated or important, LMCL response 10-30X S/N
MDL 3-5X < RL (ideally), not used for reporting
Dilution increases RL proportionally
No blank correction, blank levels may influence reporting limits by prior agreement
Applied “flags” to data quality – e.g. estimated maximum and estimated minimum probable concentrations (EMPCs)
Should always be discussed with client

31. Blank Levels – A dirty secret
Many CECs are ubiquitous as in many consumer goods and lab materials
Blanks limit detection capability in analysis such as;
PBDEs
PCBs
PFCs
BPA
Cotinine
Can control with sample size selection
Lab control not in method, but related SOPs
Ask for control charts (10 method blanks min.)

32. Blank Control Charts Use them to set large study stat. limits

33. Analyte Lists – Which Ones to Measure?
AXYS experience that what gets measured continues to get measured
Relevant POPs and CECs change over time
Legacy POPs
Replacement products for
PFCs
PBDEs
Phthalates
Aging (POPs congener distribution)
Very little measurement of metabolites and degradation products
All lack toxicity / epidemiological end point info for initial years of study
All may be endocrine disruptors
Studies should change over time

34. The Mean Percent Congener Distribution to the Sum of the 35 PCBs (Whole Weight in ng/g) for NHANES by Age Group

35. Lake Ontario % of PCB Concentrations Not Part of IADN and US/DS Lists – Drives EPA 1668 A,B,C

36. Observed Concentration
Are PFOS and PFOA the Only PFCs? 6 Months of Occurrence Data POTW Aqueous Streams
Analytes
Units
No. of Samples
No. of Detects
Percent of Detects
Observed Concentration
Avg Conc. Found
Min Conc. Found
Max Conc. Found
PFBA
ng/L
339
234 69
65.5
1.15
8540
PFBS 84
24.8
568
2.19
21000
PFDA
109
32.2
3.79
1.1
29.1
PFDoA 2
0.6
1.45
1.23
1.66
PFHpA
233
68.7
52.9
1.03
9610
PFHxA
253
74.6
328
76200
PFHxS
117
34.5
1650
2.03
190000
PFNA
210
61.9
5.62
28.1
PFOA
276
81.4
74.2
1.38
15600
PFOS
175
51.6
445
2.02
25200
PFOSA 25
7.4
15.3
1.04
61.7
PFPeA
340
216
63.5
174
29200
PFUnA 5
1.5
2.57
1.18
4.73

37. 6 Months of PFCs in POTW Biosolids
Analytes
Units
No. of Samples
No. of Detects
Percent of Detects
Observed Concentration
Avg Conc. Found
Min Conc. Found
Max Conc. Found
PFBA
ng/g
123 11
8.9
7.26
1.14
16.2
PFBS 14
11.4
9.12
2.45
32.4
PFDA 91 74
14.6
0.712
57.5
PFDoA 56
45.5
8.08
0.292
58.9
PFHpA 12
9.8
6.54
2.01
28.1
PFHxA 22
17.9
0.727
105
PFHxS 25
20.3
32.3
1.82
147
PFNA 60
48.8
9.37
0.139
39.2
PFOA 55
44.7 17
0.585
256
PFOS 85
69.1
476
0.901
7480
PFOSA 39
31.7
40.8
0.194
343
PFPeA 21
17.1
12.4
0.567
69.6
PFUnA 52
42.3
6.44
1.12
35.2

38. Minimum. Concentration Maximum Concentration
PBDEs from TNSSS – EPA 2008
Analyte
Units
Minimum. Concentration
Mean
Concentration
Maximum Concentration
% Solids %
0.43
93.53
BDE – 28
DryWt. ng/kg
2,200
160,000
BDE – 47
73,000
709,174
5,000,000
BDE – 66
1,800
110,000
BDE – 85
3,200
150,000
BDE – 99
64,000
716,362
4,000,000
BDE – 100
13,000
1,100,000
BDE – 138
1,900
40,000
BDE – 153
9,100
52,685
410,000
BDE – 154
7,700
440,000
BDE – 183
2,100
120,000
BDE – 209
3,462,942
17,000,000

39. PBDE’s by EPA 1614 – Moving to 46 congeners from Top 8 to 11 Congeners
Theory – Penta and Octa changes will eliminate most toxic PBDEs, incoming Deca controls will eliminate 209 congener
Current Situation – about 850M tons in environmental inventory in NA
Degradation of PBDE containing products giving ongoing supply
Deca plus new products production rising
Deca degradation rapid – to other PBDEs
May be in for a long PBDE story

40. Legacy POPs and New POPs PFC Beluga Study – Published ES&T June 2009 POPs = Persistency, Toxicity, Bioaccumulation + Transport
Field work: Village of Umiujaq /
Nastapoka River Estuary

41. Sample Collections: E. Hudson Bay
Study Overview
Sample Collections: E. Hudson Bay
Sediment samples (n = 8) petit ponar grabs
Macro-algae (n = 6)
Harvested beluga whales (n=25); 12 females, 12 males, 1 calf
liver, blubber, blood, milk
Fish: capelin, arctic cod, sculpin, salmon (n = 6 per species)
Chemical analysis:
Previous Work: PCBs, PBDEs, OC Pesticides
Current Study: PFCs
AXYS Extended PFC List (Carboxylates, Sulfonates, Sulfonamides)

42. Mean Values of PFCs in Beluga Blood, Liver, Milk

43. PFCs (ng/g) in Arctic Tissue

44. PFC Levels in Trophic Magnification vs. Legacy Organohalogens
ng.g-1 dry wt.
PFC Levels in Trophic Magnification vs. Legacy Organohalogens
= PCBs
= OC Pest.
= PBDEs
= PFCs
Beluga Liver

45. Contaminants of Emerging Concern – “CECs” Some New POPs + Many Endocrine Disruptors
Many not persistent but exposure constant
Many present through consumer goods – ubiquitous
Many thought to be endocrine disruptors
Endocrine System Regulates Changes in Body
Response to environment
Orchestrates body development and reproductive changes
Hormones are “Messengers” to Cells
Change cell internal and external chemistry
Many hormones and receptors, many interactions and outcomes
Endocrine Disruption Occurs when;
Hormone receptors inhibited to not function
Hormone receptors triggered by “outside” compound
Hormone System “Out of Balance”
Effect is not a “toxic” effect

46. Defining Endocrine Disruption End Points
Toxicity
Typical screening process
Long term, subtle developmental effects not screened
“Nonmonotonic Dose Response Curves” (more chemical does not equal more response – example BPA)
Effect May be at Specific Stage of Life Cycle
Requires study over long periods of life cycle
More difficult with more complex species
Low level studies required, statistically relevant study size
Limited Tools
Limited analytical and epidemiological methods at relevant levels
# of studies / study size – inconclusive data in many cases
Interpretation / vested interests

47. Topical End Points for POPs and CECs
Reproductive
Reduced fertility
Reproductive tract abnormalities
Male / Female Sex ratios
Brain Development and Behavior Issues
Thyroid impairment
I.Q. reduction
ADHD
Immune System Issues
Increased Illness
“Weaker” organism
Cancer, Diabetes, Heart Disease, Cholesterol variations, obesity etc.
Synergistic and Antagonistic Effects

48. An Interesting Study Kidd et al 2007
Environmental End Point – hormonal effects / vitellogenin gene expression on Fathead Minnows
Vitellogenin – egg yolk precursor protein expressed by female fish
Hormonal triggers (synthetic hormones, NP etc.) produce fish vitellogenin production / feminization
Study – Test (5-6 ng 17A ethinyl estradiol addition) vs. Control Lake
Year 3 produces complete Fathead Minnow feminization
Fathead population eliminated
Notes on Interpretation
End Point measured on Fathead Minnows
In other species - effects not noted
Tie to Humans?

49. Length frequency distributions of fathead minnow captured in trap nets in reference Lake 442 (A) and Lake 260 (B) (amended with 5–6 ng·L−1 of EE2 in 2001–2003) during the fall of 1999–2005
Kidd K. A. et.al. PNAS 2007;104:

50. Purpose of AXYS “Client” Requests for CEC Analysis – Example PPCPs
“Reconnaissance” or Screen in Water and Sediments based on “USGS”, 1694 or Other List
Occurrence, Levels, and Fate of PPCPs in POTW and Drinking Water Processes
Occurrence, Levels, and Fate in POTW and Water Treatment Systems for New Treatment Systems / Capital Projects
Trace back / fingerprinting of sources of PPCPs
Support of Toxicity, Fate, and End Point Studies

51. The List – Which PPCP Compounds to Analyze?
3000 potential PPCPs
Starting Point – USGS Reconnaissance List
40 PPCPS + Hormones and Sterols
Constant requests from clients for more compounds based on literature, other studies, media articles
Input from National Regulatory Bodies
Lists supplied based on volumes, persistence, toxicity / end point concerns (i.e. endocrine disruption, antibiotic resistance)
Fit for analysis – standards, surrogates, stability, appropriate instrumentation
Framework to manage addition of compounds, groups to maintain, validate, and improve

52. AXYS PHASE 3 PPCP METHOD (2008/9) Grouping of Compounds by Analytical Performance EPA Method 1694 Compounds + Extended List

53. Building the Methods Built From Back to Front Column Selection
Instrument Infusion
Response, Mode, Cone Voltage, Transitions
Retain and Repeat !!!!
Column Selection
Phase 1 only
Selection of SPE Cartridges
Multiple tests in Phase 1 only
Varying pre-wash, loading, elution
Extraction
Acidic, Basic
Validation
MDL, IPR, MS/MSD
Troubleshooting + Re-Validation

54. EPA 1694 PPCPs – Frequency and Range of Detects EPA “Targeted National Sewage Sludge Survey” All Results in dry weight ug/Kg
ANALYTE
# Detect
Minimum ug/Kg
Maximum ug/Kg
4-Epioxytetracycline 8
35.7
54.9
4-Epitetracycline 80
47.2
4,380
Erythromycin-Total 77
3.1
180
Flumequine NA
Fluoxetine 79
12.4
3,130
Gemfibrozil 76
12.1
2,650
Ibuprofen 54
99.5
11,900
Isochlortetracycline 1
3,140
Linomycin 3
13.9
33.4
Lomefloxacin 2
33.3
39.8
Metformin 6
550
1,160
Miconazole
14.2
9,210
Minocycline 32
351
8,650
Naproxen 44
20.9
1,020
Norfloxacin 29
99.3
1,290

55. Are We Measuring the Right Thing
Are We Measuring the Right Thing? Conjugates, Degradation Products, Metabolites
Current State
Analytes primarily native compounds
Most compounds “persistent” by constant presence
Limited degradation products and metabolites
No Chlorination byproducts
No conjugates
About Conjugates
Many compounds exist as conjugates from urine
Deconjugation may occur at some rate in POTW environments or on consumption
Limited info but detected in studies
Methods from Biomonitoring Available
I.e. Triclosan, BPA, Parabens, mono-phthalate esters

56. Observed Concentration
Looking for the Correct Analyte Form Triclosan Conjugates in Urine vs. Native Triclosan in Serum
Analyte
Units
No. of Samples
No. of Detects
Percent of Detects
Observed Concentration
Avg Conc. Found
Min Conc. Found
Max Conc. Found
Total
Triclosan
In Urine
ng/mL 62 52
83.9
109
1.33
1980
Native
In Serum 22 ND

57. Degradation Products / Metabolites
Many Compounds not Stable
Fit for TOF LC-MS/MS to identify degradation products
Not certain degradation products of less concern
Metabolites
In Humans and in Environment
Fit for TOF LC-MS/MS to identify degradation

58. Major Cl Transformation Products Triclosan + Free Chlorine

59. Open Forum
Questions
Follow-Ups