State of Analysis for Pharmaceuticals and Other Organic Contaminants in Multiple Media

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State of Analysis for Pharmaceuticals and Other
Organic Contaminants in Multiple Media
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Acknowledgements
M.T. Meyer, Lawrence KS mmeyer_at_usgs.gov Colleen
Rostad, Denver CO Steve Zaugg, Denver CO E.M.
Thurman, Lawrence, KS District expertise (e.g.
Gail Cordy, Betsy Frick, Kathy Lee, Pat Phillips,
Paul Stackelberg)
Dana Kolpin, Iowa City IA
dwkolpin_at_usgs.gov Larry Barber, Boulder CO Kymm
Barnes, Iowa City IA Mike Focazio, Restion VA Ed
Furlong, Denver CO Sheridan Haack, Lansing MI
The Toxics Program toxics.usgs.gov Emerging
Water Quality Issues toxics.usgs.gov/regional
/emc.html
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Objectives

• Introduction
• Extractions
• Mass Spectrometry
• Immunoassay
• Selected Results

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Potential Sources Human and Agricultural
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Sources and Source Pathways

• What is the
  contaminant
  signature of
  
  specific source types?
• What are the characteristics of the entry
  mechanism to the environment for different
  sources?

Modified from Halling-Sorenson (1998)
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Diversity of Media

• Liquid Waste (waste-storage structures)
• Manure (dry, wet, fresh, composted)
• Ambient water (SW, GW)
• Soil amended soils
• Sediment

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Issues and Problems

• Antibiotic Resistance (Pathogens).
• Transference of Resistance.
• Endocrine Disruption.
• Nutrient overloading of soil (what is in stored
  waste also goes on the field).
• Effects on Waste Treatment
• Runoff and leaching of EMC residues.

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Species Comparisons of Resistance among
Enterococci
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Enterococci Resistance in Surface Waters
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Extractions and Sample Preparation
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Solid-Phase Extraction

• Many Types of Materials
• C2-18 on Silica backbone with varying linkages
• Polymers also with hydrophillic-lipophilic
  functional groups
• Anion Exchange (WC, SC, WA, SA)
• Mixed Mode
• Immunnoaffinity
• Many manufacturers

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Extraction Technologies

• Off-line Solid-Phase Extraction (liquids)
• Cartridges (syringe, sep-pak)
• Disks
• 96-well plates
• Solid-phase microextraction
• On-line Solid-Phase Extraction (liquids)
• Prospekt cartridges
• Accelerated Solvent Extraction (solids)
• Sorbents?

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Solids - Accelerated Solvent Extraction
Pressure (atm. 2000 psi) Temp. (Ambient 200
C) Multiple cycles
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SPE Example
60 mg HLB Condition 2 mL MeOH 3 mL 0.5 N HCl 1
mL H2O Load Sample
Wash 1 mL water Elute 5 mL MeOH into test tube
Concentrate N2 to 125 mL Analyze ESI()
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Typical Manual SPE Vacuum Setup
100-1,000 ml
96-well plate, 2 ml
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Tandem Reverse Phase/Mixed Mode SPE
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Automated Off-Line SPE (cartridge based)
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Solid-Phase Microextraction
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Sample Preparation (can configure for SPE plates)
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Evaporation concentration
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Typical Concentration FactorsEnvironmental SPE

• Sample Extract Concentration
• Vol. (ml) Vol. (µL) Factor
• 100 100 1000
• 1,000 500-1000 1000-2000
• 1 µg/L 1-2 mg/L

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On-Line SPE LC/MS
1-10 ml sample, add modifiers just prior to
extraction, LC addition of IS. Extract and
analyze at same time.
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Prospekt cartridges available in many Flavors.
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Methods Development

• Special emphasis
• PhAC
• HAC

What we find in the environment depends on what
we look for and how hard we look.

• 158 Compounds in Water
• 45 Antibiotics
• 12 Prescription Drugs
• 8 Nonprescription Drugs
• 14 Hormones and Steroids
• 79 Household and
• Industrial Compounds

• 83 Compounds in Sediment
• 3 Antibiotics
• 12 Prescription Drugs
• 7 Nonprescription Drugs
• 61 Household and
• Industrial Compounds

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Mass Spectrometry
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Mass Spectrometry
Ionization GC/MS (EI, CI) LC/MS (ESI, APCI,
APPI) Types High Resolution Magnetic Sector Ion
Trap Single Quadrapole Tandem (Triple-Stage
Quadrapole) Time of Flight (TOF)
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Applications of GC/ and LC/MS

• GC/MS Volatiles
• Priority Pollutants
• Insecticides
• Hydrocarbons
• POPs
• Derivtization for more polar compounds (e.g.
  surfactants, hormones)

• LC/MS Nonvolatiles
• Hormones
• Steroids
• Pesticide Degradates
• Herbicides
• Fungicides
• Surfactants
• Pharmaceuticals
• Organometallics

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Ionization in APCI

• Vaporize the neutral pesticide with heat.
• Corona Discharge ionizes the carrier gas. N2
  e- N2 . 2e-
• Carrier gas reacts with water to form hydronium
  ion.
• Hydronium ion protonates the analyte (MH).

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APCI Source
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Ionization in Electrospray

• Ionization of the solute in solution.
• Nebulize the solution and charge the droplets.
• Desolvation of the droplets by evaporation.
• Desorption of the solution ions to gas phase ions.

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Electrospray Source
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Ionization-Continuum Diagram
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Ion Trap MS/MS
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How Q-TOF Works
Unique Feature is High Resolution of Fragment Ions
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Matrix Effects

• Suppression
• Enhancement
• Mostly occur in ESI

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Effects of Humic Material on Signal Intensity
Relative to buffered solution
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Conclusions

• A variety of choices for sample extraction and
  clean-up.
• LC/MS-MS preferable for specificity.
• Sensitivity proportional to cost.
• LC/MS ESI subject to matrix effects
• On-line SPE can be used to minimize matrix
  effects.
• Goal is to minimize sample size and matrix
  effects.

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Immunoassays
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Immunoassay Types

• Enzyme-Linked Immunosorbent Assays
• Coated
• Tubes
• 96-well plate
• Magnetic Particle
• Radioimmunoassay
• H3, C14

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Coated 96-well microtiter plate
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General Immunoassay Concept
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Tetracycline Cross-Reactivity

• Chlortetracycline 48.8
• Oxytetracycline 32.7
• Tetracycline 41.3
• Isochlortetracycline 0.3
• Epichlortetracycline 21.3

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Macrolide Cross-Reactivity
Erytrhomycin 72 Tylosin 59
Lincomycin 12
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Immunnoassay Kits

• Antibiotics (RIA, ELISA)
• Hormones (RIA, ELISA)
• 17ß-estradiol
• Ethynylestradiol
• Estrone
• Surfactants, Industrial (ELISA)
• Alkylphenols
• Alkylphenol ethoxylates
• Linear alkylbenzene sulfonate
• Bisphenol A
• Pesticides (ELISA, RIA)
• Vitellogenin and others as effects markers
• Abraxis, Beacon, SDI, R-BioPharm, Charm Sciences

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Knowledge to use IAs Effectively

• Detection levels vary from 1.5 ppt 1 ppb
• Dependability in Low and Hi DOC matrices and
  wastewater (treated and untreated) need to be
  evaluated.
• Cross-reactivity must be known.
• Coupling SPE to IA can lower detection levels but
  must be evaluated for matrix effects.
• Can be useful screens for a wide variety of
  compounds.
• Cost 300-700 for 100 tests. With QA 50 to 80
  samples/100 tests.
• With labor, supplies etc. can be 40-to
  50/analyte.

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Some General Results

• Reconnaissance Comparison
• Biosolids
• Antibiotics in swine CAFOs
• Antibiotic Residues Fish Hatcheries, CAFOs

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National Reconnaissance Studies
- Streams (1999-2000) - Ground Water (2000) -
Sources of Drinking Water (2001) - Streambed
Sediment (2002)
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GW Recon Focus on Susceptible Settings
Landfills
Areas with water reuse
Septic Systems
CAFOs
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Summary - National Recon Studies
SW GW S DW B Sed
(N139) (N47) (N74) (N51)
Non_drugs 81 15 64 50
Antibiotics 48 26 26
53 Pharms 32 6
23 100 Det metabs 69 43
19 -- DEET 74
35 19 -- Caffeine
71 11 54 24
Fluoxetine 1 4
1 100
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Metro-Effluent is a Complex Mixture of Compounds
10
8
6
?g/L
4
2
0
5-methyl-1H-benzotriazole
Alkylphenols
DEET
Plasticizers
Fragrances
Triclosan
Pharmaceuticals
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Municipal Biosolids - General Results

• Relatively little variation in the number of
  detections or concentration for a given class of
  OWCs
• Variations between products is typically an
  order of magnitude or less
• Variations demographics, time of year,
  production process

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Occurrence of Antibiotics Swine CAFO Lagoons
1998-2002
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Spatial Sampling IA (5000 feeder pigs)
10-22-03
11-18-03
07-07-03
Ctet (N, S) 1440, lt 74, lt 67,
lt Epi-Ctet 130, 178 30, lt 41,
92 Iso-Ctet 1170, 1260 3770, 3920
2570, 4600 Iso-Epi-Ctet 1120, 1160 1670,
2150 1400, 2720 Tet 71, lt
150, 171 81, 210 Anhydro-Tet lt,
lt 2, lt 2, 2 Epi-Tet
130, 52 170, 150 140,
240 Lincomycin 1, 1 9, 6
44, 38 Total-Res 4062, 2653
5875, 6397 4245, 7902
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Antibiotics in Fish Hatcheries

• Initial Recon
• - Detected in 15 of samples
• - SDM (12), OTC (4)

Follow-up (3 hatcheries) - Detected in 31 of
samples - SDM (23), OM (17), OTC (8) - Max
conc 36 mg/L - Variable persistance - SDM
(48 d), OM (28 d), OTC (20 d) - Detections in
untreated raceways
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IV. Transport and Fate
Paired Watershed Study - Fourmile Creek (IA)
low-gradient stream - Boulder Creek (CO)
high-gradient stream
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IN SUMMARY

• Extraction 1st step in analysis and cleanup.
• LC/MS-MS techniques wide variety of compounds.
• Variety of instruments. More sensitive less
  sample.
• Matrix effects an issue affects quantitation and
  detection.
• On-Line SPE with LC/MS-MS obtain 1 to 10 ppt
  levels on 10 ml sample can reduce matrix effects.

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IN SUMMARY

• Immuoassays more work needs to be done to
  determine their utility in a variety of matrices.
• Complex Mixtures (HACs, PhACs).
• Need appropriate methods for a wide variety of
  media.
• Need interdisciplinary studies to determine
  relationships, if any, between residues and
  on-site and off-site effects, treatment
  efficiency, and effectiveness of best management
  practices.

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IN SUMMARY

• Need to identify treatment and environmental
  degradates of frequently detected and high use
  compounds.
• Need sources or supplements for stable isotope
  labeled compounds.

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