1. EXPERIENCES IN THE ANALYSIS OF PHARMACEUTICALS AND PERSONAL CARE PRODUCTS IN POTW AND ENVIRONMENTAL SAMPLES Coreen Hamilton AXYS Analytical Services Ltd., Sidney, BC, Canada Presented at NEMC, Washington DC August 10, 2010

2. Overview  Overview of Analytical Method  Goal and Scope  Which PPCPs?  PPCP Method  Analytical challenges with complex matrices  Options for hormones and sterols analysis  Axys’ experiences - some results  Are we measuring the right compounds?

3. Goals and Scope of Method A fully validated method suitable for routine use, providing results of known accuracy and precision. A fully validated method suitable for routine use, providing results of known accuracy and precision. Applicable to samples with complex matrix such as wastewaters, biosolids and soil. Applicable to samples with complex matrix such as wastewaters, biosolids and soil. Analysis of a large variety of target compounds. Analysis of a large variety of target compounds.

4. PPCP Analysis – A Starting Point 2001 USGS Stream Reconnaissance 95 ORGANIC WASTEWATERCONTAMINANTS 41 Antibiotics, Prescription / Non – Prescription Drugs 6 PAHS 15 Steroids and Hormones 7 Insecticides 5 NP and OP Compounds 18 Industrial Chemicals Triclosan, BPA, and DEET

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

6. What’s On The PPCP Target List 121 “Pharmaceutical and Personal Care Products” Antimicrobial drugs from the macrolide, ß- lactam, penicillin, quinoline, sulfonamide and tetracycline classes Antimicrobial drugs from the macrolide, ß- lactam, penicillin, quinoline, sulfonamide and tetracycline classes Selected analgesic, anti-depressant, anti- cholesterol, anti-convulsant, anti-asthmatic, diabetic and blood pressure drugs Selected analgesic, anti-depressant, anti- cholesterol, anti-convulsant, anti-asthmatic, diabetic and blood pressure drugs Caffeine and 1,7-dimethylxanthine, cotinine Caffeine and 1,7-dimethylxanthine, cotinine Triclosan and triclocarban Triclosan and triclocarban

7. Desired Method Attributes Isotope Dilution / Recovery Corrected Isotope Dilution / Recovery Corrected DLs in low ng/L or ng/g range DLs in low ng/L or ng/g range Positive Identification/Accurate Quantification Positive Identification/Accurate Quantification Validated by Accepted Procedures Validated by Accepted Procedures Rugged Rugged Large List of Targets Large List of Targets Right “List of Targets” Right “List of Targets”

8. Overview of Analytical Method Modified EPA 1694. Two extractions: pH 2 and pH 10 Modified EPA 1694. Two extractions: pH 2 and pH 10 Labeled standards added before extraction Labeled standards added before extraction Solid samples extracted into aqueous matrix. Solid samples extracted into aqueous matrix. Extraction/concentration by SPE Extraction/concentration by SPE 5 LC/MS/MS runs 5 LC/MS/MS runs Internal Standard Quantification Internal Standard Quantification

9. Modifications to Method 1694 Analysis of 32 additional “List 5 “ compounds. Analysis of 32 additional “List 5 “ compounds. Addition of 18 compounds to Lists 3 List 4 Addition of 18 compounds to Lists 3 List 4 33 extra labeled standards added before acid extraction 33 extra labeled standards added before acid extraction No modifications to sample extraction and cleanup procedures. No modifications to sample extraction and cleanup procedures. An additional LC/MS/MS run on acid extract – ESI positive An additional LC/MS/MS run on acid extract – ESI positive

10. AXYS PPCP METHOD EPA Method 1694 Compounds + Extended List PPCPc by LC-MS/MS 121 Compounds 49 labeled Surrogates List 1 PPCPs Acidic Extract +ve ESI 49 Compounds 49 Compounds 11 Surrogates List 2 TCY’s Acidic Extract +ve ESI 14 Compounds 1 Surrogate List 3 PPCPs Acidic Extract -ve ESI 12 Compounds 9 Surrogates List 4 PPCPs Basic Extract +ve ESI 14 Compounds 11 Surrogates List 5 PPCPs Acidic Extract +ve ESI 32 Compounds 17 Surrogates

11. Standards and Surrogates Authentic standards for each target compound Authentic standards for each target compound Stable isotope labeled standards used as internal standards for all target compounds (labeled analogs not available for all analytes) Stable isotope labeled standards used as internal standards for all target compounds (labeled analogs not available for all analytes) Labeled injection standards (atrazine, amitriptyline or 2,4,5-TPP) to quantify recovery of internal standards – key tool for detection of matrix enhancement or suppression Labeled injection standards (atrazine, amitriptyline or 2,4,5-TPP) to quantify recovery of internal standards – key tool for detection of matrix enhancement or suppression

12. LC/MS/MS Quantification Calibration using 5 points and internal standard quantification Calibration using 5 points and internal standard quantification Linear or quadratic calibration with 1/x weighting Linear or quadratic calibration with 1/x weighting Bracketing calibration during run (initial and final calibration verification) Bracketing calibration during run (initial and final calibration verification) Monitor “recovery” of injection standard to detect matrix suppression or enhancement Monitor “recovery” of injection standard to detect matrix suppression or enhancement

13. Surrogate Recoveries as a Function of Sample Matrix MATRIX TYPE NO. OF SAMPLES Biosolid67 Compost9 Soil/Sediment75 Vegetation11 Peat Moss 31 Aqueous Samples 370 Aqueous SPM 66

14. Acetominophen Surrogate Performance

15. Caffeine Surrogate Performance

16. Ciprofloxacin Surrogate Performance

17. Trimethoprim Surrogate Performance

18. Sulfamethazine Surrogate Performance

19. Erythromycin Surrogate Performance

20. Tools for Analyzing High Matrix Samples Multiple Internal Standards Multiple Internal Standards Sample Size Management Sample Size Management Larger Size SPE Cartridge Larger Size SPE Cartridge Monitor LC-MS/MS response for evidence of matrix effects: Monitor LC-MS/MS response for evidence of matrix effects: - anomalous surrogate recovery - absolute response of injection standards - dilution and re-run

21. Challenges with POTW Samples Complex matrix can affect extraction, cleanup or MS/MS quantification Complex matrix can affect extraction, cleanup or MS/MS quantification Range of analyte concentrations within a sample Range of analyte concentrations within a sample Physical nature of samples : “wet” in biosolids, particulate in aqueous samples Physical nature of samples : “wet” in biosolids, particulate in aqueous samples

22. Results for POTW Samples POTW influent, effluent and biosolids from multiple sources POTW influent, effluent and biosolids from multiple sources Data from samples analyzed over a 6 month period Data from samples analyzed over a 6 month period Provides a small snapshot of “Detects” and concentrations Provides a small snapshot of “Detects” and concentrations Target analyte: large list or small list? Target analyte: large list or small list?

23. Frequent Flyers: List 1 >60% BiosolidsInfluent/Effluent % DetectsMax ng/g% DetectsMax ng/L Azithromycin956530685550 Caffeine581000064218000 Carbamazepine986030735690 Ciprofloxacin100475005843300 Clarithromycin716177011900 Dehydronifedipine352566132 Diltiazem86947713080 Diphenhydramine1005730747350 Erythromycin-H2O90312772990 Fluoxetine94313056146 Miconazole97921031855 Norfloxacin56102001318500 Ofloxacin9758100443240 Sulfamethoxazole41651705460 Thiabendazole8037063102 Trimethoprim39273635130

24. High Flyers: >100 ng/g or ng/L BiosolidsInfluent / Effluent %DetectsMax ng/g% DetectsMax ng/L 1,7-Dimethylxanthine8237003873100 Acetaminophen712100029251000 Cefotaxime0ND172830 Digoxigenin32571124 Digoxin25601117 Lincomycin37118278 Penicillin G1723621 Penicillin V15914121 Sarafloxacin1198051260 Sulfadiazine214019153 Sulfadimethoxine56214107 Sulfamerazine33612126 Sulfanilamide8156004415 Sulfathiazole1216255 Tylosin138197 Virginiamycin1046921050

25. High Flyers - > 500 ng/g or ng/L BiosolidsInfluent / Effluent %DetectsMax ng/g% DetectsMax ng/L 1,7-Dimethylxanthine8237003873100 Acetaminophen712100029251000 Cefotaxime0ND172830 Digoxigenin32571124 Digoxin25601117 Lincomycin37118278 Penicillin G1723621 Penicillin V15914121 Sarafloxacin1198051260 Sulfadiazine214019153 Sulfadimethoxine56214107 Sulfamerazine33612126 Sulfanilamide8156004415 Sulfathiazole1216255 Tylosin138197 Virginiamycin1046921050

26. Occurrence of List 4 Analytes N Biosolids % Detects Max ng/g N Infl/ Effl % Detects Max ng/L Albuterol11322317341604 Amphetamine41001476647672 Atenolol41003570471990 Atorvastatin450196641233 Cimetidine1139097801733911700 Clonidine40ND70340 Codeine11427328194477590000 Cotinine114476901906211000 Enalapril40ND7041 Hydrocodone41001170335110000 Metformin11317184016858485000 Oxycodone40ND70565720000 Ranitidine1136022501734816800 Triamterene41006287047512

27. Occurrence of List 3 Analytes N Biosolids % Detects Max ng/g N Infl/ Eff % Detects Max ng/L 2-Hydroxy- ibuprofen6529390009652208000 Bisphenol A650ND96125200 Furosemide6531303096419800 Gemfibrozil1758231902096811000 Glipizide6521196726 Glyburide6514801964190 Hydrochlorothiazide6520470096658030 Ibuprofen17775257002105375700 Naproxen17269108001626950000 Triclocarban1721004410001626413700 Triclosan172971330001624921100 Warfarin17510.52072051

28. Occurrence of List 2 Analytes N Biosolid % Detect Max ng/g N Infl/ Effl % Detects Max ng/L 4-Epianhydrochlortetracyc1110ND990ND 4-Epianhydrotetracycline1114921601015250 4-Epichlortetracycline11149741050ND 4-Epioxytetracycline1119931000ND 4-Epitetracycline11196495010211412 Anhydrochlortetracycline1117708991110 Anhydrotetracycline111701960990ND Chlortetracycline1146101015671500 Demeclocycline11152001050ND Doxycycline114935090157215660 Isochlortetracycline11153140105671 Minocycline92518650973739 Oxytetracyclin11448221015761110 Tetracycline114975270157312700

29. Other “Hits” - List 5 Analyte Biosolid Max ng/g Infl / Effl Max ng/LAnalyte Biosolid Max ng/g Infl / Effl Max ng/L 10-hydroxy- amitriptyline2368Norfluoxetine6016 Amitriptyline448133Norverapamil9512 Amlodipine45333Paroxetine19325 Benzoylecgonine23880Prednisolone1510107 Cocaine71100Promethazine62ND DEET2060100Propoxyphene2331 Desmethyldiltiazem2865Propranolol71 Diazepam219Sertraline559102 MeprobamateND756TheophyllineND41400 Methylprednisolone19647Valsartan1035030 Metoprolol66706Verapamil7031

30. Example Results San Francisco Estuary Institute Pilot 2006 Map of sampling locations in the South Bay. Samples were collected at sites SB01-10.

31. Survey Results and Comparison to Literature Values

32. Delaware River Basin Sampling Locations Phase 2 PPCP Survey

33. OTC Prescription Antibiotics DRBC 2007 survey

34. Methods for Hormones and Sterols Two Options 1. Method 1698 GC/HRMS analysis as TMS derivatives GC/HRMS analysis as TMS derivatives Hormones and Sterols Hormones and Sterols 2. Modified Method 1694 LC-MS/MS analysis LC-MS/MS analysis Hormones only Hormones only

35. HORMONES and STEROLS METHOD Base of EPA Method 1698 17 Hormones and 9 Sterols LLE + Derivatization HRMS (1698) HRMS (1698) GC/LRMS Option All Targets (not in 1698) 1698 HRMS All Targets 1 or 2 Runs (High Sterols in Biosolids Create in Biosolids CreateSplit) Sterols by GC/LRMS Hormones by HRMS Practical Option

36. LC-MS/MS METHOD – 17 HORMONES Modified EPA 1694 AcidicExtraction+ LC/MS/MS LC/MS/MS +ESI 10 Targets 5 Surrogates -ESI 7 Targets 2 Surrogates

37. Sterol Target Analytes Campesterol Cholesterol * Coprostanol Desmosterol Desogestrel epi-coporostanol β-Sitosterol β -Stigmastanol Stigmasterol

38. Hormone Target Analytes ESI + Allyl trenbolone Androstenedione Androsterone Desogestrel Estriol Mestranol Norethindrone * Norgestrel * Progestrone * Testosterone

39. Hormone Target Analytes ESI - Dihydroequilin Equilenin Equilin 17β-Estradiol 17α-Estradiol * Estrone 17 β-Ethinylestradiol *

41. HRMS HORMONES and STEROLS – Concentration Range of Analytes HRMS HORMONES and STEROLS – Concentration Range of Analytes  Hormones low level, many sterols very high level in POTW biosolids and influent  Affects blank acceptance criteria  5-10 ng for most compounds  500 for cholesterol and β-sitosterol  Adjustments required to prevent “NQ” on high level sterols (cholesterol and β- sitosterol most frequent )  Extract split used to maintain QC  Over spike with Sterol surrogates at beginning of process  1/20 to 1/100 split after clean-up  2 instrument runs  Method QC adjusted to match  GC/MS and LC-MS/MS Options

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

43. Degradation Products / Metabolites Many compounds not stable – in humans and in environment Many compounds not stable – in humans and in environment Metabolites or degradation products may be more prevalent than parent PPCPs Metabolites or degradation products may be more prevalent than parent PPCPs TOF LC-MS/MS to identify these compounds TOF LC-MS/MS to identify these compounds Are these of concern? Are these of concern?

44. SAMPLE HANDLING / PRESERVATION / HOLD TIME STUDY Preservatives and Chlorine Quenchers Preservatives and Chlorine Quenchers Arrest microbiological activity: cold or frozen Arrest microbiological activity: cold or frozen Likely no single preservative for all compounds Likely no single preservative for all compounds Some chlorine quenching options - Thiosulfate, Ascorbic Acid (preferred). Some chlorine quenching options - Thiosulfate, Ascorbic Acid (preferred). Containers Containers HDPE, Glass (Amber), Silanization HDPE, Glass (Amber), Silanization One container solution? One container solution? Analytes not grouped by container behavior Analytes not grouped by container behavior AXYS uses HDPE and Glass AXYS uses HDPE and Glass

45. Next Steps More labeled standards as they become available More labeled standards as they become available Additional target compounds including metabolites Additional target compounds including metabolites Removal of compounds from list based on occurrence and/or effects data Removal of compounds from list based on occurrence and/or effects data Application to biological tissues Application to biological tissues

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