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Lee Blum, Ph.D. Testing for Bisphenol A and other Endocrine Disruptor Chemicals

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Bisphenol A <ul><li>Why NMS Labs interested in testing for Bisphenol A: </li></ul><ul><ul><li>Growing public concern </li></ul></ul><ul><ul><ul><li>National Toxicology Program - 2008 “Some concern” </li></ul></ul></ul><ul><ul><ul><li>Endocrine Society Scientific Statement </li></ul></ul></ul><ul><ul><ul><ul><li>2009 Recognition of chemicals that interfere with the biosynthesis, metabolism or activity of hormones. </li></ul></ul></ul></ul><ul><ul><ul><li>FDA - 2010 “Some concern” </li></ul></ul></ul><ul><ul><li>Recognition of widespread human exposures </li></ul></ul><ul><ul><ul><li>One of the world's highest production volume chemicals, with over 2.2 million tonnes (6.4 billion pounds) produced annually. ( ScienceDaily Jan. 13, 2010 ) </li></ul></ul></ul><ul><ul><ul><li>Releases to the environment exceed 1 million pounds per year. (U.S. EPA) </li></ul></ul></ul><ul><ul><ul><li>Detectable in more than 92% of the U.S. population. ( Calafat, et al, 2008) </li></ul></ul></ul>

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Bisphenol A <ul><li>CDC NHANES Study 2003-2004 (Calafat, et al., 2008) </li></ul><ul><ul><li>Urinary Total Bisphenol A in U.S. population (n = 2517) </li></ul></ul>Population Median (25 th – 95 th percentile) All 2.7 ng/mL (1.3 – 15.9) 6 – 11 years 3.7 ng/mL (1.7 – 16.0) 12 – 19 years 4.2 ng/mL (1.9 – 16.5) 20 – 59 years 2.7 ng/mL (1.2 – 15.5) 60 + years 1.9 ng/mL (0.8 – 13.3) Females 2.4 ng/mL (1.2 – 15.7) Males 3.2 ng/mL (1.4 – 16.0)

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Bisphenol A NTP – The Study & The Results

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Bisphenol A – It’s Everywhere

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Bisphenol A - Exposures <ul><li>Air, dust and water </li></ul><ul><li>Primarily through diet </li></ul><ul><ul><li>Majority in food and beverages </li></ul></ul><ul><ul><ul><li>Epoxy resins - Internal container coatings </li></ul></ul></ul><ul><ul><ul><li>Polycarbonate plastic – bottles (baby and water), food containers and tableware </li></ul></ul></ul><ul><li>Others </li></ul><ul><ul><li>Dental sealants or composites </li></ul></ul><ul><ul><li>Processing of PVC (constituent of some PVC plastics) </li></ul></ul><ul><ul><li>Thermal paper </li></ul></ul><ul><ul><li>Recycling of thermal paper </li></ul></ul><ul><ul><li>Epoxy based paints and lacquers </li></ul></ul><ul><ul><li>Workers involved in the manufacture of BPA and BPA containing products </li></ul></ul>

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Bisphenol A - Toxicokinetics <ul><li>Absorption through ingestion, inhalation and skin. </li></ul><ul><li>Metabolism - an efficient first-pass metabolism in the liver after oral administration and is rapidly converted to BPA-glucuronide. </li></ul><ul><li>Virtually completely eliminated within 24 hours following acute exposures. </li></ul><ul><ul><li>Study by Stahlhut et al. (2009) found that urine BPA levels of individuals who fasted for several hours were not as low as expected. </li></ul></ul>

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Bisphenol A <ul><li>Purposes of testing: </li></ul><ul><ul><li>Support of Medical Research </li></ul></ul><ul><ul><ul><li>Provide resources for bioanalytical testing </li></ul></ul></ul><ul><ul><li>Support of Environmental and Occupational Medicine </li></ul></ul><ul><ul><ul><li>Clinical evaluations </li></ul></ul></ul><ul><ul><ul><li>Environmental and workplace monitoring </li></ul></ul></ul>

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BPA – Procedure in Urine <ul><li>Analysis in urine </li></ul><ul><ul><li>Sample volume is 1.0 mL of urine </li></ul></ul><ul><ul><li>Internal standard D16-BPA </li></ul></ul><ul><ul><li>Calibrators are prepared with each batch of samples </li></ul></ul><ul><ul><ul><li>0.25 to 10.0 ng/mL </li></ul></ul></ul><ul><ul><li>Hydrolysis </li></ul></ul><ul><ul><li>Liquid/liquid extraction </li></ul></ul><ul><ul><li>Derivatization with PFPA (pentafluoropropionic anhydride) </li></ul></ul><ul><ul><li>Analysis by GC – EI Mass spectrometry with SIM mode of analysis </li></ul></ul><ul><ul><ul><li>Mass ions monitored: </li></ul></ul></ul><ul><ul><ul><ul><li>BPA 505 m/z (Quant); 520 m/z; 506 m/z </li></ul></ul></ul></ul><ul><ul><ul><ul><li>D16-BPA 516 m/z (Quant); 534 m/z; 517 m/z </li></ul></ul></ul></ul>

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BPA - Instrumentation Hewlett Packard/Agilent 5973 MSD

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BPA – Collection Concerns <ul><li>Specimen Collection </li></ul><ul><ul><li>Urine samples are collected in standard urine collection cups and refrigerated as soon as possible. Samples should be transferred to polypropylene vials within 4 hours of collection and frozen. Store the specimens frozen (-20 o C) and ship on dry ice. </li></ul></ul><ul><ul><li>Ref : Laboratory Procedure Manual for Bisphenol A [and other environmental phenols], Organic Analytical Toxicology Branch, Division of Laboratory Sciences, National Center for Environmental Health, CDC, Atlanta, GA May 25, 2005. </li></ul></ul>

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BPA – Linearity in Urine Observed Concentrations (ng/mL) Target Value Day 1 Day 2 Day 3 10.0 ng/mL 10.6 10.4 10.2 5.0 ng/mL 4.90 5.02 4.99 2.5 ng/mL 2.44 2.43 2.50 1.0 ng/mL 0.98 0.98 0.98 0.25 ng/mL 0.25 0.25 0.25

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BPA – Linearity in Urine

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BPA - Within Day Precision in Urine N = 5 x 3 Days 0.25 ng/mL 0.5 ng/mL 7.5 ng/mL Day 1 Mean 0.30 ± 0.015 0.44 ± 0.01 6.64 ± 0.1 %CV 5.83% 1.26% 0.82% % Accuracy 104% 87.2% 88.5% Day 2 Mean 0.25 ± 0.026 0.41 ± 0.008 6.77 ± 0.1 %CV 10.4% 1.99% 1.69% % Accuracy 101% 82% 90.3% Day 3 Mean 0.23 ± 0.015 0.44 ± 0.02 6.38 ± 0.5 %CV 6.48% 3.59% 7.24% % Accuracy 93.6% 88.0% 85.1%

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BPA - Between Day Precision in Urine N = 5 x 3 Days 0.25 ng/mL 0.5 ng/mL 7.5 ng/mL Mean 0.25 ± 0.02 0.43 ± 0.02 6.6 ± 0.30 %CV 8.91% 3.87% 4.63% % Accuracy 100% 86.0% 87.9%

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Urine Sample Endogenous (ng/mL) Spiked (ng/mL) Target (ng/mL) Measured (ng/mL) Deviation from Target 1 0.15 1.0 1.15 1.12 -2.61% 2 1.75 2.5 4.25 4.05 -4.71% 3 2.61 5.0 7.61 6.87 -9.72% 4 0.41 10.0 10.4 9.26 -11.0% 5 3.89 15.0 18.9 19.0 0.53% 6 0.31 0.25 0.56 0.54 -3.57% 7 1.78 1.0 2.78 2.69 -3.24% 8 0.47 2.5 2.97 2.81 -5.39% 9 1.30 5.0 6.30 6.07 -3.65% 10 0.16 1.0 1.16 1.04 -10.3% 11 0.24 2.5 2.74 2.62 -4.38% 12 4.82 5.0 9.82 9.06 -7.74% 13 0.90 0.25 1.15 1.08 -6.09% 14 0.57 1.0 1.57 1.50 -4.46% 15 0.57 2.5 3.07 2.92 -4.89% 16 1.78 5.0 6.78 6.63 -2.21% 17 2.70 0.25 2.95 3.01 2.03% 18 4.28 1.0 5.28 5.26 -0.38% 19 1.08 2.5 3.58 3.06 -14.5% Mean Endogenous 1.57 ± 1.46 ng/mL Mean Bias -5.07 ± 4.15%

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BPA – Storage Stability in Urine

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BPA – Storage Stability in Urine

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BPA – Storage Stability in Urine

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BPA – Patient Urine Samples Samples Tested 36 Median 3.2 ng/mL Range <0.25 to >10 ng/mL

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BPA – 0.25 ng/mL in Urine

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BPA – Typical Urine Sample

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BPA – Procedure in Blood/Serum/Plasma <ul><li>Analysis in Blood/Serum/Plasma </li></ul><ul><ul><li>Sample volume is 1.0 mL </li></ul></ul><ul><ul><li>Internal standard D16-BPA </li></ul></ul><ul><ul><li>Calibrators are prepared with each batch of samples </li></ul></ul><ul><ul><ul><li>0.25 to 10.0 ng/mL </li></ul></ul></ul><ul><ul><li>Solid Phase Extraction (SPE) </li></ul></ul><ul><ul><li>Derivatization with PFPA (pentafluoropropionic anhydride) </li></ul></ul><ul><ul><li>Analysis by GC – EI Mass spectrometry with SIM mode of analysis </li></ul></ul><ul><ul><ul><li>Mass ions monitored: </li></ul></ul></ul><ul><ul><ul><ul><li>BPA 505 m/z (Quant); 520 m/z; 506 m/z </li></ul></ul></ul></ul><ul><ul><ul><ul><li>D16-BPA 516 m/z (Quant); 534 m/z; 517 m/z </li></ul></ul></ul></ul>

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BPA – Linearity in Serum

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BPA – Linearity in Blood

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BPA - Within Day Precision in Serum N = 5 0.25 ng/mL 0.75 ng/mL 7.5 ng/mL Mean 0.21 ± 0.01 0.80 ± 0.06 7.61 ± 0.26 %CV 5.12% 6.94% 3.42% % Accuracy 85.6% 106% 102%

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BPA – Spiked Plasma Samples Plasma Sample Endogenous (ng/mL) Spiked (ng/mL) Target (ng/mL) Measured (ng/mL) Deviation from Target 1 0.22 0.25 0.47 0.47 0.00% 2 0.22 2.50 2.72 2.63 -3.31% 3 0.67 0.50 1.17 1.15 -1.71% 4 0.67 5.00 5.67 5.48 -3.35% 5 0.26 1.00 1.26 1.30 3.17% 6 0.26 10.0 10.3 10.0 -2.53% 7 0.11 0.75 0.86 0.92 6.98% 8 0.11 7.50 7.61 7.52 -1.18% 9 2.10 3.00 5.10 5.31 4.12% 10 2.10 9.00 11.1 11.3 1.62% Mean Endogenous 0.67 ± 0.83 ng/mL Mean Bias 0.38 ± 3.49 %

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BPA – Spiked Blood Samples Blood Sample Endogenous (ng/mL) Spiked (ng/mL) Target (ng/mL) Measured (ng/mL) Deviation from Target NaF 0.08 0.25 0.33 0.36 9.09% NaF 0.08 2.50 2.58 2.86 10.8% K Oxalate 0.17 0.50 0.67 0.70 4.48% K Oxalate 0.17 5.00 5.17 6.06 17.2% Na EDTA 0.18 1.00 1.18 1.32 11.9% Na EDTA 0.18 10.0 10.2 12.2 19.4% K EDTA 0.12 0.75 0.87 0.83 -4.60% K EDTA 0.12 7.50 7.62 9.00 18.1% Na Heparin 0.22 3.00 3.22 3.30 2.48% Na Heparin 0.22 9.00 9.22 10.1 9.54% Mean Endogenous 0.15 ± 0.05 ng/mL Mean Bias 9.85 ± 7.54%

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BPA – Storage Stability in Serum

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BPA – 0.25 ng/mL in Serum

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BPA – 0.75 ng/mL in Blood

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Endocrine Disrupting Chemicals <ul><li>Phthalate Metabolites in Urine </li></ul><ul><ul><li>Mono-n-butyl phthalate </li></ul></ul><ul><ul><li>Mono-ethylhexylphthalate </li></ul></ul><ul><ul><li>Mono-(2-ethyl-5-hydroxyhexyl) phthalate </li></ul></ul><ul><ul><li>Mono-(2-ethyl-5-oxohexyl) phthalate </li></ul></ul><ul><li>Chlorinated Hydrocarbons in Serum/Plasma/Blood and Adipose Tissue </li></ul><ul><ul><li>e.g. DDT, DDE and DDD; and others </li></ul></ul><ul><li>Herbicides in Serum/Plasma/Blood and Urine </li></ul><ul><ul><li>e.g. Atrazine and others </li></ul></ul><ul><li>Metals in Serum/Plasma/Blood and Urine </li></ul><ul><ul><li>e.g. Arsenic, Cadmium, Lead, Manganese, Mercury, Zinc and others </li></ul></ul>

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Hormone Testing <ul><li>Estrogen in Serum/Plasma </li></ul><ul><ul><li>Estrone (E1) </li></ul></ul><ul><ul><li>Estradiol (E2) </li></ul></ul><ul><ul><li>Estriol (E3) </li></ul></ul><ul><li>Testosterone in Serum/Plasma </li></ul><ul><ul><li>Testosterone, Free, Total, and Bioavailable </li></ul></ul><ul><ul><li>Dihydrotestosterone (DHT) </li></ul></ul><ul><li>Thyroid Hormones in Serum/Plasma </li></ul><ul><ul><li>T3 (Triiodothyronine), Free </li></ul></ul><ul><ul><li>T4 (Thyroxine), Free </li></ul></ul><ul><li>Coming Soon an Expanded Thyroid Panel </li></ul>

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[email_address] Phone: 1-800-522-6671 Lee Blum, Ph.D.