Lee Blum, Ph.D.

Testing for Bisphenol A and other Endocrine Disruptor

Chemicals

Bisphenol A• Why NMS Labs interested in testing for

Bisphenol A:– Growing public concern

• National Toxicology Program - 2008 “Some concern”• Endocrine Society Scientific Statement

– 2009 Recognition of chemicals that interfere with the biosynthesis, metabolism or activity of hormones.

• FDA - 2010 “Some concern”– Recognition of widespread human exposures

• 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)

• Releases to the environment exceed 1 million pounds per year. (U.S. EPA)

• Detectable in more than 92% of the U.S. population. (Calafat, et al, 2008)

Bisphenol A

Population Median (25th – 95th 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)

• CDC NHANES Study 2003-2004 (Calafat, et al., 2008) – Urinary Total Bisphenol A in U.S. population (n = 2517)

Bisphenol A

NTP – The Study & The Results

Bisphenol A – It’s Everywhere

Bisphenol A - Exposures• Air, dust and water• Primarily through diet

– Majority in food and beverages• Epoxy resins - Internal container coatings• Polycarbonate plastic – bottles (baby and water),

food containers and tableware

• Others– Dental sealants or composites– Processing of PVC (constituent of some PVC

plastics)– Thermal paper– Recycling of thermal paper – Epoxy based paints and lacquers – Workers involved in the manufacture of BPA and

BPA containing products

Bisphenol A - Toxicokinetics• Absorption through ingestion,

inhalation and skin.• Metabolism - an efficient first-pass

metabolism in the liver after oral administration and is rapidly converted to BPA-glucuronide.

• Virtually completely eliminated within 24 hours following acute exposures.– Study by Stahlhut et al. (2009) found that urine

BPA levels of individuals who fasted for several hours were not as low as expected.

Bisphenol A

• Purposes of testing:– Support of Medical Research

•Provide resources for bioanalytical testing

– Support of Environmental and Occupational Medicine •Clinical evaluations•Environmental and workplace

monitoring

BPA – Procedure in Urine• Analysis in urine

– Sample volume is 1.0 mL of urine– Internal standard D16-BPA– Calibrators are prepared with each batch of

samples• 0.25 to 10.0 ng/mL

– Hydrolysis– Liquid/liquid extraction– Derivatization with PFPA

(pentafluoropropionic anhydride)– Analysis by GC – EI Mass spectrometry with

SIM mode of analysis• Mass ions monitored:

– BPA 505 m/z (Quant); 520 m/z; 506 m/z– D16-BPA 516 m/z (Quant); 534 m/z; 517 m/z

BPA - Instrumentation

Hewlett Packard/Agilent 5973 MSD

BPA – Collection Concerns

• Specimen Collection – 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 (-20oC) and ship on dry ice. 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.

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

BPA – Linearity in Urine

BPA Linearity - Urine

y = 1.0395x - 0.0907

R2 = 0.999

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Target Concentration (ng/mL)

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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/mLDay 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%

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%

UrineSample

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%

BPA – Storage Stability in Urine

BPA Stability in Urine

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Room Temp

BPA – Storage Stability in Urine

BPA Stability in Urine

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BPA – Storage Stability in Urine

BPA Stability in Urine

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Day 0 Day 1 Day 2 Day 7 Day 14 Day 30

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Refrig Temp 3oC

Frozen Temp -10oC

BPA – Patient Urine Samples

Samples Tested 36

Median 3.2 ng/mL

Range <0.25 to >10 ng/mL

BPA – 0.25 ng/mL in Urine

BPA – Typical Urine Sample

BPA – Procedure in Blood/Serum/Plasma• Analysis in Blood/Serum/Plasma

– Sample volume is 1.0 mL – Internal standard D16-BPA– Calibrators are prepared with each batch

of samples• 0.25 to 10.0 ng/mL

– Solid Phase Extraction (SPE)– Derivatization with PFPA

(pentafluoropropionic anhydride)– Analysis by GC – EI Mass spectrometry with

SIM mode of analysis• Mass ions monitored:

– BPA 505 m/z (Quant); 520 m/z; 506 m/z– D16-BPA 516 m/z (Quant); 534 m/z; 517 m/z

BPA – Linearity in Serum

BPA – Linearity in Blood

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%

BPA – Spiked Plasma Samples

PlasmaSample

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%

BPA – Spiked Blood Samples Blood

SampleEndogenous

(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%

BPA – Storage Stability in Serum

BPA – 0.25 ng/mL in Serum

BPA – 0.75 ng/mL in Blood

Endocrine Disrupting Chemicals

• Phthalate Metabolites in Urine– Mono-n-butyl phthalate– Mono-ethylhexylphthalate– Mono-(2-ethyl-5-hydroxyhexyl) phthalate– Mono-(2-ethyl-5-oxohexyl) phthalate

• Chlorinated Hydrocarbons in Serum/Plasma/Blood and Adipose Tissue– e.g. DDT, DDE and DDD; and others

• Herbicides in Serum/Plasma/Blood and Urine– e.g. Atrazine and others

• Metals in Serum/Plasma/Blood and Urine– e.g. Arsenic, Cadmium, Lead, Manganese,

Mercury, Zinc and others

Hormone Testing

• Estrogen in Serum/Plasma– Estrone (E1)– Estradiol (E2)– Estriol (E3)

• Testosterone in Serum/Plasma– Testosterone, Free, Total, and Bioavailable– Dihydrotestosterone (DHT)

• Thyroid Hormones in Serum/Plasma– T3 (Triiodothyronine), Free– T4 (Thyroxine), Free

• Coming Soon an Expanded Thyroid Panel

lee.blum@nmslabs.comPhone: 1-800-522-6671

Lee Blum, Ph.D.