Measuring Phthalate Metabolites in Biological Matrices

Transcription

1 Measuring Phthalate Metabolites in Biological Matrices Antonia M. Calafat rganic Analytical Toxicology Branch Division of Laboratory Sciences National Center for Environmental Health Workshop on Quality of Analytical Data in HBM November 30, 2011 Brussels, Belgium National Center for Environmental Health Division of Laboratory Sciences

2 verview Analytical Chemistry & Biomonitoring Quality Control/Quality Assurance Analyte vs Exposure Biomarker

3 Biomonitoring Assessment of internal dose by measuring the parent chemical (or its metabolite or reaction product) in human samples (e.g., blood, urine) Integrates all sources/routes of exposure Trace concentrations (vs environmental levels)

4 Biomonitoring & Analytical Chemistry Complex & expensive instrumentation State-of-the-art laboratory facilities Highly trained personnel Adequate analytical standards & reagents

5 ptimal Characteristics of an Analytical Method Sensitive Specific/Selective Accurate Precise/Reproducible Rugged Cost effective *Biomonitoring Minimal sample volume* Simple* Multianalyte* Compromise High throughput* Automation QA/QC program*

6 Analytical Steps Sample workup Deconjugation Preconcentration Extraction Separation Chromatography Quantification Isotope dilution mass spectrometry ther Matrix & chemical influence the choice of analytical method

7 Matrix and Chemical Influence Analytical Method Matrix Pre-concentration Environmental chemical Sample workup Deconjugation step Pre-concentration Separation HPLC vs GC Detection Mass spectrometry vs others

8 Pre-concentration Method Solid phase extraction (SPE) ff-line vs on-line Solid phase micro extraction (SPME) Accelerated Solvent extraction Supercritical fluid extraction Liquid-liquid extraction (LLE) thers

9 Advantages of on-line SPE In-house systems (additional valve & pump) Reduced sample volume Specific populations Reduced amount of reagents and solvents Decreased cost Decreased potential exposure to chemicals Decreased waste Minimal manual manipulation of samples Improved reproducibility Unattended operation Coupled to HPLC-MS/MS

10 ur current analytical method uses less sample, is more efficient and allows for higher throughput than our previous methods BEFRE Relatively large volume of sample & solvents/reagents Quite labor-intensive 1 ml sample NW ~1/10 volume Unattended operation 0.1 ml sample extraction extraction evaporation reconstitution 1 ml extract 0.1 ml injection injection 25 μl (250 μl sample) 100 μl

11 Quality Control/Quality Assurance Programs Internal procedures Blanks QCs (blind, bench, replicates) Deconjugation QCs Proficiency testing Internal Inter-laboratory comparisons

12 Quality Control Materials Matrix-matched specimens ne to three concentrations Spike (free) vs conjugated species Characterized to establish precision QC concentrations evaluated using modified Westgard rules

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14 Current Interlaboratory Programs G-EQUAS DEHP oxidative metabolites MnBP MiBP MBzP Environmental & occupational levels

15 Analyte vs Exposure Biomarker Many analytes can be measured Additional information is needed to demonstrate the utility of measured analytes as exposure biomarkers We do not measure exposures, but concentrations

16 Analytical Chemistry vs Biomonitoring Analyte Validated method Biomarker Adequate facilities & instrumentation Qualified personnel QA/QC (e.g., laboratory blanks) Available analytical standards Analyte metabolism & toxicokinetics Biomarker selection Variability in concentrations Matrix factors Sampling factors Timing/place of collection Field blanks

17 Biomarker & Matrix Selection Biomarker choice Most abundant/relevant compound for target population Minimize exposure misclassification Matrix choice Urine: non-persistent chemicals Blood: persistent chemicals ther matrices? Endogenous matrix components can affect the analytical results o Phthalates (esterases) Stability, collection issues Calafat and Needham. Int J Androl. 2008, 31(2):139-43

18 Selection of Exposure Biomarkers: DINP Example DINP metabolites: MNP (~2%) & MCP (~11%) MNP (minor metabolite): insensitive biomarker of DINP background exposures Unweighted number of participants (weighted percentage) MNP urinary concentrations MCP urinary concentrations Detectable Non-detectable Total Detectable 347(12.9%) 2 (0.02%) 349 (12.9%) Nondetectable 2100 (82.4%) 99 (4.7%) 2199 (87.1%) Total 2447 (95%) 101 (5%) Select most abundant/relevant biomarker to minimize exposure misclassification 82.4 % of persons classified as exposed to DINP are misclassified based on urinary concentrations of MNP only Calafat et al. EHP 2011, 119:50-5

19 Phthalate Metabolite Exposure to Phthalates Median urinary concentrations (µg/l) a NHANES NHANES NHANES NHANES NHANES DEP MEP DBP MBP/MiBP / / / /8.0 BBzP MBzP DEHP MEHP MEHHP MEHP MECPP DINP MCP MNP <LD <LD DIDP MCNP a N ~ 2600; representative population (6+ years ); spot sample;

20 Choosing the Appropriate Biological Matrix for Biomonitoring Chemical dependent Population dependent (age, health status, etc.) Laboratory instrumentation dependent

21 Matrix Urine for non-persistent chemicals Blood for persistent chemicals ther matrices?

22 D 4 -MEHP, ng/ml Endogenous Matrix Components can Affect the Analytical Results R R lipases H R P450 H R Spiked m ilk, untreated Spiked m ilk, H3P4 Spiked m ilk, HCH Spiked milk, TCCA Spiked w ater Unspiked m ilk Time, h Enzyme activity in milk, blood, meconium, amniotic fluid and saliva Acid to quench enzyme activity, but use involves safety concerns Calafat et al. J Chromatogr B (2004) 805:49-56.

23 Concentration (ng/ml) Matrix Influence on Biomarkers Measures Spiked urine and amniotic fluid analyzed for oxidative & hydrolytic phthalate metabolites Hydrolysis xidation DEHP MEHP MEHHP + others Urine A Urine B AF A Measure 1 Measure 2 MEHHP AF B Measure 1 Measure 2 MEHP Urine A Urine B Measure only oxidative phthalate metabolites AF A AF B

24 What About Meconium? Exploratory matrix Distribution of target analytes Collection protocols Avoid contamination of meconium with urine Non-persistent chemicals Set the time window of collection Meconium vs other Control meconium-diaper contact Wet/dry weight Comparisons of biomarkers concentrations in meconium in epidemiologic studies should be conducted cautiously Mainly semi-quantitative data Limited use

25 LSGM concentrations (µg/l) Daily variation of phthalate metabolite urinary concentrations mep mbp mbzp mehp 4 3 Silva et al. EHP 2000, 112: Morning Midday Evening

26 Concentration (µg/g creatinine) Variability in Biomarker Urinary Concentrations 8 adults (50% female) Collected all urine voids for 7 days Variability in concentrations of phthalate metabolites MEP (PCP use): between-person variability accounted for > 75% of total variance MEHHP (diet): within-person variability contributed 69 83% of total variance Spot samples intra-day variability: MEHHP (51%) MEP (21%) Preau et al. EHP 2010, 118(12): MEP S1 S2 S3 S4 S5 S6 S7 S MEHHP Day of week

27 Sampling Strategies Does a single sample adequately characterize an individual s average exposure for a given time period? 24-h vs spot collections Varies depending upon the biomarker, exposure scenario and the population studied For chronic exposures, probably For episodic exposures, maybe, depending upon type (e.g., diet), frequency and magnitude of exposure Time of collection and last urination for spot collections Age-related variability Can we overcome variability? Multiple urine collections per person Cost (storage, analysis) & compliance considerations Pooling several spot samples

28 Mean MBP urinary level (µg/l) Despite Variability, Biomonitoring Data Show Clear Exposure Differences: Case of Dibutyl Phthalate Medications taken chronically can contribute to DBP exposures that approaches the NAEL measured in animal studies 2000 Mesalamine users (N = 6) Non-users (N = 7874) DBP Hauser et al. EHP 2004, 112: ; Hernandez-Diaz et al. EHP 2009, 117:185-9

29 Phthalates metabolism Exposure DIESTER R R Hydrolysis Metabolism MNESTER R H Conjugation xidative metabolism R H Excretion (R/R ) H CH H H

30 Long & short term stability of glucuronide conjugates of phthalate metabolites in urine riginal level 4 o C (day 3) 25 o C (day 3) A Mean % glucuronide MEP MCPP MECPP riginal level -70 o C (1 yr, 4 FT cycles) -70 o C (1 yr) -70 o C (2 yr, 5 FT cycles) -70 o C (2 yr) -70 o C (3 yr, 6 FT cycles) -70 o C (3 yr) MEHP MBP MEHHP MEHP MBzP B MEP MCPP MECPP MEHP MBP MEHHP MEHP MBzP

31 DEHP metabolism C H 3 H 2 C H 2 C C H 3 H C H 2 C H (CH 2 ) 3 C H 3 C H 3 H C H 2 C H (CH 2 ) 3 C H 3 H C 2-Ethylhexanol C 2 2-Ethylhexanol C H 2 C C C H 2 C H (CH 2 ) 3 C H 3 C H 2 C H (CH 2 ) 3 C H 3 H C H 2 C H (CH 2 ) 3 C H 3 C H H H C C Mono(2-ethylhexyl)- C 2 phthalate Di(2-ethylhexyl)- C H 3 (MEHP) phthalate (DEHP) 5.9% Phthalic acid C H 3 C H 2 C H 2 H H C C H 3 H C H 2 C H 3 H C H 2 C H 3 H C H 2 C H 3 H R C H 2 C H (CH 2 ) 3 C H 3 2-(2-Hydroxyethyl)- hexylphthalate R C H 2 C H (CH 2 ) 3 C H 3 2-(1-Hydroxyethyl)- hexylphthalate R C H 2 C H (CH 2 ) 3 C H 2 2-Ethyl-6-hydroxyhexylphthalate R C H 2 C H (CH 2 ) 2 C H C H 3 2-Ethyl-5-hydroxyhexylphthalate (MEHHP) R C H 2 C H 23.3% C H 2 C H C H 2 C H 3 2-Ethyl-4-hydroxyhexylphthalate C H 2 R C H 2 C H CH (CH 2 ) 3 C H 3 2-Carboxymethylhexylphthalate (2cx-MMHP) HC R C H 2 C H (CH 2 ) 3 C H 3 2-Carboxy-hexylphthalate 4.2% C H 3 C R C H 2 C H (CH 2 ) 3 C H 3 2-(1-xyethyl)- hexylphthalate R C H 2 C H R C H 2 C H From Koch et al Arch Toxicol. 79: C H 2 C H 3 CH 2 2-Ethyl-3-carboxypropylphthalate C H 2 C H 3 CH (CH 2 ) 3 CH 2-Ethyl-5-carboxypentylphthalate (MECPP) C H 2 C H 3 R C H 2 C H 18.5% C H 2 C H 3 R C H 2 C H (CH 2 ) 2 CH 2-Ethyl-4-carboxybutylphthalate (CH 2 ) 2 C C H 3 2-Ethyl-5-oxyhexylphthalate (MEHP) C H 2 C H 3 R C H 2 C H 15.0% C H 2 C C H 2 C H 3 2-Ethyl-4-oxyhexylphthalate

32 Using Toxicokinetics to Explain Unusual Results Pilot using Avon Longitudinal Study of Parents and Children (ALSPAC) specimens collected in in the UK DEHP Metabolites NHANES (20+ years) Median (µg/l) Maternal ALSPAC pool (µg/l) a MEHP MEHHP MEHP MECPP [MEHP] ALSPAC ~ 50* [MEHP] NHANES [oxidative DEHP] ALSPAC ~ [oxidative DEHP] NHANES [MEHP] total (ALSPAC) ~ [MEHP] free (ALSPAC) Contamination with DEHP/MEHP during collection or storage a N =20 individual spot specimens; Holmes et al. Int.J.Child Health Hum. Develop. 2010, 3:85.

33 Exposure during Specimen Collection Pregnant women provided samples at time of delivery Phthalate metabolites in urine (median) [MEHP] ~ 25 * [MEHP] background levels [MEHHP, MEHP] ~ 5 * [MEHHP, MEHP] background levels [ther phthalate metabolites] unremarkable All women had an intravenous line for glucose, water & electrolytes balance support DEHP is a PVC plasticizer In this case, DEHP metabolites concentrations can t be used as surrogates for DEHP exposure during pregnancy Results replicated (Vandentorren et al. Environ Res. 2011) Yan X et al Hum Ecol Risk Assess 15:

34 Final Thoughts: Analytical Method Adequate instrumentation & facilities Maintenance Qualified & highly specialized/trained personnel Adequate analytical standards Quality control/quality assurance program Quality control Interlaboratory comparisons

35 Final Thoughts: Matrix & Biomarker Choice Persistence of biomarker Persistent (blood) Non-persistent (urine) Variability in concentrations for non-persistent chemicals Multiple samplings needed? Metabolism variation (e.g., age, genetics) Most representative biomarker Matrix composition may affect biomarker measures Matrix collection may affect biomarker measures Matrix availability at a given life stage Volume Ability to collect matrix (e.g., fetus access)

36 Final Thoughts: Collection & Storage Matter Even with validated analytical methods, toxicokinetics knowledge & adequate sampling Collection (how/when/where?) Hospital /medical settings Collection protocols (e.g., use of wipes, preservatives) Integrity of specimen Representativeness (absorbent collection materials) Cross-matrix contamination (meconium/urine; AF/blood) Stability of biomarker & matrix (e.g., conjugates, DBS) Contamination Measure trace levels of parent chemical (if ubiquitous & unidentified sources exist) Add analytes post-collection use of archived samples Field blanks, blind replicates Calafat and Needham EHP 117:1481-5

37 Take Home Messages Biomonitoring must use validated analytical methods Many analytes can be measured, but not all of them are good exposure biomarkers Used correctly, high-quality biomonitoring data will strengthen the exposure assessment

38 Carmen Dunbar Lily Jia Kayoko Kato Josh Kramer Freddy Lu Jim Preau Ella Samandar Manori Silva Sherry Ye Xiaoliu Zhou Acknowledgements Past group members Larry Needham

39 THANK YU! For more information please contact Centers for Disease Control and Prevention 1600 Clifton Road NE, Atlanta, GA Telephone, CDC-INF ( )/TTY: Web: The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. National Center for Environmental Health Division of Laboratory Sciences