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TRANSCRIPT
ISBM_2013_P6.4 | page 1Manchester | 2013-09-09
A company of Bayer and LANXESS
Gabriele Leng and Wolfgang Gries Institute of Biomonitoring, Leverkusen, Germany
Holger KochInstitute for Prevention and Occupational Medicine, Bochum, Germany
Gabriele Leng and Wolfgang Gries Institute of Biomonitoring, Leverkusen, Germany
Holger KochInstitute for Prevention and Occupational Medicine, Bochum, Germany
DETERMINATION OF BIS(2-PROPYLHEPTYL)PHTHALATE (DPHP) EXPOSURE IN THE GENERAL POPULATION
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• plasticizer for technical applications
• substitution product for Di-(2-ethylhexyl)phthalate (DEHP)• alternative to Diisononylphthalate (DiNP) or Diisodecylphthalate (DIDP)
• Aim:
DPHP - APPLICATIONS
Development of an analytical method for the sensitive and selective determination of DPHP body burden (metabolites in urine)Examine which analytical way meets the tasks:
LC-MS/MS (in general standard method for phthalate metabolites) OR
GC-HRMS, GC-MS/MS
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DPHP METABOLISM
OH-MPHP: Mono-2-(propyl-6-hydroxy-heptyl)-phthalatecx-MPHxP: Mono-2-(propyl-6-carboxy-hexyl)-phthalateoxo-MPHP: Mono-2-(propyl-6-oxoheptyl)-phthalate
Bis(2-propylheptyl)phthalate
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DPHP – METHOD OVERVIEW GC-MS HPLC-MS/MS
1 ml Urine-enzymatic hydrolysis: + d4-internal standard; pH 6.5, glucuronidase-incubation
like GC-MS
Acidification at pH 2 with hydrochloric acid like GC-MS
Extraction with tert-butylmethylether like GC-MS
Evaporation in nitrogen to dryness like GC-MS
Disolution in acetonitrile Disolution in methanol
Selective derivatization on carboxylic group with 1,1,1,3,3,3- hexafluoro-2-isopropanol (HFIP) + N,N‘-diisopropylcarbodiimide
./.
1 M sodium hydrogen carbonate ./.
Extraction in iso-octan ./.
1 µL split-splitless; GC-MS-NCI detection 10 µL
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LOD AND LOQ
Analyte GC-HRMS* GC-MS/MS LC-MS/MS*
Cx-MPHxP 0.05 0.05 0.1
OH-MPHP 0.1 0.1 0.2
Oxo-MPHP 0.08 0.1 0.2
Analyte GC-HRMS* GC-MS/MS LC-MS/MS*
Cx-MPHxP 0.15 0.15 0.3
OH-MPHP 0.3 0.3 0.5
Oxo-MPHP 0.25 0.3 0.5
LOD (µg/L urine)*
LOQ (µg/L urine)**
* LOD: Calibration curve method** LOQ: 3 x LOD
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PRECISION DATA
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POOL URINE SPIKED WITH 1 µg/L OF EACH METABOLITE
LC-MS/MS GC-HRMS after HFIP-derivatization
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Aim: To examine if Cx-MPHxP, OH-MPHP and Oxo-MPHPare main DPHP-metabolites in humans To get information about the elimination kinetics of DPHP in humans
Performed by Holger Koch at IPAapproved by Ethical commission University of Bochum (registration number 4022-11, 24.5.2011)
5 male volunteersOral intake of about 50 mg D4-DPHP Urine collected during the first 48 hours after intake in intervals Analysis of DPHP metabolites
HUMAN ELIMINATION KINETIC STUDY
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• 24 % of applied dose was found during the first 48 h • main metabolite: oxo-MPHP > OH-MPHP >> cx-MPHxP
HUMAN ELIMINATION KINETIC STUDY
volunteer volunteer
0++ 0++0 – 24 h 0 – 48 h
Elim
inat
ion
% o
f dos
e
Elim
inat
ion
% o
f dos
e
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ELIMINATION OF OXO-MPHP-D4 IN 5 VOLUNTEERS
0
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Human elimination kinetic study:
DPHP-specific metabolites in urine: Major: Mono-2-(propyl-6-oxoheptyl)-phthalate (oxo-MPHP) Major: Mono-2-(propyl-6-hydroxy-heptyl)-phthalate (OH-MPHP)
Very minor: Mono-2-(propyl-6-carboxy-hexyl)-phthalate (cx-MPHxP)
DPHP-metabolites are eliminated within 48 h24 % of applied oral dose is found
LESSONS LEARNT SO FAR
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DiNP – Diisononylphthalate
DiNPs (DINP1 and DINP2) and DiDP consist of different isomers
DiDP – Diisodecylphthalate
DiNP-metabolites:7OH-Mono-methyloctylphthalate (OH-MiNP) 7oxo-Mono-methyloctylphthalate (oxo-MiNP) 7carboxy-Mono-methylheptylphthalate (cx-MiNP)
DiDP and DPHP – metabolites:OH-Mono-isodecylphthalate (OH-MiDP) oxo-Mono-isodecylphthalate (oxo-MiDP) Carboxy-Mono-isodecylphthalate (cx-MiDP)
DPHP and DIDP are both C10 phthalates that are not separated by HPLCDiNP1 contains branched C10 - side chains (up to 10 %)
FACTORS CONTRIBUTING TO FALSE POSITIVE DPHP VALUES
DINP1, CAS No. 68515-48-0, 1,2-benzenedicarboxylic acid, di-C8-10-branched alkyl esters, C9-richDINP2, CAS No. 28553-12-0, Diisononylphthalate
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DIDP/DPHP METABOLITES IN 12 WORKERS WITH PLASTISOL (DINP AND DIDP) EXPOSURE
HPLC-MS/MS GC-HRMS
Sum of cx Sum of OH Sum of oxo cx-MPHxP OH-MPHP oxo-MPHP
Median (µg/L) 15.7 44.9 6.1 <LOQ <LOQ 0.26
Mean (µg/L 28.3 127.7 16.9 <LOQ <LOQ 0.31
Range (µg/L) 2.1-99.7 7.7-337 1.1-49.2 <LOQ <LOQ <LOQ-0.72
Detection rate (%)
100 100 100 0 0 58
Samples provided by Holger Koch, IPA
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DPHP ANALYSIS BY LC-MS/MSChromatograms of urine sampleof DiDP/DiNP exposed person, overlaid with a 1 µg/L standard of DPHP metabolites
Result:sum of metabolites from DiDP + DiNP + DPHP is found, i.e. no selective determination of DPHP-body burden possible
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DPHP ANALYSIS BY GC-HRMS
Chromatograms of urine sampleof DiDP/DiNP exposed person, overlaid with a 1 µg/L standard of DPHP metabolites
Result:DPHP-metabolites can be separated from DiDP + DiNPmetabolites, i.e. selective determination of DPHP-body burden is possible
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40 urine samples of general population
Analysis done by GC/HRMS
Results: in 35 urine samples DPHP metabolites < LOQ
In 15 urine samples DPHP metabolites > LOQ – see next table
DPHP IN GENERAL POPULATION
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0
DPHP IN 15 SAMPLES > LOQ
oxo-MPHP - in all 15 samples (mean: 0.40 µg/l; max: 0.93 µg/l)
OH-MPHP - in 3 samples (mean: 0.41 µg/l)
cx-MPHxP - in 0 sample
Crea (g/L) Oxo-MPHP (µg/L)
OH-MPHP (µg/L)
Cx-MPHP (µg/L)
1.46 0.32 < 0.30 < 0.15
2.39 0.25 < 0.30 < 0.15
3.74 0.46 < 0.30 < 0.15
0.52 0.30 < 0.30 < 0.15
2.6 0.28 < 0.30 < 0.15
1.43 0.28 < 0.30 < 0.15
2.35 0.37 0.51 < 0.15
3.48 0.27 < 0.30 < 0.15
2.83 0.93 0.38 < 0.15
1.39 0.27 < 0.30 < 0.15
1.59 0.35 < 0.30 < 0.15
1.57 0.38 < 0.30 < 0.15
1.87 0.41 < 0.30 < 0.15
1.5 0.37 < 0.30 < 0.15
3.69 0.69 0.34 < 0.15
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CONCLUSIONS DPHP-ANALYSIS
HPLC-MS/MS only adequate for prescreening
GC-HRMS (or GC-MS/MS) necessary for selective DPHP analysis
Oxo-MPHP found in 15 out of 40 urine samples of general population,Low range
More information:Gries, Ellrich, Küpper, Ladermann, Leng: Analytical method for the sensitive determination of major di-(2-propylheptyl)-phthalate metabolites in human urine, Journal of Chromatography B, 908, 128-136, 2012
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Human kinetic study: Holger Koch, Institute for Prevention and Occupational Medicine of the German Social Accident Insurance –Institute for the Ruhr-Universität Bochum (IPA)
Method development: part of project on human biomonitoring as a cooperation between BMU (Federal Ministry for the Environment, Nature, Conservation and Nuclear Safety)
and VCI (German Chemical Industry Association)
Toxicological advise & project lead: Rainer Otter, BASF SE
ACKNOWLEDGEMENT
My biomonitoring team