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6.4 Leng

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  • 1. DETERMINATION OF BIS(2-PROPYLHEPTYL)PHTHALATE (DPHP) EXPOSURE IN THE GENERAL POPULATION Gabriele Leng and Wolfgang Gries Institute of Biomonitoring, Leverkusen, Germany Holger Koch Institute for Prevention and Occupational Medicine, Bochum, Germany ISBM_2013_P6.4 | page 1 Manchester | 2013-09-09 A company of Bayer and LANXESS
  • 2. DPHP - APPLICATIONS • plasticizer for technical applications • substitution product for Di-(2-ethylhexyl)phthalate (DEHP) • alternative to Diisononylphthalate (DiNP) or Diisodecylphthalate (DIDP) • Aim: 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 ISBM_2013_P6.4 | page 2 Manchester | 2013-09-09
  • 3. DPHP METABOLISM Bis(2-propylheptyl)phthalate OH-MPHP: Mono-2-(propyl-6-hydroxy-heptyl)-phthalate cx-MPHxP: Mono-2-(propyl-6-carboxy-hexyl)-phthalate oxo-MPHP: Mono-2-(propyl-6-oxoheptyl)-phthalate ISBM_2013_P6.4 | page 3 Manchester | 2013-09-09
  • 4. 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 ISBM_2013_P6.4 | page 4 Manchester | 2013-09-09
  • 5. LOD AND LOQ LOD (µg/L urine)* 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 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 LOQ (µg/L urine)** Analyte * LOD: Calibration curve method ** LOQ: 3 x LOD ISBM_2013_P6.4 | page 5 Manchester | 2013-09-09
  • 6. PRECISION DATA ISBM_2013_P6.4 | page 6 Manchester | 2013-09-09
  • 7. POOL URINE SPIKED WITH 1 µg/L OF EACH METABOLITE LC-MS/MS ISBM_2013_P6.4 | page 7 Manchester | 2013-09-09 GC-HRMS after HFIP-derivatization
  • 8. HUMAN ELIMINATION KINETIC STUDY Aim: To examine if Cx-MPHxP, OH-MPHP and Oxo-MPHP are main DPHP-metabolites in humans To get information about the elimination kinetics of DPHP in humans Performed by Holger Koch at IPA approved by Ethical commission University of Bochum (registration number 4022-11, 24.5.2011) 5 male volunteers Oral intake of about 50 mg D4-DPHP Urine collected during the first 48 hours after intake in intervals Analysis of DPHP metabolites ISBM_2013_P6.4 | page 8 Manchester | 2013-09-09
  • 9. HUMAN ELIMINATION KINETIC STUDY 0++ 0++ 48 h 0– Elimination % of dose Elimination % of dose 0 – 24 h volunteer volunteer • 24 % of applied dose was found during the first 48 h • main metabolite: oxo-MPHP > OH-MPHP >> cx-MPHxP ISBM_2013_P6.4 | page 9 Manchester | 2013-09-09
  • 10. ELIMINATION OF OXO-MPHP-D4 IN 5 VOLUNTEERS 0 ISBM_2013_P6.4 | page 10 Manchester | 2013-09-09
  • 11. LESSONS LEARNT SO FAR 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 h 24 % of applied oral dose is found ISBM_2013_P6.4 | page 11 Manchester | 2013-09-09
  • 12. FACTORS CONTRIBUTING TO FALSE POSITIVE DPHP VALUES DiNP – Diisononylphthalate DiDP – Diisodecylphthalate DiNPs (DINP1 and DINP2) and DiDP consist of different isomers 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 HPLC DiNP1 contains branched C10 - side chains (up to 10 %) DINP1, CAS No. 68515-48-0, 1,2-benzenedicarboxylic acid, di-C8-10-branched alkyl esters, C9-rich DINP2, CAS No. 28553-12-0, Diisononylphthalate ISBM_2013_P6.4 | page 12 Manchester | 2013-09-09
  • 13. 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 ISBM_2013_P6.4 | page 13 Manchester | 2013-09-09
  • 14. DPHP ANALYSIS BY LC-MS/MS Chromatograms of urine sample of 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 ISBM_2013_P6.4 | page 14 Manchester | 2013-09-09
  • 15. DPHP ANALYSIS BY GC-HRMS Chromatograms of urine sample of DiDP/DiNP exposed person, overlaid with a 1 µg/L standard of DPHP metabolites Result: DPHP-metabolites can be separated from DiDP + DiNP metabolites, i.e. selective determination of DPHP-body burden is possible ISBM_2013_P6.4 | page 15 Manchester | 2013-09-09
  • 16. DPHP IN GENERAL POPULATION 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 ISBM_2013_P6.4 | page 16 Manchester | 2013-09-09
  • 17. DPHP IN 15 SAMPLES > LOQ Crea (g/L) Oxo-MPHP (µg/L) OH-MPHP (µg/L) 0 CxMPHP (µg/L) 1.46 0.32 < 0.30 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 (mean: 0.40 µg/l; max: 0.93 µg/l) < 0.15 2.39 oxo-MPHP - in all 15 samples ISBM_2013_P6.4 | page 17 Manchester | 2013-09-09 OH-MPHP - in 3 samples (mean: 0.41 µg/l) cx-MPHxP - in 0 sample
  • 18. 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 ISBM_2013_P6.4 | page 18 Manchester | 2013-09-09
  • 19. ACKNOWLEDGEMENT 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 My biomonitoring team ISBM_2013_P6.4 | page 19 Manchester | 2013-09-09