6.4 Leng

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


• 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


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


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


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


PRECISION DATA


POOL URINE SPIKED WITH 1 µg/L OF EACH METABOLITE

LC-MS/MS GC-HRMS after HFIP-derivatization


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


• 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


ELIMINATION OF OXO-MPHP-D4 IN 5 VOLUNTEERS

0


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


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


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


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


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


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


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


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


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

6.4 Leng

Technology

Transcript of 6.4 Leng