1. Biomonitoring of 4,4´-methylenediphenyl diisocyanate
(MDI) with new specific biomarker MDA-Val-Hyd
Gabriele Leng and Wolfgang Gries
Institute of Biomonitoring, Leverkusen, Germany
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A company of
Bayer and LANXESS
2. MDI - AROMATIC ISOCYANATE
–N=C=O
Aromatic isocyanate: MDI
MDI (Diphenylmethan-4,4'-diisocyanat)
substance for the production of polyurethanes, special plastics, lacquer, glues,
paints, foams
Polymeric: foam for heat resistance materials, car seats
Monomeric: shoe sole, coatings, composition leather
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3. ISOCYANATES – HEALTH ASPECTS
Acute
• Irritations of skin and mucosa
• Sensitization of respiratory tract
Chronic
• Isocyanate asthma
Limiting values for MDI in Germany:
•
•
MAK: 50 µg/m³ air
BLW: 10 µg/L urine
Limiting values for MDI in UK:
•
Biological monitoring guidance value (BMGV) for isocyanates:
1 µmol/mol creatinine (ca. 4 µg/g creatinine)
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5. MDI – ANALYTICAL METHODS
1. Amines (urine):
•
•
Short term parameter, covers body dose of last 2.5 - 3.5 days
not isocyanate-specific as no differentiation between isocyanate and amine
2. Hemoglobin-adducts (blood):
•
•
•
Long term parameter, covers body dose of last 3 months
not isocyanate-specific as no differentiation between isocyanate and amine
bond at cystein
3. Albumin adducts (blood):
•
•
•
Long term parameter, covers body dose of last 3 weeks
Isocyanate specific
bond at lysin
4. Globin adducts (blood):
•
•
•
•
Long term parameter, covers body dose of last 3 months (cumulative)
Isocyanate specific
bond at valin; named: MDA-Val-Hyd
No human data available; knowledge derived from rats
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6. WHY IS SPECIFICITY SO IMPORTANT?
Fact: at many work places there is a potential simultaneous exposure to
the isocyanate MDI as well as to the aromatic amine MDA
(Methylenedianiline)
Problem: Both substances are very different from toxicological point of
view:
MDA – Focus cancer (cat. 2)
MDI – Focus sensitization / allergy
Urine marker shows sum of MDI + MDA body burden
Globin-marker shows selectively MDI – body burden
Therefore:
Globin-marker MDA-Val-Hyd is a specific marker for MDI!
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7. KNOWN FROM THR RAT
Globin-adduct (Valin)
Hemoglobin-adduct (Cystein)
MAK: 0.050 mg/m³!
Sabbioni, Chem. Res. 2000
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8. …AND HUMANS?
Questions to be answered:
Is MDA-Val-Hyd formed in humans following MDI-exposure?
In case it is, is it possible to measure it very sensitive?
Approach:
1. Development of a sensitive as well as robust analytical method for the
determination of MDA-Val-Hyd
2. Applications:
occupational medicine
environmental medicine
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9. GLOBIN ADDUCT OF MDI
O
HN
Synthesis of standard (Ehrenstorfer, Germany):
5-Isopropyl-3[4-(4-aminobenzyl)phenyl]hydantoin
N
O
ABP-Val-Hyd = MDA-Val-Hyd
NH
CD
HN
N
D
3
Synthesis of internal standard (Ehrenstorfer, Germany):
5-Isopropyl-3[4-(4-aminobenzyl)phenyl]hydantoin-d8
O
3
CD
CD
2
ABP-Val-Hyd-d8 = MDA-Val-Hyd-d8
O
NH
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2
10. METHOD DEVELOPMENT
Step
Details
Erythrocytes –
separation
Centrifugation of EDTA-blood sample, washing of
erythrocytes with NaCl, water adding
Hemolysis of
erythrocytes
- 20 °C
Globin-isolation
Solution is denaturated first with HCl/isopropanol, then
with ethylacetate
Acid hydrolysis
+ HCl, + ISTD (Aim: Cleavage of MDA-Val-Hyd from
terminal valin)
Extraction /
derivatization
Chloroform / Heptafluoro butyric acid anhydrid (HFBA)
Gaschromatography
Agilent GC HP 5890, Rtx 5 MS 20 m x 0.32 mm x 0.5 µm
Detection
Waters AutoSpec Ultima; GC-HRMS-NCI
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11. DETECTION OF MDA-Val-HYD
Substance
m/z
Rt (min)
MDA-Val-Hyd-HFBA
499.1331
ca. 8:50
MDA-Val-Hyd-HFBA-d8
507.1833
ca. 8:50
LOQ: 0.05 ng/g MDA-Val-Hyd/l blood (0.155 pmol/g globin)
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12. PRECISION DATA OF MDA-HYD
56 spiked globin samples, analyte concentrations: 0.1, 0.5, 1.0 and 2.0 ng/g:
Precision in series:
relative standard deviation between 3.0 and 9.3 %
Precision from day to day (on 5 days):
relative standard deviation between 8.3 and 9.8 %
Fact: excellent precision data due to application of deuterated internal
standard (similar fragmentation, compensation of analytical variabilities)
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16. BACKGROUND LEVELS
1. No known exposure:
40 persons without any known MDI-exposure
Biomonitoring < LOQ
2. Low inhalative exposure:
Application with protection clothes of one-component-PU-foam (containing
10 % free MDI) for 20 minutes; no dermal contact; only inhalative
2 - 5 µg MDI/m³ air (MAK: 50 µg/m³)
Biomonitoring < LOQ
3. Provocation experiments:
3 - 20 µg MDI/m³ air
Biomonitoring > LOQ
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17. SUMMARY MILESTONES
1. Development of a sensitive and robust analytical method (GC-HRMS)
successfully managed
2.
Specific MDI-marker in humans is MDA-Val-Hyd
• Blood marker (adduct) covering body burden of the last 3 months
• Differentiation between isocyanate and aromatic amine body burden
1. Application in MDI-exposed workers: MDA-Val-Hyd detected
2. Background level of MDI
No
More information in our publication:
Gries, Leng: Analytical determination of specific 4,4´-methylene diphenyl diisocyanate hemoglobin adducts in human blood.
Analytical and Bioanalytical Chemistry (published online), 2013
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18. ACKNOWLEDGEMENT
Synthesis of reference standards:
International Isocyanate Institute
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)
My biomonitoring team
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