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Outline
Introduction Free formaldehyde in the presence of serum albumin and in blood HPLC-MS method development formaldehyde in blood Inhalation study with formaldehyde in rats
Introduction (1)
Formaldehyde (FA) is suspected of being associated with leukemia. Possible mechanism: FA enters the blood after inhalation and is able to reach the bone marrow.
The most occurring exposure route for FA is inhalation. Previous inhalation studies
have shown no raised FA level in the blood of exposed animals.
Introduction (2)
Assumption: either FA cannot enter the blood via inhalation or blood concentrations
return to their physiological value very fast (no concentration increase detectable).
Outline
Introduction Free formaldehyde in the presence of serum albumin and in blood HPLC-MS method development formaldehyde in blood Inhalation study with formaldehyde in rats
10 m 86
20 m 84
99 m 85
149 m 83
21 h 72
21 h 73
86
= minutes, h = hours.
Outline
Introduction Free formaldehyde in the presence of serum albumin and in blood HPLC-MS method development formaldehyde in blood Inhalation study with formaldehyde in rats
HPLC-MS method development formaldehyde in blood (1) Analytical challenge: FA is an endogenous compound. Exogenous and endogenous FA should be distinguished.
Solution
Labeled 13C-FA will be administered to rats during the inhalation study Carbon stable isotopes natural abundance
12 6C 13 6C
98.9% 1.1%
Proposed method: derivatization with DNPH and High Performance Liquid Chromatography Mass Spectrometry (HPLCMS) after hexane extraction. Deprotonated FA-DNPH (m/z 209) derivative will be detected.
ratio of the deprotonated DNPH derivative (m/z 209/210) will be determined using MS. Naturally this ratio is 10.8 and it will decrease when 13C-FA enters the blood via inhalation. Molecular formula and isotope pattern FA-derivative
Natural: C7H6N4O4
Labeled: [13C]1C6H6N4O4
HPLC-MS method development formaldehyde in blood (4) Analytical challenge: FA is rapidly metabolized and/or rapidly binds to FA-acceptors in blood => low recovery
Solution
Experimental procedure was optimized => Derivatization within 3 minutes Low recovery could not be solved completely, repeatability was optimized: Fast and repeatable sample preparation (next to exposed rats) Isotopic distribution is not affected by recovery
HPLC-MS method development formaldehyde in blood (5) Analytical challenge: sensitivity => exogenous (labeled) FA should be detected as sensitively as possible
Solution Addition of 13C-FA-DNPH signal to base natural FADNPH signal LOD for endogenous formaldehyde in blood 100 g/L LOD for exogenous formaldehyde in blood 30 g/L. Sensitive region for low fraction 13C-FADNPH label
Peak area ratio m/z 209/210 vs. Fraction 13C-label
12
Fraction 13C-label
Outline
Introduction Free formaldehyde in the presence of serum albumin and in blood HPLC-MS method development formaldehyde in blood Inhalation study with formaldehyde in rats
Kleinnijenhuis, A.J., Staal, Y.C.M., Duistermaat, E., Engel, R., Woutersen, R.A., 2012. The determination of exogenous formaldehyde in blood of rats during and after inhalation exposure (Submitted).
-Isotopic distribution at different concentration levels: RSD was 2.1% or lower from 11-433 g/L -Relative difference <5% when fraction was lower than 0.5
Fraction 13C-label
Before exposure: 2.7 mg/L During exposure 3 hrs: 2.6 mg/L During exposure 6 hrs: 2.0 mg/L After exposure 10 minutes: 2.1 mg/L After exposure 30 minutes: 1.8 mg/L
=> It is unlikely that FA enters the blood of rats after inhalation and as such may cause leukemia.
Acknowledgements TNO Triskelion: Roel Engel Yvonne Staal Hans Muijser Evert Duistermaat TNO: Ruud Woutersen FormaCare: Heinz-Peter Gelbke
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