Analysis of formaldehyde in blood of rats during and after inhalation exposure Sponsor: Research laboratory: P.O. Box 844 3700 AV Zeist The Netherlands Presenter: Dr. A.J. Kleinnijenhuis T +31 88 866 28 00 info@tno.triskelion.nl www.triskelion.nl
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. FormaCare requested TNO Triskelion to investigate whether FA can enter the blood stream via inhalation. FA is an endogenous compound with a concentration of 2-3 mg/l in blood and has a short half life. 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). TNO Triskelion developed a novel, sensitive method for selective detection of exogenous FA in blood. To distinguish exogenous FA and endogenous FA it was decided to expose rats to 13 C-FA and monitor the isotopic ratio in blood to determine whether exogenous 13 C- FA had entered the blood after inhalation exposure. Additionally, free FA was determined in the presence of serum albumin and in blood.
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
Free formaldehyde in the presence of SA (1) Approach for determination of free FA: Separation of free and bound FA on an analytical High Performance Liquid Chromatography (HPLC) column Post-column derivatization with acetyl acetone UV-detection of the derivate The recovery of FA from physiological saline was 100-104 % at 1-10 mg/l level with RSD < 1%. Due to good recovery and repeatability method is suitable for quantitative analysis. Free FA in the presence of protein was determined. FA can form adducts with proteins. Serum albumin => most abundant protein.
Free formaldehyde in the presence of SA (2) Free FA in the presence of bovine serum albumin (BSA): The concentration of FA was 1-5 mg/l The concentration of BSA was 45 g/l in physiological saline The mixtures were incubated for 24 hours At t=0 the recovery was 88% and gradually decreased to 75% after 24-hr incubation. => Up to 25% of the FA binds to BSA
Free formaldehyde in the presence of SA (3) Recovery of FA from a 5.11 mg/l FA solution in 45 g/l BSA in DPBS. Time 1 0 9 m 19 m 29 m 39 m 312 m 24 h 24 h Recovery (%) 88 84 82 79 78 80 76 76 Recovery of FA from a 2.62 mg/l FA solution in 45 g/l BSA in DPBS. Time 1 0 9 m 19 m 29 m 39 m 178 m 22 h 22 h Recovery (%) 88 84 82 82 82 81 73 74 Recovery of FA from a 1.06 mg/l FA solution in 45 g/l BSA in DPBS. Time 1 0 10 m 20 m 99 m 149 m 21 h 21 h Recovery (%) 86 86 84 85 83 72 73 1 m = minutes, h = hours.
Free formaldehyde in the presence of SA (4) Free FA in the presence of rat serum albumin (RSA): The concentration of FA was 1 and 10 mg/l (n=5) The concentration of RSA was 45 g/l The mixtures were incubated for 24 hours At t=0 the recovery was 95% with RSD < 4% and was still >90% after 24-hr incubation. => FA hardly binds to RSA (< 5%)
Free formaldehyde in the presence of SA (5) Free FA in the presence of human serum albumin (HSA): The concentration of FA was 1 and 10 mg/l (n=5) The concentration of HSA was 45 g/l The mixtures were incubated for 24 hours At t=0 the recovery was 77% with RSD < 5% and was 50% after 24-hr incubation. => Up to 50% of the FA binds to HSA
Free formaldehyde in the presence of SA (6) Unexpectedly, there is a difference in FA binding between BSA, RSA and HSA Binding independent of FA concentration RSA hardly binds FA, BSA up to 25% and HSA up to 50% Possible explanation: Structural differences, although tertiary structure is highly conserved Different posttranslational modifications
Free formaldehyde in blood (7) The FA concentration in blank rat blood plasma was low. The FA concentration in blank blood plasma and hemolysate slightly decreased during 1-hr incubation at RT Whole blood, blood plasma and hemolysate were fortified with 5 mg/l FA and incubated for 1 hr at RT. Fortified FA was relatively stable in blood plasma. Fortified FA concentration rapidly decreased in hemolysate. After fortification of whole blood, no free FA was detected in blood plasma or hemolysate. FA seems to rapidly migrate to the red blood cells and be degraded/bound much faster than the already present endogenous FA.
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 13 C-FA will be administered to rats during the inhalation study Carbon stable isotopes natural abundance 12 6C 98.9% 13 6C 1.1%
HPLC-MS method development formaldehyde in blood (2) HPLC-MS Proposed method: derivatization with DNPH and High Performance Liquid Chromatography Mass Spectrometry (HPLC- MS) after hexane extraction. Deprotonated FA-DNPH (m/z 209) derivative will be detected.
HPLC-MS method development formaldehyde in blood (3) To distinguish exogenous and endogenous FA in blood the isotopic 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 13 C-FA enters the blood via inhalation. Molecular formula and isotope pattern FA-derivative Natural: C 7 H 6 N 4 O 4 Labeled: [ 13 C] 1 C 6 H 6 N 4 O 4
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 13 C-FA-DNPH signal to base natural FA- DNPH signal LOD for endogenous formaldehyde in blood 100 µg/l LOD for exogenous formaldehyde in blood 30 µg/l. Sensitive region for low fraction 13 C-FA- DNPH label Peak area ratio m/z 209/210 12 10 8 6 4 2 0 Peak area ratio m/z 209/210 vs. Fraction 13C-label 0 0.2 0.4 0.6 0.8 1 1.2 Fraction 13C-label
HPLC-MS method development formaldehyde in blood (6) Recovery from physiological saline was between 89 and 93% at 1-10 mg/l FA concentration with RSD < 3%. Recovery from physiological saline containing 45 g/l rat serum albumin (RSA) was between 75 and 79% at 1-10 mg/l FA concentration with RSD < 6%. Initially fortified FA was not recovered from blood at all or very variable results were obtained using different approaches. The most critical sample preparation parameter that was identified: Time needed for each sample preparation step
HPLC-MS method development formaldehyde in blood (7) Final method: Add 200 µl whole blood to 500 µl EDTA solution (anti-coagulant and hemolysis) Add 200 µl hemolysate to 5 ml 2,4-DNPH solution in acetonitrile containing perchloric acid React for 15 min at RT and vortex Centrifuge 10 min at 1500 rpm Analyze with HPLC-MS Total dilution: 91 times dilution The recovery of FA from whole blood was 35-45 % at 2-6 mg/l level with RSD < 7%. Due to good repeatability method is suitable for quantitative analysis
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
Inhalation study with formaldehyde in rats (1) HPLC-MS method validation and an inhalation study with FA in rats were performed under GLP (Good Laboratory Practice) Add 200 µl whole blood to 500 µl EDTA solution (anti-coagulant and hemolysis) Add 200 µl hemolysate to 5 ml 2,4-DNPH solution in acetonitrile containing perchloric acid React for 15 min at RT and vortex Centrifuge 10 min at 1500 rpm Analyze with HPLC-MS 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).
Inhalation study with formaldehyde in rats (2) Method validation set up (isotopic distribution): -Isotopic distribution determined for 6 different rats: 10.91 (SD 0.37) -Two times one side 95% confidence interval: 2 x 1.96 x SD: -Significantly different isotopic ratio: 9.46 (fraction between 1 and 1.5%). -Fraction 1%: theoretic isotopic ratio 9.77 -Fraction 1.5%: theoretic isotopic ratio 9.32 -Isotopic distribution at different concentration levels: RSD was 2.1% or lower from 11-433 µg/l -Relative difference <5% when fraction was lower than 0.5 Peak area ratio m/z 209/210 12 10 8 6 4 2 Peak area ratio m/z 209/210 vs. Fraction 13C-label 0 0 0.2 0.4 0.6 0.8 1 1.2 Fraction 13C-label
Inhalation study with formaldehyde in rats (3)
Inhalation study with formaldehyde in rats (4) Method validation set up (FA quantification in blood): Linear calibration curve 2.5-250 µg/l (r > 0.996) Recovery FA 35-45% Repeatability < 7% from endogenous 6 mg/l fortification level Low recovery reflects amount of free FA in blood, no correction Calibration curve FA-DNPH (HPLC-MS) 6.0E+05 Peak area m/z 209 => 209 (arbitrary units) 5.0E+05 4.0E+05 3.0E+05 2.0E+05 1.0E+05 0.0E+00 0 50 100 150 200 250 300 Concentration FA-DNPH, expressed as FA (µg/l)
Inhalation study with formaldehyde in rats (5) Method validation set up (Stability FA in blood extract): During storage at 2-10 C the concentration of FA in blank blood extract increased 60% Fortified FA concentration did not increase (relative difference within 10%) Co-extracted FA donors from blood that reacted with excess reagent? The FA concentration in study samples was corrected for the residence time in autosampler (storage time).
Inhalation study with formaldehyde in rats (6) Inhalation study set up: Sprague Dawley rats (12 wks old) were exposed nose-only for 6 hrs to 10 ppm FA in air. Mean body weight 401 g. Blood was sampled from the tail tip before (t=0), during (t = 3 and 6 hrs) and after exposure (10 and 30 minutes), n=10. Samples were prepared for analysis using the validated method next to the exposed rats. After exposure the rats were decoupled from the exposure unit and coupled to clean air unit. Simulated air samplings were taken to dismiss possible 13 C-FA contamination via the air. The FA air concentration was monitored.
Inhalation study with formaldehyde in rats (7) Results: The air concentration was as expected. Temperature was 22.2 C (SD 1.0) and RH was 54.0 % (SD 3.5). The determined isotopic ratios did not differ significantly from validation set (10.91): Before exposure: 10.66 During exposure 3 hrs: 10.96 During exposure 6 hrs: 10.85 After exposure 10 minutes: 11.07 After exposure 30 minutes: 10.70
Inhalation study with formaldehyde in rats (8) Results: There was no increase in blood FA concentration during exposure. The overall mean determined concentration was 2.3 mg/l. 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
Inhalation study with formaldehyde in rats (9) Discussion and conclusion: The analytical method was capable of detecting 1.5% exogenous 13 C-FA (30 µg/l at endogenous 2 mg/l blood FA concentration) using the isotopic ratio of the DNPH derivative m/z 209/210 Less than 1.5% of the FA present in rat blood was exogenous during and after exposure No effect was detected of inhalation exposure to 10 ppm FA. => 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? Questions?