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 [email protected] 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 13C-FA and monitor the isotopic ratio in blood to determine whether exogenous 13CFA 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 110 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. 0 9m 19 m 29 m 39 m 312 m 24 h 24 h 88 84 82 79 78 80 76 76 Time1 Recovery (%) Recovery of FA from a 2.62 mg/L FA solution in 45 g/L BSA in DPBS. 0 9m 19 m 29 m 39 m 178 m 22 h 22 h 88 84 82 82 82 81 73 74 Time1 Recovery (%) Recovery of FA from a 1.06 mg/L FA solution in 45 g/L BSA in DPBS. 0 10 m 20 m 99 m 149 m 21 h 21 h 86 86 84 85 83 72 73 Time1 Recovery (%) 1m = 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 13C-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 (HPLCMS) 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 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 • LOD for endogenous formaldehyde in blood ≈ 100 µg/L • LOD for exogenous formaldehyde in blood ≈ 30 µg/L. Sensitive region for low fraction 13C-FADNPH label Peak area ratio m/z 209/210 vs. Fraction 13C-label 12 Peak area ratio m/z 209/210 • Addition of 13C-FA-DNPH signal to base natural FADNPH signal 10 8 6 4 2 0 0 0.2 0.4 0.6 0.8 Fraction 13C-label 1 1.2 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 Peak area ratio m/z 209/210 vs. Fraction 13C-label 12 Peak area ratio m/z 209/210 -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 10 8 6 4 2 0 0 0.2 0.4 0.6 0.8 Fraction 13C-label 1 1.2 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) Peak area m/z 209 => 209 (arbitrary units) 6.0E+05 5.0E+05 4.0E+05 3.0E+05 2.0E+05 1.0E+05 0.0E+00 0 50 100 150 200 Concentration FA-DNPH, expressed as FA (µg/L) 250 300 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 13C-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 13C-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: Ruud Woutersen FormaCare: Heinz-Peter Gelbke ? TNO Triskelion: Roel Engel Yvonne Staal Hans Muijser Evert Duistermaat Questions?
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