Analysis of formaldehyde in blood of rats during and

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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