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Journal of Analytical Toxicology, Vol. 31, April 2007
Measurement of Aflatoxin and Aflatoxin Metabolites in Urine by
Liquid Chromatography-Tandem Mass Spectrometry"*
Robert A. Everley 1,2, Frederic t. CineP, Dongliang Zhang 1,
Peter F. ScholP, John D. Groopman 3, and Timothy R. Croley 1,2,*
1Commonwealth of Virginia, Division of Consolidated Laboratories,
600 N. 5th Street, Richmond, Virginia 23219; 2Virginia Commonwealth
University, Department of Chemistry, 1001 W. Main Street, P.O. Box
842006, Richmond, Virginia 23284-2006; 3Department of Environmental
Health Sciences, Bloomberg School of Public Health, Johns Hopkins
University, Baltimore, Maryland 21205-2103
I Abstract
Automated immunoaffinity solid-phase extraction followed by
liquid chromatography-tandem mass spectrometry and chemical
analogue internal standardization is employed to detect and
quantify the aflatoxins AFB1, AFB2, AFG1, AFG2, and the metabolites
AFM1 and AFP 1 in urine. The dynamic range of the method is nearly
three orders of magnitude with limits of detection in the low
femtogram on column range. The method was validated over a 12-day
period by eight analysts. This method is suitable for agricultural,
forensic, and public health laboratories during an accidental
outbreak or a chemical terrorism event where a rapid and accurate
diagnosis of aflatoxicosis is needed.
Introduction
Because of the ever increasing threat of terrorist attacks
around the globe and, more specifically, the threat of a chem- ical
terrorism attack, analytical chemistry laboratories that would aid
in forensic investigations and public health domains must be
prepared to provide quality laboratory results quickly and
efficiently. In such an event, the number of victims could be large
and the type of warfare agent may not be immediately obvious. To
this end, analytical methods that can provide rapid and sensitive
confirmation and quantitation of the agent are vital in determining
which agent is used, each individual's de- gree of exposure, and
the extent of the population that is ex- posed (1).
* Results from this research were presented at the 54th ASMS
Conference on Mass Spectrometry. t Author to whom correspondence
should be addressed: Timothy R. Croley, Commonwealth of Virginia,
Division of Consolidated Laboratories, 600 N. 5th St. Richmond, VA
23219. E-mail: [email protected].
Of the many toxins that could be used in an attack, those
previously weaponized are of particular interest. One example is
aflatoxins, which were weaponized by the Iraq government during the
first Gulf War. According to a United Nations Spe- cial Commission
(UNSCOM) report, the Salman Pak weapons facility in Iraq produced
2200 L of aflatoxins loaded in 122-mm rockets, 400-1b bombs, and
SCUD missiles (2).
Aflatoxins are secondary metabolites of the fungi Aspergillus
flavus, from which their name (A. fla.) is derived. The pre-
dominant aflatoxins AFB1, AFB2, AFG1, and AFG2 are desig- nated B
and G because of their blue and green fluorescent color observed
under UV illumination. The most studied and most hepatotoxic
aflatoxin is AFB1 (LDs0 1.16 mg/kg in rat) (3), for which the World
Health Organization (WHO) suggests there is no safe dose (4).
Moreover, these compounds are known to be mutagenic and
teratogenic. Clinical symptoms of aflatoxin exposure include
abdominal pain, rash, and gas- trointestinal bleeding (5,6). The
commonality of these symp- toms with those seen in other illnesses
prevent them from providing unambiguous identification of their
cause, which further emphasizes the need for an analytical method
that provides more definitive information and enables a conclusive
diagnosis.
The work presented here builds upon previous investigations of
aflatoxins in urine (7,8) in an effort to improve both the sen-
sitivity and dynamic range of those methods. To improve upon the
speed of previous methods, an automated immunoaffinity solid-phase
extraction (SPE) method has been developed in conjunction with
liquid chromatography-tandem mass spec- trometry (LC-MS-MS)
analysis to take advantage of the sen- sitivity, specificity, and
ease of quantitation the technique provides. Figure 1 shows the
structures for the four parent aria- toxins of interest and the
metabolites of AFB1 that were chosen for this study.
150 Reproduction (photocopying) of editorial content of this
journal is prohibited without publisher's permission.
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Journal of Analytical Toxicology, Vol. 31, April 2007
Experimental
Chemicals and materials High-performance liquid chromatography
(HPLC) grade
methanol, acetonitrile, and formic acid were purchased from
Fisher Scientific (Fairlawn, N J). Deionized water was purified in
house to yield organic-free 18.3 M I x cm water using an E- pure
purification system (Barnstead International Dubuque, IA).
Aflatoxin reference standards (AFB1, AFB2, AFG1, AFG2, AFM1, and
AFP 1) were purchased from Sigma-Aldrich Chem- ical Company (St.
Louis, MO).
Standard preparation and characterization Aflatoxins and the
internal standard AFB2 were dissolved in
acetonitrile and diluted in 85:15 MeOH/H20 (v/v) to a final con-
centration of 1 ng/pL. Eight calibration standards, low and high
quality controls (QCs), and a urine blank containing in- ternal
standard were stored in 50-mL polypropylene conical tubes at 4~
Calibration standards were prepared in 1 mL of pooled human urine
spiked with 25 pL of the stock solution of AFB2. To all urine
samples (unknowns, QCs, blanks, and stan- dards) an equal amount of
85:15 MeOH/H20 was added. For ex- ample, the highest concentration
standard was made by mixing (per 1 mL of urine) 250 pL of the 1
ng/pL aflatoxin mixture, and 25 pL of the 1 ng/pL internal standard
for a total volume of 1.275 mL. To the other samples the same
amount of urine and internal standard were used, but with varying
amounts of aflatoxin standard and 85:15 MeOH/H20 while maintaining
a total volume of 1.275 mL. The final concentrations of the urine
standards were: 0.392, 0.784, 3.92, 7.84, 19.6, 58.8, 118, and 196
ng/mL for the calibration standards, 1.96 and 157 ng/mL for the QC
low and high, respectively, and the internal standard concentration
in all samples was 19.6 ng/mL.
Extraction Urine (1 mL) and water (1 mL) were added to glass
tubes (10
x 75 ram), and empty tubes of the same size (for elution)
were
AFBI AJ"~
^F01 AF(J2
AI~ AFp I
Figure I. Structures of the four parent aflatoxins and the two
metabolites of AFBI monitored in this study.
inserted into the Gilson 215 Liquid Handler (Middleton, WI) for
automated extraction. Custom-made immunoaffinity columns, (3-mL
barrel, 400 ng aflatoxin equivalent binding capacity, Vicam,
Watertown, MA) were used for the extraction. The buffer was
discarded followed by rinsing the column twice with water, leaving
a small amount of water on top of the resin. The columns were then
conditioned with water (2 x 2 mL). After the diluted urine sample
was loaded, the column was washed with water (2 x 2 mL), followed
by an air push (3 s) using the Gilson solenoid valve. The analytes
were eluted from the columns with 85:15 MeOH/H20 with 1% (v/v)
formic acid (2 x 0.5 mL), followed by an air push (30 s). The
extract was then transferred to an autosampler vial for LC-MS-MS
analysis.
LC-MS--MS Chromatography was performed with an Agilent 1100
HPLC
(Wilmington, DE) equipped with a 3-pro, 2.0- x 150-ram
phenyl-hexyl column (Phenomenex, Torrance, CA) at 50~ Injections of
the extract (1 pL) were made on the HPLC using a mobile phase
consisting of H20 (0.1% formic acid) (solvent A) and acetonitrile
(0.1% formic acid) (solvent B). The mobile phase gradient is given
in Table I.
Samples were analyzed by positive ion electrospray-MS-MS
spectrometry operating in multiple reaction monitoring (MRM) mode
on an API 4000 LC-MS-MS system (Applied Biosystems, Foster City,
CA). The MS settings are listed in Table II. Individual compound
specific parameters (i.e., declus- tering potentials, entrance
potentials, and collision cell exit po- tentials) were optimized
for each analyte.
Data analysis and recovery The product ion abundances of the
analyte and internal stan-
dard were used to calculate analyte/internal standard ratios for
quantitation. Linear regression analysis with "l/x" weighting was
used for curve fitting. All data processing was performed
Table I. Instrument Parameters for LC
Time (min) Rate (mL/min) %B
0.00 0.325 30 10.00 0.325 30 10.10 0.325 95 11.50 0.325 95 11.51
0.325 30 15.50 0.325 30
Table II. Instrument Parameters for MS
Analyte Precursor CE Product
AFP1 299 33.0 271 AFM1 329 33.0 273 AFG 2 331 35.0 313 AFB 2 315
37.0 287 AFG 1 329 39.0 243 AFB 1 313 33.7 285
151
-
automatically using the Analyst 1.4 software (Applied Biosys-
tems). Extraction efficiencies were calculated as a percentage of
the ratio of extracted analyte peak area and the non-extracted
standard peak area. Four extractions over a period of two days were
performed for this study, with all at a concentration of 7.84
ng/mL. Four injections of non-extracted standard at an equal
concentration were also made. The peak areas for the extracted and
non-extracted samples were averaged and this average was used to
calculate the ratio for each analyte.
Animal study Urine samples were obtained from two male F344
rats
(173-176 g body weight). AFB1 (91 pg/kg body weight) or the
vehicle (dimethylsulfoxide) were administered by intraperi- toneal
injection (150 IJL) on two consecutive days, and the rats were
housed in metabolic cages. Urine was collected for ap- proximately
18 h after the second dose and stored at -20~ Urine aliquots (1 mL)
were treated with 250 pL of 85:15 MeOH/H20, and 1 mL of this
mixture was then extracted. The animal study was conducted in
accordance with Johns Hopkins University's Animal Care and Use
Committee requirements, which comply with the National Research
Council's Guide for the Care and Use of Laboratory Animals.
Results and Discussion
The parent aflatoxins AFB], AFB2, AFG], and AFG2, and two phase
I oxidative metabolites of AFB], AFM], and AFP] (9), were selected
for analysis. Both AFP1 and AFM] are excreted in human urine (10)
and studies have demonstrated that AFM] is the most abundant AFB1
metabolite found in the urine of rats and humans (11). Furthermore,
research involving AFM1 has shown that urinary AFM1 levels reflect
exposure in humans (12). Metabolites of AFB2, AFG1, and AFG2 were
not included in this study because these three compounds are
generally not observed in the absence of AFB1, and AFB1 is the most
oc- current aflatoxin found in food (13).
Urine (vs. blood or serum) was chosen as the sample matrix
primarily due to its ease of collection. If victims are in rea-
sonably stable condition, they can provide a urine sample without
the assistance of a medical professional. Minimizing the workload
of hospital staff is critical in a scenario such as a terrorist
attack when hospitals will likely be overwhelmed.
AFB2 was chosen as a chemical analogue internal standard because
it behaves similarly to the other aflatoxins both in the
immunoaffinity column (IAC) and in the analytical method. In
addition, AFB2 is chromatographically well-resolved from the other
analytes and less toxic than AFB1 and AFG]. Chemical analogue
internal standardization was employed for two rea- sons. First,
isotopically labeled standards were only available for AFB2 and
were prohibitively expensive (~ $2000 for 8.3 IJg AFB2 3H). Second,
the upper limit of linearity (ULOL) was de- termined by occupying
the available binding sites of antibody in the column. The use of
isotopically labeled internal standards for each aflatoxin would
result in more aflatoxin being added to the immunoaffinity column,
which would lower the ULOL and,
152
Journal of Analytical Toxicology, Vol. 31, April 2007
therefore, decrease the dynamic range of the method. If AFB2
were in a real world sample, a t-test could be per-
formed to determine if the internal standard peak areas in un-
known samples are significantly higher than the mean AFB2 peak area
in the calibration standards. If this were the case, then because
all of the compounds of interest are equally suit- able as chemical
analogue internal standards, an aflatoxin not present in the sample
would be added to a separate aliquot of unknown and then
re-extracted. A second alternative would be to measure the
concentration of AFB~ by the method of stan- dard additions.
However, both of these methods would be dif- ficult if the
concentration of AFB2 was at or near the limit of detection (LOD),
so a third alternative, analyzing each un- known in duplicate (one
with internal standard, one without), could be employed.
Immunoaffinity extraction of aflatoxins in various matrices has
been reviewed (14) and involves non-covalent binding of the toxins
to monoclonal antibodies in aqueous environment followed by release
upon denaturing of the antibodies using high organic content
solvents. After a comparison with C18 SPE in this laboratory, the
immunoaffinity method was chosen because of its increased recovery,
selectivity, and cleanliness of extracts. Because the extraction
columns were originally man- ufactured for food analysis, the
antibody used was designed to target only the parent aflatoxins;
however, because of the struc- tural similarities between the
parent and metabolites, the metabolites were efficiently extracted
as well. For this reason, the columns could readily be used for
clinical samples.
Table III. Extraction Efficiency for all Five Analytes Measured
at 7.84 ng/mL in Urine (n = 4)
Analyte Extraction Efficiency (%)
AFB 1 87.4 + 8.2 AFM1 81.9 + 3.0 AFP~ 80.3 _+ 4.9 AFG1 93.2 _+
8.5 AFG 2 84.5 _+ 8.8
2~4
2~e4
2~4
b4
t 4e4
60WO
4~00
2~00
AFM~
] AFBI I AFG' I
AFP I | AFG:
s '
:~ :~e: : : : : : : : : : : : : : : : : : : : : :fa :;6 :g/~ u~e
::tra:
-
Journal of Analytical Toxicology, Vol. 3t, April 2007
Automated extraction was utilized as a means to increase sample
throughput. The extraction took 1.67 min/sample, cor- responding to
36 samples/h. The efficiency of the automated extraction was
measured at a concentration of 7.84 ng/mL in urine. The extraction
efficiencies for each of the five com- pounds are shown in Table
III. The results ranged from 80 to 93% and were in agreement with
manufacturer specifications, which were defined for food
matrices.
The best chromatographic resolution was obtained using a
phenyl-hexyl column. Figure 2 depicts the separation of all six
aflatoxins used in this study extracted fromhuman urine at a
concentration of 19.6 ng/mL. The MS-MS fragmentations of the
aflatoxins are shown in Figure 3. It can be seen that both B
aflatoxins and the AFB1 metabolites fragment by losing the carbonyl
on the cylcopentanone group, and that AFMt further breaks down by
losing the two adjacent CH2 groups on the
/31~ ,114,
'9 A '
~Oo4i , J~ i
Slt~I ,,3~+
+t.. ,,,++ '."+ .. + t , - i
t,.,++ I: , + ,+.++ ,~~ ,,~,+ I, ,m,,j
+ mPo.~ + "-0+ ~,, T ,+:
11 ...................... J.._+w.'l.
................................ ~ ............................. "
1+
D
m, l l
~,., ~+..++. m. .~ ~, m . - mo m-++. . .+ m~"
iil I~I.O!
-+I ii i' | + ,+ ++++., + ++T+ ,++ +.+ , . ++, + +
+_.++...++,J++,. ++,. +.
im~M)
:+ l' E ,"~ / ,..t /
:=i/ ! i= t:.: l / " ,.,,-i I
10~oJ I
=i / , '~t / ",
~+-m+. ~+++- +.+ ++..~.....-..++--,++-- +...+++,+.+.+.++.....
+..++.+.++..+ +..++ . ,~(Nm)
,-! C .... + F J ,.!
9 [ 11~4 i
|+ i ! '-~ - ,-i ! -~ Mm.~
1~01 i ~ mr , 1~118 ~ i I 1 i i~ o ml _ ~ ___
Figure 3. MS-MS fragmentation spectra for each aflatoxin with
arrows indicating the precursor ion: AFB; (A), AFB+ (B), AFG1 (C),
AFG+ (D), AFM1 (E), and AFP; (F).
153
-
ring. The G aflatoxins differ by containing a lactone group in
place of the pentanone ring structure. These compounds frag- ment
by the loss of water and the further loss of the outer car- bonyl
group and adjacent portion of the ring, as well as the loss of H2.
The fragmentation patterns suggest that the site of pro- tonation
for each aflatoxin was the inner carbonyl group. These
fragmentation patterns were based upon interpretation of the MS-MS
spectra because isotopically labeled experiments were not performed
for reasons stated previously.
The instrument LOD (S/N = 3:1) for each compound is given in
Table IV. LOD values were reported in femtograms (fg) on column to
avoid ambiguity inherent in using units of concen- tration such as
parts per billion (ppb). This ambiguity made it difficult to
determine a relative LOD in papers where the per- tinent
information to calculate the LOD on column was not in- cluded
(15,16). By providing the LOD as fg on column, the values reported
in this manuscript are independent of the volume of urine extracted
and/or injection volume, and they re- flect the amount of sample
that was detected by this method. Table IV shows the LOD for each
analyte in terms of grams on- column and in moles. An LOD of 100 fg
is equivalent to a 1-~L injection of non-extracted standard at 100
fg/l~L. These values meet the goal of developing a sensitive method
as these detec- tion limits are more than 10 times lower than those
previously reported (17,18) for aflatoxins in urine. The limit of
quantita- tion (LOQ) (S/N = 10:1) was calculated in urine using the
same procedure stated previously. The LOQ was determined to be 392
fg on-column for all analytes.
A wide dynamic range is needed in the analysis of chemical
Table IV. LOD for Each Analyte Defined as a Signal-to- Noise of
3:1
Analyte Femtograms on Column Attomoles
AFP~ 50 168 AFM~ 100 305 AFG2 100 302 AFG~ 100 305 AFBI 50
160
i LIk 5
Concentration (n~/raL) Figure 4. An eight-point calibration plot
from 0.392 to 196 ng/mL in urine using linear regression with "l/x"
weighting.
Journal of Analytical Toxicology, Vol. 31, April 2007
warfare agents for two reasons. First, because there are no
studies in the literature showing the range of concentrations in a
victim exposed to weaponized aflatoxin, having a dynamic range of
nearly three orders of magnitude increases the likeli- hood that
this method will cover relevant concentrations. Second, the
exposure of victims after an event will not be uni- form because of
their different proximities to the attack epi- center. To achieve
this goal, custom-made extraction columns with a larger bed size
were employed. The dynamic range of the method was from the LOQ at
0.392-196 pg on-column for each analyte. Procedures that required
time consuming con- centration steps, which may allow for the
detection of lower aflatoxin levels, were avoided to increase assay
throughput [i.e., 1 mL of urine was extracted into 1 mL of MeOH/H20
with 1% (v/v) formic acid]. Figure 4 shows a calibration curve for
all five toxins in human urine. The dynamic range pre- sented here
meets the aforementioned goal of covering a wider range than those
previously reported (8,17).
As seen in Figure 4, a slight deviation from linearity begins to
occur at the highest point. This is due to the large aflatoxin/an-
tibody ratio at this concentration. This was examined by ana-
lyzing non-extracted standards that showed increased linearity at
this same concentration and at even higher concentrations (data not
presented), which verified that our ULOL was dictated by the
binding capacity of the extraction column and not by the
instrument. At concentrations above the ULOL, the number of
AFG+ QCL A
A 9 9
8 9 9
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . & . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
AFGI QCH B
+,,~ i ,
w w
Figure 5. Quality control plots for AFG~, QC Low at 1.96 ng/mk
(A)and QC High at 157 ng/mL (g). The central line represents the
mean, the dashed line is two standard deviations, and the outer
line is three stan- dard deviations from the mean.
154
-
Journal of Analytical Toxicology, Vol. 31, April 2007
binding sites became a limiting factor and some unbound aria-
toxin was removed during the wash step of the extraction. This was
an important consideration when determining the ULOL of an
extraction method using immunoaffinity columns.
The method was validated by analyzing a calibration curve, two
quality controls (low and high), and a urine blank spiked with
internal standard. This experiment was repeated 20 times over a
period of 12 days with no more than 2 sets being analyzed in a
single day. Eight analysts conducted the experiments
Table V. Validation Results Depicting the Precision and Accuracy
of the Method.
Lower Upper Concentration Mean % 95% Limit 95% Limit
Analyle (ng/mL) (ng/mL) Accuracy (ng/mL) (ng/mL)
AFB 1 1.96 2.01 103 1.95 2.08 AFB 1 157 153 97.5 149 157 AFM1
1.96 2.01 102 1.95 2.07 AFM~ 157 152 97.0 148 156 AFG1 1.96 2.07
106 2.02 2.13 AFG~ 157 152 97.0 150 155 AFP1 1.96 2.03 104 1.97
2.09 AFP1 157 151 96.5 147 155 AFG2 1.96 2.06 105 2.02 2.11 AFG2
157 146 93.5 144 150
~k,~ i i
i f,*sl
AFMI A
AFF
AFB 2
]~ AFB,
1re(m|)
AFB 2
1o . 12 S3 14 IS
B
1 = 3 4 s" T ; * ~o . ,2 1] 14 is ' r im* ( id l )
Figure 6. Chromatograms from the animal study, dosed rat (A) and
con- trol rat (B).
during the 12-day period. Linear regression with "l/x" weighting
was used for each analyte to account for het- eroscedasticity in
the data. The calibration standards were an- alyzed in a random
order and the curves for each aflatoxin had an average correlation
coefficient > 0.995. The quality of the method was represented
by quality control low and high plots of AFG1 (Figure 5). Similar
results were obtained for the four other toxins. The results of the
accuracy and precision of the method for each analyte are shown in
Table V. The range of the
percent accuracy of the means for all five ana- lytes was from
97.0 to 105.6%, and the highest % relative standard deviation (RSD)
found was 6.67%. In all but one case, the mean was s one standard
deviation away from the true value. No significant contributions
from carryover
% RSD were seen in the blank samples. After validation, the
method was further
6.67 tested by analyzing the urine of an AFB1 ex- 5.18 posed
rat. The dose administered to the rat (91 6.59 IJg/kg body weight)
was well below the LDs0 5.58 for rats and corresponded to a dose of
several 5.68 milligrams for a human adult. A dose of 2-6 3.o4
mg/day was observed during an outbreak of 6.30 aflatoxicosis in
western India (19), and similar 5.16 amounts were consumed in a
recent outbreak 4.79 in Kenya (20). It was expected that roughly
5.07
equal amounts of the two metabolites AFM] and AFP1, and a small
amount of unmetabo-
lized ARB1, would be excreted. No aflatoxins other than the in-
ternal standard were detected in the negative control rat urine
sample. AFBI, AFMI, and AFP] were detected in urine from the
AFBl-dosed rat at 1.38, 48.8, and 41.4 n~mL, respectively; 4 mL of
urine was collected and indicated total excretion of 5.52, 195.2,
and 165.6 ng, respectively, during the 18-h period. The creatinine
levels of the two urines were not measured; thus, no comparison of
the metabolite concentrations with literature values were made.
However, relative to AFP], the amount of AFM 1 was more abundant,
which is consistent with previous studies of AFB1 exposure in rats
(11,21). Figure 6 shows the chromatograms for the negative control
and AFB1 dosed rat urine samples.
It was also expected that other metabolites of aflatoxin would
be present in the dosed rat urine. Precursor and product ion masses
of other AFB] metabolites (18) were used to build a sep- arate MP~
method to detect the presence of these compounds. Metabolites such
as AFB-diol and AFQ] were found. The presence of AFQI was further
confirmed by matching the retention time with a chromatogram
obtained previously when AFQ] was com- mercially available (data
not presented). This method was suit- able for monitoring other
metabolites on a qualitative level, but quantitative analysis was
difficult because AFM] and AFP 1 are the only commercially
available AFB] metabolite standards.
Conclusions
The LC-MS-MS method described uses less urine and has lower
limits of detection than previously reported methods.
155
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Journal of Analytical Toxicology, Vol. 31, April 2007
Other advantages of this approach are the specificity of both
immunoaffinity extraction and tandem MS and no require- ment for
derivatization. The dynamic range of 0.392-196 pg on column,
combined with an automated extraction, yield rapid results over a
wide range of exposure. This method is well suited to aid forensic
and public health laboratories during the investigation of a
terrorist attack by providing confirmation of military or civilian
exposure to weaponized aflatoxin. In ad- dition, the method
presented here can be utilized to diagnose aflatoxicosis (22)
caused by consumption of a food supply con- taminated intentionally
by the hands of terrorists or acciden- tally during an outbreak
(19,23,24).
Acknowledgments
The authors would like to thank Erin Carson, Mike Martin, Chris
Nixon, Jeff Snow, Shane Wyatt, and Jessica Zuckschw- erdt, who
contributed to the validation process, and Janet Pruitt for the
insightful discussions. The authors would also like to thank Dr.
Torn York for his comments to the manuscript. Funding for the
analytical work (R.E., EC., D.Z., and T.C.) was funded by the
Centers for Disease Control and Prevention grant # U90/CCU317014.
The animal study (P.S. and J.G.) work was funded by the NIH grants
# P01 ES06052 and #P30 ES03819.
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