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I Abstract
Automated immunoaffinity solid-phaseextraction followed by
liquid chromatography-tandem massspectrometry and chemical
analogue internal standardization is employed to detect and
quantify the aflatoxins AFB1, AFB2,AFG1, AFG2, and the
metabolites AFM1 and AFP1 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 diagnosisof aflatoxicosis is needed.
Introduction
Because of the ever increasing threat of terrorist attacks
around the globe and, more specifically, the threat of a chemical 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 degree of exposure, and the extent of the population that is exposed (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: tim.croley@dgs.virginia.gov.
150
Experimental
Chemicals and materials
AFBI
^F01
Rate(mL/min)
%B
0.325
0.325
0.325
0.325
0.325
0.325
30
30
95
95
30
30
0.00
10.00
10.10
11.50
11.51
15.50
AJ"~
AF(J2
AI~
AFpI
Figure I. Structuresof the four parent aflatoxins and the two metabolites
of AFBI monitored in this study.
Analyte
Precursor
CE
Product
AFP1
AFM1
AFG2
AFB2
AFG1
AFB1
299
329
331
315
329
313
33.0
33.0
35.0
37.0
39.0
33.7
271
273
313
287
243
285
151
automatically using the Analyst 1.4 software (Applied Biosystems). Extraction efficiencieswere 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 intraperitoneal injection (150 IJL)on two consecutive days, and the rats
were housed in metabolic cages. Urine was collected for approximately 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.
ExtractionEfficiency(%)
AFB 1
AFM1
AFP~
AFG1
AFG 2
87.4 + 8.2
81.9 + 3.0
80.3 _+4.9
93.2 _+8.5
84.5 _+8.8
AFM~
2~4
2~e4
2~4
b4
t 4e4
AFP I |
AFG'
AFBI
I
AFG:
'
60WO
4~00
2~00
:~:~e:::::::::::::::::::::
2007
9'
'
~Oo4i
,J~i
Slt~I
,,3~+
..+t..,,,++
+
t , -'."+
i
t,.,++
,+.++
I:,~~
,,~,++
"-0+
I,
T
~,,
11...................... J.._+w.'l................................. ~
,m,,j
mPo.~ +
,+:
m,ll
.............................
iil
" 1+
:+
l'
,"~
,..t
i'
.-
mo m - + + . . . +
m~"
t:.:l
" ,.,,-i
10~oJ
=i
'~t
//
,
",
....+
,.!
11~4 i
|+i
! '-~
- ,-i
Mm.~
! -~
1~01 i
~,
im~M)
m..~
! i=
ii-+I
,-!
~,., ~+..++.
:=i//
I~I.O!
mr,
1~118~ i
ii~
ml
___
AFG+ QCL
Femtogramson Column
Attomoles
AFP~
AFM~
AFG2
AFG~
AFBI
50
100
100
100
50
168
305
302
305
160
.................................................
& ............................................
AFGI QCH
+,,~ i,
i LIk
5
(n~/raL)
Figure 4. An eight-point calibration plot from 0.392 to 196 ng/mL in
urine using linear regressionwith "l/x" weighting.
Concentration
154
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 standard deviations from the mean.
binding sites became a limiting factor and some unbound ariatoxin was removed during the wash step of the extraction. This
was an important consideration when determining the ULOLof
an extraction method using immunoaffinitycolumns.
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 dayswith no more than 2 sets being analyzed
in a single day. Eight analysts conducted the experiments
s"
T
;
'rim* (idl)
1o
12
S3
~o
,2
1]
14
14
IS
is
Figure 6. Chromatogramsfrom the animal study,dosed rat (A) and control rat (B).
Conclusions
The LC-MS-MS method described uses less urine and has
lower limits of detection than previously reported methods.
155
Acknowledgments
The authors would like to thank Erin Carson, Mike Martin,
Chris Nixon, Jeff Snow, Shane Wyatt, and Jessica Zuckschwerdt, 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.
References
1. J.R. Barr. Biological monitoring of human exposure to chemical
warfare agents. J. Anal. Toxicol. 28:305 (2004).
2. R.A. Zilinskas. Iraq's biological weapons. The past as future? J.
Am. Med. Assoc. 278:418-424 (1997).
3. G.N. Wogan, G.S. Edwards, and P.M. Newberne. Structure-activity relationships in toxicity and carcinogenicity of aflatoxins and
analogs. Cancer Res. 31:1936-1942 (1971 ).
4. E. Anklam, J. Stroka, and A. Boenke. Acceptance of analytical
methods for implementation of EU legislation with a focus on mycotoxins. Food Control 13" 173-183 (2002).
5. J.M. Cullen and P.M. Newberne. Acute hepatotoxicity of aflatoxins. In The Toxicology ofAflatoxins: Human Health, Veterinary,
and Agricultural Significance, D.L. Eaton and J.D. Groopman, Eds.
Academic Press, San Diego, CA, 1994, pp 3-26.
6. R.M. Willis, J.J.Mulvihill, and J.H. Hoofnagle. Attempted suicide
with purified aflatoxin. Lancet 315:1198-1199 (1980).
7. A. Kussak, B. Andersson, K. Andersson, and C.A. Nilsson. Determination of aflatoxicol in human urine using immunoaffinity
column clean-up and liquid chromatography. Chemosphere 36:
1841-1848 (1998).
8. B.I. Vazquez, C.A. Fente, C.M. Franco, A. Cepeda, G. Mahuzier,
and P. Prognon. Preliminary study on fluorimetric detection of
aflatoxins Q1, P1, and B1 using heptakis-di-O-methyl-~-cyclodextrin as post-column HPLC reagent. Anal Commun. 36:5-7
(1999).
9. D.L. Eaton, H.S. Ramsdell, and G.E. Neal. Biotransformation of
aflatoxins. In The Toxicology of Aflatoxins: Human Health,
Veterinary, and Agricultural Significance, D.L. Eaton and
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