An enzyme-linked immunosorbent assay for the detection of esfenvalerate metabolites in human urine

Article abstract of DOI:10.1021/tx990091h

The pyrethroids are one of the most heavily used insecticide classes in the world. Sensitive and rapid analytical techniques are needed for assessments of human exposure to these compounds. Highly sensitive and selective ELISAs for glycine conjugates of e

Full text of DOI:10.1021/tx990091h

NOVEMBER 1999
VOLUME 12, NUMBER 11
© Copyright 1999 by the American Chemical Society
Articles
An En zym e-Lin k ed Im m u n osor ben t Assa y for th e
Detection of Esfen va ler a te Meta bolites in Hu m a n Ur in e
Guomin Shan,† Ingrid Wengatz,‡ Donald W. Stoutamire,† Shirley J . Gee,† and
Bruce D. Hammock*,†
Department of Entomology, University of California, Davis, California 95616, and Center for
Environmental Studies, University of Nevada, Las Vegas, Nevada 89154-4009
Received May 24, 1999
The pyrethroids are one of the most heavily used insecticide classes in the world. Sensitive
and rapid analytical techniques are needed for assessments of human exposure to these
compounds. Highly sensitive and selective ELISAs for glycine conjugates of esfenvalerate key
metabolites phenoxybenzoic acid (PBA) and s-fenvalerate acid (sFA) were developed. Rabbits
were immunized with either N-(3-phenoxybenzoyl)-4-amino-L-phenylalanine-fetuin or N-[(S)-
4-chloro-2-(methylethyl)benzeneacetyl]-4-amino-L-phenylalanine-fetuin, and all sera were
screened against numerous coating antigens. The antibodies with the least interference and
best sensitivity were optimized and characterized. The I50s for sFA-glycine and PBA-glycine
in buffer were found to be 0.40 ( 0.12 µg/L (1.47 ( 0.44 nmol/L) and 0.42 ( 0.18 µg/L (1.56 (
0.67 nmol/L), respectively. Both assays exhibited high selectivity. Little or no cross reactivity
to the parent compound and other metabolites was measured. The matrix effects of urine were
investigated. Solid-phase extraction (SPE) strategies were used in an attempt to decrease the
matrix effects and increase the sensitivity of the overall method. The limit of quantitation
(LOQ) for both sFA-glycine and PBA-glycine in urine with SPE is 1.0 µg/L (3.70 nmol/L). These
assays could be used as markers of exposure for monitoring biological samples.
act on the axons in the peripheral and central nervous
systems by interacting with sodium channels in mam-
mals and insects. A prolonged opening of the sodium
channel immediately evokes a repetitive nerve action and
causes a series of neurotoxic effects such as hyperactivity,
tremor, ataxia, convulsion, and possible paralysis (4-6).
Production personnel and farmworkers are exposed to
pyrethroids during manufacture and application. Al-
though these compounds are claimed to be safe for
humans (7), after exposure, reversible symptoms of
poisoning such as headaches, dizziness, nausea, irritation
In tr od u ction
Pyrethroids such as esfenvalerate, cypermethrin, and
deltamethrin, a group of highly potent insecticides with
relatively low mammalian toxicity, have been widely used
in agriculture, forestry, horticulture, public health, and
households all around the world (1-3). These neurotoxins
* To whom correspondence should be addressed: Department of
Entomology, University of California, Davis, CA 95616. Phone: (530)
752-7519. Fax: (530) 752-1537. E-mail: bdhammock@ucdavis.edu.
† University of California.
‡ University of Nevada.
10.1021/tx990091h CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/14/1999

1034 Chem. Res. Toxicol., Vol. 12, No. 11, 1999
Shan et al.
F igu r e 1. Structures of esfenvalerate and its metabolites.
of the skin and nose, and paraesthesia have been
reported (8, 9). Moreover, the chronic toxic effects in
humans are not clear yet. Studies on chronic exposure
effects by Hallenbeck and Cunningham-Burns (10) have
suggested that fenvalerate may have caused lymph node
and spleen damage as well as carcinogenisis. Recently,
Repetto (11) demonstrated that pyrethroids have sup-
pressive effects on the immune system. In studies on the
estrogenic potential of pyrethroids in human breast
carcinoma cell, Go et al. (12) suggest that pyrethroids
should be considered hormone disruptors. Therefore, it
is desirable that sensitive and efficient analytical meth-
ods be developed for both toxicological and epidemiologi-
cal monitoring (13).
Studies of pyrethroid metabolism in mammals have
revealed that pyrethroids are metabolized rapidly by
oxidation and hydrolytic cleavage of the ester linkage,
followed by conjugation (14-17). Fenvalerate was shown
to be metabolized in mammals to yield fenvalerate acid
(FA)1 and 3-phenoxybenzoic acid (PBA), which are con-
jugated in part to glucuronic acid and glycine, and then
excreted renally (Figure 1) (3, 18-21). Although no
studies regarding the conjugates of pyrethroid metabo-
lites in humans have been reported, it has been well
established that conjugation with one of a variety of
amino acids (such as glycine) is an important biotrans-
formation pathway for xenobiotics containing a carboxylic
acid group (22, 23).
Current analytical methods for pyrethroid metabolites
in urine rely upon multistep sample cleanup procedures,
including hydrolysis, extraction, and derivatization. The
samples are then analyzed using either HPLC (20) or gas
chromatography with mass spectrometry (20, 24-27).
These methods are time-consuming and expensive, and
often do not fulfill the requirements of routine analysis
regarding sensitivity and practicality for effective moni-
toring. A promising method for rapid assessment of
exposure to agrochemicals is the use of immunoassays
that detect key urinary biomarkers of exposure (28). It
has been demonstrated that immunoassays are highly
sensitive and selective analytical tools for identifying
trace chemicals such as pesticide residues and their
F igu r e 2. Structures of haptens 1-5.
metabolites (29). Recently, several immunoassays have
been developed for identifying pesticide metabolites as
biomarkers of exposure, such as hydroxytriazines and
dealkylated triazines (30), naphthalene mercapturates
(31), and triazine mercapturates (32).
In the study presented here, we describe the develop-
ment of immunoassays for the detection of glycine
conjugates of the esfenvalerate metabolites PBA and sFA,
which might be used as biomarkers of human exposure
to esfenvalerate. The interference of urine as a matrix
and the sample preparation method were investigated
and will be discussed.
Exp er im en ta l P r oced u r es
Ch em ica ls. The standards cypermethrin, fluvalinate, fen-
valerate, and permethrin were obtained from Riedel de Haen
(Seelze, Germany). Racemic 4-chloro-R-(1-methylethyl)benzene-
acetic acid was generously supplied by E. I. duPont De Nemours
& Co. (Wilmington, DE) and resolved to give the (R)- and (S)-
isomers as described previously (33). Esfenvalerate was syn-
thesized as described by Shan et al. (33) with a purity of >99%
based on analytical data. Bovine serum albumin (BSA), oval-
bumin (OVA), fetuin, Tween 20, 3,3′,5,5′-tetramethylbenzidine
(TMB), and goat anti-rabbit IgG conjugated to horseradish
peroxidase (HRP) were purchased from Sigma Chemical Co. (St.
Louis, MO). Other chemical reagents were purchased from
Aldrich Chemical Co. (Milwaukee, WI), Fisher Scientific (Pitts-
burgh, PA), and Lancaster Synthesis, Inc. (Windham, NH). TLC
was performed on 0.2 µm silica gel 60-F254 glass plates from
E. Merck (Darmstadt, Germany). Flash chromatographic sepa-
rations were carried out on 40 µm average particle size Baker
silica gel, packed in glass columns with a diameter that gives a
column height/diameter ratio of ∼7. The f notation denotes a
stepwise solvent gradient.
In str u m en ts. NMR data were obtained using a General
Electric QE-300 spectrometer (Bruker NMR, Billerica, MA).
Chemical shift values are given in parts per million downfield
from internal tetramethylsilane. Melting points were deter-
mined on
a Thomas-Hoover Uni-Melt apparatus (Thomas
Scientific, Swedesborogh, NJ ) and are uncorrected. Fast atom
bombardment high-resolution mass spectra (FAB-HRMS) were
obtained on a ZAB-HS-2F mass spectrometer (VG Analytical,
Wythenshawe, U.K.), using xenon (8 keV, 1 mA) for ionization
and 3-nitrobenzyl alcohol or glycerol as the matrix. Polyethylene
glycol was added to the matrix as a mass calibrant. ELISAs
were carried out with 96-well microtiter plates and read with a
microplate reader (Molecular Devices, Menlo Park, CA) in dual-
wavelength mode (450-650 nm).
1
Abbreviations: Ab, antibody; Ag, antigen; BSA, bovine serum
albumin; cAg, coating antigen; EDC, 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide hydrochloride; ELISA, enzyme-linked immunosorbent
assay; FA, fenvalerate acid; sFA, esfenvalerate acid; FAB-HRMS, fast
atom bombardment high-resolution mass spectroscopy; GAR-HRP,
goat anti-rabbit immunoglobulin conjugated to horseradish peroxidase;
I
50, concentration of analyte giving 50% inhibition; LOQ, limit of
quantitation; NHS, N-hydroxysuccinimide; OVA, ovalbumin; PB, phe-
noxybenzyl; PBA, phenoxybenzoic acid; PBS, phosphate-buffered
saline; PBST, phosphate buffered saline with 0.05% Tween 20; SPE,
solid-phase extraction; TMB, tetramethylbenzidine.
Ha p ten Syn th esis a n d Ver ifica tion . Syntheses of the
haptens were carried out as outlined in Schemes 1 and 2. All

ELISA for Esfenvalerate Metabolites
Chem. Res. Toxicol., Vol. 12, No. 11, 1999 1035
N-[(S)-4-Ch lor o-r-(1-m e t h yle t h yl)b e n ze n e a ce t yl]gly-
cin e (8). (S)-4-Chloro-R-(1-methylethyl)benzeneacetyl chloride
was prepared from the corresponding acid (2.12 g, 0.01 M) by
the same procedure as described above for phenoxybenzoyl
chloride. This was added dropwise over 10 min to a vigorously
stirred ice-cooled solution of glycine (1.5 g, 0.02 M) and 4-(di-
methylamino)pyridine (4 mg) in 10 mL of 2 N KOH. After an
additional 15 min, the reaction mixture was neutral. The ice
bath was removed, and additional base solution (4.5 mL) was
added dropwise over the course of 10 min to keep the mixture
slightly basic. After an additional 15 min, the reaction mixture
was filtered through Celite to remove a small amount of
flocculant precipitate, acidified (3 N HCl), and extracted with
CH2Cl2. The extracts were washed with water, dried over
MgSO4, and stripped to a colorless gum. The residue was
chromatographed on 40 g of silica gel (CH2Cl2 f 3% HOAc in
EtOAc). Stripping product fractions gave a gum containing some
acetic acid, which showed only one spot by TLC: Rf ) 0.46 (3%
HOAc in EtOAc). Crystallization from 6 mL of n-butyl chloride
diluted with 2 mL of hexane gave 1.76 g (65%) of fine white
needles (mp 137-139 °C). A second crop of 102 mg (mp 136-
138 °C) was obtained from the concentrated filtrate: [R]D24
Sch em e 1
Sch em e 2
1
(combined solids) 29.1 (c 1.17 CHCl3); H NMR (CHCl3) δ 0.70
(d, J ) 6.7 Hz, 3 H, CH3), 1.03 (d, J ) 6.5 Hz, 3 H, CH3), 2.35
(m, J ) 6.5, 10.3 Hz, 1 H, Me2CH), 2.93 (d, J ) 10.3 Hz, 1 H,
Me2CHCH), 3.88 (dd, J ) 5.0, 18.5 Hz, 1 H, HCHCO), 4.08 (dd,
J ) 5.4, 18.5 Hz, 1 H, HCHCO), 6.38 (t, J ) 5.0 Hz, 1 H, NH),
7.26 (s, 4 H, Ar); 13C NMR (DMSO-d6) δ 20.2 (CH3), 21.1 (CH3),
31.1 (Me2C), 40.7, 58.7, 128.0 (2C), 130.0 (2C), 131.3, 139.0,
171.2, 172.9; FAB-HRMS m/z calcd for C13H17ClNO3 ([M + H]+)
270.09, found 270.09.
reactions were straightforward, and yields were moderate to
good. The syntheses of haptens 1-5 (Figure 2) are outlined in
the previous paper describing an immunoassay for esfenvalerate
(33).
N-(3-P h en oxyben zoyl)-4-n itr o-L-ph en ylalan in e (6). 3-Phe-
noxybenzoic acid (1.48 g, 6.9 mmol) in 3 mL of CHCl3 was
treated under N2 with SOCl2 (0.73 mL, 10 mmol) and DMF (0.5
µL). After heating at 60-65 °C for 1 h, stripping, addition of
hexane, and restripping, the acid chloride was obtained as a
pale yellow liquid. This was added dropwise over the course of
a few minutes to a vigorously stirred solution of 4-nitro-L-
phenylalanine (1.5 g, 6.57 mmol) and potassium carbonate (1
g, 7.2 mmol) in 10 mL of water followed by dropwise titration
with 10% sodium hydroxide over the course of 10 min to pH 9.
After 10 min, the odor of acid chloride was no longer detectable.
The mixture was acidified (3 N HCl) and extracted with ethyl
acetate, and the organic phase was filtered, washed with water,
and stripped to a yellow gum. Flash chromatography on 35 g of
silica gel (CH2Cl2 f EtOAc f 3% HOAc/EtOAc) followed by
crystallization from toluene gave 1.34 g (48%) of white solid:
[R]2D4 -38.6 (c 0.755, EtOH); mp 140-146 °C. The hot melt
recrystallized and remelted at 164-165 °C. The TLC of the melt
[Rf ) 0.45 (3% HOAc/THF)] was unchanged: 1H NMR (CDCl3)
δ 3.32 (dd, J ) 13.9, 5.9 Hz, 1 H, HCH), 3.48 (dd, J ) 13.9, 5.8
Hz, 1 H, HCH), 5.09 (dt, J ) 7.1, 5.8 Hz, 1 H, CHCOOH), 6.63
(d, J ) 7.2 Hz, 1 H, NH), 6.99-8.13 (m, 13 H, Ar); 13C NMR
(DMSO) δ 36.0, 53.6, 117.1, 118.9 (2C), 121.6, 122.3, 123.2 (2C),
123.9, 130.1, 130.1 (2C), 130.4 (2C), 135.6, 146.3, 146.5, 156.2,
156.6, 156.8, 172.6.
N-(3-P h en oxyben zoyl)glycin e (9). Crude stripped 3-phe-
noxybenzoyl chloride, prepared as described above from the acid
(1.5 g, 7 mmol), was added in portions to a vigorously stirred
ice-cooled solution of glycine (0.578 g, 7.7 mmol) in 7 mL of 1 N
KOH along with equivalent portions of 2 N KOH over the course
of 30 min. Additional caustic was added over the course of 15
min to keep the aqueous phase slightly basic. The homogeneous
mixture was acidified (3 N HCl) and extracted with ether. The
combined extract was washed with water and stripped to pale
green oil. Crystallization from EtOAc/n-butyl chloride gave 0.88
g (46%) of a white solid (mp 148-150 °C). Chromatography of
the filtrate (silica gel, CH2Cl2 f EtOAc) recovered an additional
8%: TLC Rf ) 0.6 (3% HOAc in EtOAc); 1H NMR (DMSO-d6) δ
3.88 (d, J ) 5.8 Hz, 2 H, CH2), 7.03-7.67 (m, 9 H, Ar), 8.89 (t,
J ) 5.8 Hz, 1 H, NH), 12.6 (s, 1 H, COOH); 13C NMR (DMSO-
d6) δ 41.4 (CH2), 117.3, 118.9 (2C), 121.7, 122.4, 123.8, 130.2
(3C), 135.9, 156.4, 156.9, 165.8 [C(O)N], 171.3 [C(O)]; FAB-
HRMS m/z calcd for C15H14NO4 ([M + H]+) 272.09, found 272.09.
Ha p ten Con ju ga tion . Conjugates were synthesized using
a
diazotization, activated ester method and the periodate
method (34). To obtain immunogens, haptens 5 and 7 were
conjugated to fetuin. Coating antigens were made by coupling
haptens 1-5 and 7 and PBA to BSA and/or OVA. The conjuga-
tions of haptens 1-5 with BSA or OVA were described previ-
ously (33).
N-(3-P h en oxyben zoyl)-4-a m in o-L-p h en yla la n in e (7). A
solution of 6 (0.522 g, 1.28 mmol) and 10% palladium-carbon
catalyst (4.5 mg) in 95% ethanol (20 mL) was stirred under
hydrogen at 50 °C for 2.5 h and 25 °C for 3 h. The resulting
suspension was diluted to 100 mL with ethanol, heated to 75
°C to dissolve solids, and filtered hot through Celite. Dilution
with 60 mL of hexane and standing overnight gave 0.366 g (76%)
of a white solid (mp 217-218 °C). A second crop of 92 mg (19%,
mp 214-216 °C) was obtained from the filtrate: [R]2D4 (com-
bined solids) -54 (c 1.08, DMF); TLC Rf ) 0.16 (2.5% HOAc in
15:85 CH2Cl2/EtOAc); 1H NMR (DMSO-d6) δ 2.84 (dd, J ) 13.6,
10.6 Hz, 1 H, HCHAr), 2.97 (dd, J ) 13.7, 4.4 Hz, 1 H, HCHAr),
4.44 [m, J ) 7.9, 4.4 Hz, 1 H, HCC(O)], 6.41-7.6 (m, 13 H, Ar),
8.63 (d, J ) 8.0 Hz, 1 H, NH); 13C NMR (DMSO-d6) δ 35.9
(CH2Ar), 55.2 (CHCOOH), 114.2 (2C), 117.6, 119.1 (2C), 121.7,
122.6, 124.1, 125.2, 129.8 (2C), 130.3, 130.4 (2C), 136.2, 147.2,
156.6, 157.0, 165.9 [C(O)N], 173.7 (COOH); FAB-HRMS m/z
calcd for C22H21N2O4 ([M + H]+) 377.1501, found 377.1520.
Ha p ten Con ju ga tes 7-BSA a n d -OVA. A 100 µL aliquot
of butyl nitrite solution (123 µL/2 mL of DMSO) was added
dropwise to a stirred solution of 9.4 mg (0.025 mmol) of hapten
7 in 1.05 mL of 37.5 mM H2SO4 in DMSO at 16 °C. Stirring
was continued for 10 min, and the solution was divided in two
equal aliquots. One aliquot was added to a solution of BSA and
the other to a solution of OVA. Each protein solution consisted
of 20 mg of protein dissolved in 5 mL of borate buffer (0.1 M,
pH 9.4). The stirring was continued overnight at 4 °C. The
solutions became yellow in color. The reaction mixtures were
purified using a dextran-desalting column and phosphate-
buffered saline (PBS, pH 7.5) as the eluent. The fractions of
purified conjugates were easily detected because of their orange
color. These fractions were pooled and stored in aliquots at -80,
-20, and 4 °C.

1036 Chem. Res. Toxicol., Vol. 12, No. 11, 1999
Shan et al.
Ha p ten Con ju ga tes 5-F etu in a n d 7-F etu in . To a stirred
solution of 24 mg (0.004 mmol) of fetuin in 4 mL of PBS was
added 400 µL of aqueous NaIO4 (100 mM). Stirring was
continued for 20 min at room temperature. The solution was
then dialyzed overnight at 4 °C against 1 mM sodium acetate
buffer (pH 4.4). The dialyzed solution was transferred and
aliquoted into two vials. To each vial was added 30 µL of 200
mM carbonate buffer (pH 9.7), followed by 18 mg (0.05 mmol)
of each hapten (5 or 7), each in 1 mL of ethanol. The mixtures
were stirred for 3 h at room temperature (after 1 h, 1 mL of
THF was added to each to prevent precipitation of the haptens).
Finally, 100 µL of freshly prepared NaBH4 solution (4 mg/mL
of water) was added to each vial, and the solutions were
incubated overnight at 4 °C. The conjugates were purified and
stored as described above.
in DMSO per 25 mL of acetate buffer (pH 5.5)] per well was
pipetted into each well. Ten to fifteen minutes later, the color
development was stopped by adding 50 µL of 2 M H2SO4 per
well. The plates were then read in a dual-wavelength mode,
subtracting the absorbance at 650 nm from the absorbance at
450 nm. All experiments were conducted using three or four
well replicates. Standard curves were obtained by plotting
absorbance against the logarithm of analyte concentration, and
they were fitted to the four-parameter logistic equation y ) {(A
- D)/[1 + (x/C)B]} + D, where A is the maximum absorbance at
zero analyte concentration, B is the curve slope at the inflection
point, C is the concentration of analyte giving 50% inhibition
(I50), and D is the minimum absorbance at infinite concentration.
Assa y Op tim iza tion . The assay conditions were optimized
in such a way that the I50s were minimized. This goal was
achieved by screening antibodies and antigens in
a two-
Ha p ten Con ju ga tes 8-BSA a n d 9-BSA. Compound 8 and
9 were coupled to BSA by the N-hydroxysuccinimide ester
method of Langone and Van Vunakis (35). The acid hapten
(0.015 mmol) was dissolved in 150 µL of dry DMF with
equimolar NHS and 0.020 mmol of 1,3-dicyclohexylcarbodiimide.
After the solution had been stirred for 3.5 h at room tempera-
ture, the precipitated dicyclohexylurea was removed by cen-
trifugation, and the supernatant was added to 20 mg of BSA
solution in 2.5 mL of distilled water and 0.5 mL of DMF. The
reaction mixtures were stirred gently at 4 °C overnight. The
solution was then dialyzed exhaustively against PBS and stored
at -20 °C until it was used.
Con ju ga tion of P BA-BSA. PBA (0.025 mmol) was dis-
solved in 2 mL of dry DMF, and then 6 mg (0.05 mmol) of NHS
and 5.8 mg (0.03 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide hydrochloride (EDC) were added. The reaction
mixture was stirred overnight at room temperature. BSA (50
mg) was dissolved in 10 mL of PBS and 2 mL of carbonate buffer
(pH 9). The activated hapten was added dropwise to the protein
solution. The mixture was stirred for 30 min at room temper-
ature, and for 7 h at 4 °C. The solution was then dialyzed against
PBS over the course of 72 h at 4 °C and stored at -20 °C.
Im m u n iza tion . Immunizations were performed following the
protocol reported previously (36). Three female New Zealand
white rabbits were immunized intradermally with each immu-
nogen (5- and 7-fetuin). After the initial immunization with
100 µg of the immunogen in PBS, emulsified in Freund’s
complete adjuvant (1:1 v/v), further injections using Freund’s
incomplete adjuvant were given. Booster injections followed in
3-week intervals. The rabbits were bled 10 days after each
immunization. Among the three rabbits immunized with 7-fe-
tuin, two rabbits (numbers 18 and 20) died prior to the final
bleed. After coagulation of the blood, the serum was isolated
after centrifugation for 10 min at 4 °C. Sera were collected prior
to immunization to provide a control serum that shows no
related humoral immune response. Antisera were screened for
their specific antibody titer. Final bleeds were used for ELISA
development.
dimensional titration for the best dilution of coating antigen and
antiserum. Then, the competitive inhibition curves were meas-
ured for different antibody and antigen combinations, and the
one with the lowest I50 was selected for further assay develop-
ment.
Cr oss Rea ctivity. The optimized assays were submitted to
cross-reactivity studies by using the standard solution of the
analytes, other structurally related compounds (see Table 4),
and 20 free amino acids. The cross reactivity (CR) was obtained
from the metabolite standard and the related compound I50
values from the same plate, where % CR ) (I50 of the metabolite/
I50 of the tested compound) × 100.
Solven t Effects. Standard curves of analytes 8 and 9 were
prepared in PBS buffer containing 0, 10, 20, 40, 60, and 80%
(v/v) methanol to determine the effects of solvent.
Ma tr ix Effects. (a ) p H. Preparing analytes in phosphate
buffers at pH 4, 6, and 8 tested the effect of pH. All other assay
conditions were as described above.
(b) Ion ic Str en gth . The effects of ionic strength on the
quantitation of 8 and 9 were studied by preparing analyte
standard solutions in 1×, 3×, 5×, and 8× PBS all at pH 7.5.
Other assay conditions were the same as described above.
(c) Ur in e. The effects of the urine matrix on the quantitation
of 8 and 9 were evaluated by preparing analyte standard
solutions in a buffer with different concentrations (0, 1, 2, 4,
and 10%) of urine. Four urine samples tested in this study were
from healthy individuals without known exposure to pyre-
throids.
Solid -P h a se Extr a ction (SP E). The SPE method used in
this study was modified from a previously reported method for
the extraction of atrazinemercapturic acid from human urine
using a C18 column (32). The C18 column (10 cm3/500 mg; part
no. 1211-3027, Varian Sample Preparation Products, Harbor
City, CA) was preconditioned with 3 mL of methanol, deionized
water, and 0.1 M phosphate buffer (pH 2.2). The urine samples
(10 mL) were mixed with equal volumes of 0.1 M phosphate
buffer (pH 2.2) and loaded on the column, eluting with 2-4 mL/
min flows. Columns were then washed with 0.1 M phosphate
buffer (pH 2.2), deionized water, and deionized water containing
20% methanol. After being dried under high vacuum for 15 min,
the column was eluted with 3 mL of 100% methanol. The volume
was adjusted to 4 mL with methanol, diluted 1:50 with 20%
methanolic buffer, and analyzed with an ELISA.
En zym e-Lin k ed Im m u n osor ben t Assa y (ELISA). BSA
and OVA conjugates were used as coating antigens diluted with
a coating buffer [50 mM sodium carbonate/bicarbonate buffer
(pH 9.6)]. Microtiter plates were usually coated overnight at 4
°C with 100 µL of the hapten-protein conjugate per well. After
the plates had been washed with PBST {phosphate-buffered
saline with Tween 20 [8 g/L NaCl, 1.15 g/L Na2HPO4, 0.2 g/L
KH2PO4, 0.2 g/L KCl, and 0.05% Tween (v/v)]}, the surface of
the wells was blocked with 200 µL of a BSA solution (0.5% in
PBS) by incubating for 30 min at room temperature. After
another washing step, 100 µL of PBS-diluted antiserum per well
(for determination of antibody titer) or 50 µL of PBS-diluted
antiserum per well and 50 µL of standard solution per well were
dispensed into the wells and incubated for 60 min at room
temperature. Following another washing step (four times), 100
µL of GAR-HRP per well (diluted 1:3000 in PBS) was added to
each well, and the wells were incubated for 60 min at room
temperature. The plate was washed four times, and 100 µL of
substrate solution [3.3 µL of 30% H2O2 and 400 µL of 0.6% TMB
Resu lts a n d Discu ssion
Ha p ten Design a n d Syn th esis. No study has re-
ported which conjugate is predominant for pyrethroid
metabolites in human urine. In mammals, the major
“detoxication” reaction for carboxylic acid-containing
xenobiotics is conjugation, with either an amino acid for
forming a peptide or glucuronic acid for forming a
glycoside (22, 23). In general, larger and more compli-
cated cyclic acids tend to be excreted as glycosides, but
simpler acids such as benzoic and arylacetic acids are

ELISA for Esfenvalerate Metabolites
Chem. Res. Toxicol., Vol. 12, No. 11, 1999 1037
Ta ble 1. Su m m a r y of Titr a tion Testsa
Ab/immunogen 1-BSA 2-BSA 3-BSA 3-OVA 4-BSA 4-OVA 5-BSA 5-OVA 7-BSA 7-OVA 8-BSA 9-BSA PBA-BSA
b
15/5-fetuin
16/5-fetuin
17/5-fetuin
19/7-fetuin
+
+
+
-
+
+
+
-
+++
++
+++
-
++
+
+++
-
+++
++
+++
-
+
+
++
-
++++c ++++c
+++c +++c
++++c ++++c
-
-
-
-
-
-
++
++
++++
-
-
-
-
-
-
-
-
-
++++c ++++c
+++
+++
a
b
The data shown are at an antibody dilution of 1:16000 and a coating antigen concentration of 1 mg/L. Legend: -, <0.25; +, 0.25-
0.50; ++, 0.50-0.75; +++, 0.75-1.00; and ++++, >1.00. Hapten structures of 1-5 are given in Figure 2 and of 7-9 in Scheme 1.
c Homologous systems.
Ta ble 2. Selected Com p etitive ELISA Da ta
more commonly eliminated in the form of amino acid
coating
antigen
I50
(µg/L)
0.74a
1.2
8.2
0.49
0.49
10
conjugates (22, 23). Therefore, phenoxybenzoic acid, a
primary breakdown metabolite of pyrethroids, and fen-
valerate acid may predominantly undergo amino acid
conjugation in humans. Glucuronidation is also an al-
ternative conjugation pathway for these two acids; how-
ever, the possible internal transesterification makes
these conjugates equilibrium mixtures, which are difficult
to separate and detect. In contrast, the glycine conjugates
are more stable and measurable. In this study, glycine
conjugates of PBA and sFA were chosen as target
analytes.
immunogen
analyte
antiserum
Ab 15
5-fetuin
sFA-glycine (8)
3-BSA
4-BSA
5-BSA
3-BSA
4-BSA
5-BSA
8-BSA
3-BSA
4-BSA
5-BSA
8-BSA
7-BSA
7-OVA
9-BSA
PBA-BSA
Ab 16
Ab 17
Ab 19
2.6
0.24
0.34
2.1
1.5
2.5
3.6
1.2
0.36
To generate a specific antibody for an analyte, it is
important to alter the structure of the target compound
as little as possible when it is modified for coupling to a
carrier protein. In this respect, it is prudent to attach
the handle as distally as possible from the partial
structure that best defines the target. This was ac-
complished in the previous study for developing an
esfenvalerate immunoassay by attachment of the handle
at the 4-position of the terminal phenyl group of fenval-
erate (haptens 1 and 2). Similarly, in this study, hapten
handles for glycine conjugates were attached to the amino
acid, leaving the target acid unmodified (in haptens 5
and 7). Syntheses of haptens were carried out using the
chiral isomers where appropriate to facilitate purification
as well as to make the chiral hapten corresponding to
the major (active) isomer in esfenvalerate insecticide.
4-Nitro-L-phenylalanine having the same chirality as the
natural L-phenylalanine was used as the intermediate
in an effort to minimize handle recognition. The synthesis
route for hapten 7 is summarized in Scheme 1. The acid
chloride of 3-phenoxybenzoic acid was reacted through a
modified Schotten-Baumann reaction with 4-nitro-L-
phenylalanine to give the N-acylated nitro intermediate
6. Subsequent catalytic reduction of the nitro group to
the amine gave the corresponding amino hapten 7.
The syntheses of the target metabolites 8 and 9 are
summarized in Scheme 2. These were also prepared via
Schotten-Baumann reactions of the respective acid chlo-
rides with glycine and isolated by chromatography and
crystallization. The structures of these compounds as well
as all of the haptens were confirmed by NMR and mass
spectroscopy.
7-fetuin
PBA-glycine (9)
a
Each set of data represents the average of four experiments.
colored protein conjugates. The color can be seen as an
indicator of a successful coupling reaction. The colored
products are also easily detected when purified by gel
chromatography.
Scr een in g of th e Ser a a n d Assa y Op tim iza tion .
A checkboard titration system was used for screening of
antibody and antigen combinations (38). The antisera of
four rabbits were tested against 13 coating antigens, and
the data are summarized in Table 1. In general, all
antisera exhibited higher titers in homologous systems
than in heterologous systems. In a homologous system,
the fact that the hapten in the coating antigen and the
hapten in the immunogen are identical is the primary
reason for a higher titer. Antibodies raised against
5-fetuin only recognized coating antigens containing the
FA portion, and no titer was measured with PB-contain-
ing antigens. In contrast, antibodies raised against
7-fetuin had a very high titer with PB-containing coating
antigen 7-BSA or -OVA, 9-BSA, and PBA-BSA, and
no recognition with any other antigens that were tested.
The combinations of antibody and/or coating antigen with
higher titers (OD > 0.5 in Table 1) were further screened
by competitive assay. Since no difference (except 4-BSA
or -OVA) was observed in antibody titers between
coating antigens with different carrier proteins (BSA or
OVA), only BSA conjugates of 3-5 were used for further
screening with Ab 15-17 against compound 8. 7-BSA,
9-BSA, and PBA-BSA were tested for inhibition experi-
ments with Ab 19 against compound 9.
Ha p ten Con ju ga tion . Fetuin was chosen as a carrier
protein for haptens 5 and 7. For the conjugation of the
haptens to fetuin, which is a glycoprotein, the NaIO4
method was used. As discussed by Wengatz et al. (37),
the following advantages were anticipated: (1) the
increased carbohydrate content of the carrier increases
the antigenicity of the immunogen; (2) the carbohydrate
moiety allows the immunogen to be fairly hydrophilic
despite loading with a lipophilic hapten; and (3) bovine
fetuin is commercially available. The diazotization method
was used in this study to prepare coating antigens. This
conjugation method has the advantage of producing
All combinations that were tested exhibited a very low
I50 (<10 µg/L) against their target analytes (Table 2),
which ranged from 10.0 to 0.30 µg/L. The homologous
systems (Ab 15-17 vs cAg 5-BSA; Ab 19 vs 7-BSA) had
higher I50s than the heterologous systems. For example,
with Ab 16, the I50 for analyte 8 in a homologous system
is about 20 times higher than that in heterologous
systems. These results are consistent with studies of

1038 Chem. Res. Toxicol., Vol. 12, No. 11, 1999
Ta ble 3. Effects of Solven t (Meth a n ol)a
Shan et al.
analyte
Ab/cAg
% MeOHb
ABSmax (A)
slope (B)
I50 (C) (µg/L)
ABSmin (D)
sFA-glycine (8)
Ab 17/3-BSA
0
10
20
40
60
80
0
10
20
40
60
80
0.51
0.52
0.55
0.64
0.66
0.67
0.55
0.60
0.65
0.72
0.81
0.77
1.5
1.1
1.2
1.1
0.94
0.95
0.80
0.82
0.80
0.77
0.71
0.55
0.28 ( 0.03c
0.18 ( 0.02
0.31 ( 0.04
0.99 ( 0.12
4.9 ( 0.26
22 ( 1.8
0.45 ( 0.08
0.30 ( 0.02
0.25 ( 0.03
0.33 ( 0.03
1.0 ( 0.07
4.2 ( 0.18
0.02
0.01
0.03
0.03
0.01
0.03
0.01
0.01
0.02
0.03
0.02
0.04
PBA-glycine (9)
Ab 19/PBA-BSA
a
ELISA conditions: 0.5 µg/mL coating antigen 3-BSA, 1:16000 antiserum 17, 0.5 µg/mL coating PBA-BSA, 1:10000 Ab 19, and 1:3000
goat anti-rabbit IgG-HRP. Concentration of MeOH in standard solution (PBS-MeOH). c Mean value ( SD. Each set of data represents
the average of three experiments.
b
Ta ble 4. Cr oss Rea ctivities of Esfen va ler a te Meta bolites
ELISAs for the triazines (39) and arylureas (40). Re-
cently, Goodrow and Hammock (41) have reported the
advantages of using heterologous haptens for immunogen
and coating/tracer haptens and their uses for selective
and sensitive assay design and development. In this
study, only Ab 17/cAg 3-BSA and Ab 19/cAg PBA-BSA
were used for further assay development to detect 8 and
9, respectively.
a n d Oth er Rela ted Com p ou n d sa
Solven t Effects. Methanol was evaluated for solvent
effects in these two assays. The results (Table 3) show
that methanol apparently influenced assay sensitivity
and absorbance. The maximum absorbance (without
analyte) for both assays was enhanced with increasing
methanol concentrations. However, the I50 was increased
when the solvent concentration exceeded 40% (for 9) and
20% (for 8) with decreasing slope values. Selection of the
optimum MeOH concentration was based on the I50
values, the slope, and the ratios of maximum and
minimum absorbances for standard curves (A/D). Gener-
ally, a lower I50, at a higher MeOH concentration, and a
high slope are desirable. In the study presented here, 20%
MeOH was chosen in both assays for the analyte dilution
and the standard solution preparation.
Cr oss Rea ctivity. Esfenvalerate, cypermethrin, flu-
valinate, esfenvalerate metabolites, other structurally
related compounds, and 20 free amino acids were tested
for cross reactivities (Table 4). Both antibodies 17 and
19 are highly specific for their target analytes 8 and 9,
respectively. In all cases, the interference of the parent
compound, other metabolites, and other tested com-
pounds was negligible. Although PBA, PB alcohol, phe-
noxybenzoate, and three other tested pyrethroids contain
the PB group, which is present in the immunogen hapten,
they did not interfere in the assay for hapten 9 (CR <
0.5%). However, this antibody (Ab 19) binds to PBA-
BSA very well (high titer in Table 1), indicating the
important contribution of the handle (NH) recognition
in the antibody specificity. The amide group, generated
during coupling, in the PBA-BSA conjugate makes this
antigen hapten structurally more like the target conju-
gates. Because free PBA and sFA are possible metabolites
in urine (18, 21), it is particularly significant that assays
for both 8 and 9 have no cross reactivity with PBA and
FA. This makes these assays very useful for selective
detection of esfenvalerate metabolites.
a
Cross reactivity was calculated as (I50 of esfenvalerate/I50 of
analyte) × 100%. Each set of data represents the average of three
experiments.
in solution (Figures 3B and 4B). At an ionic strength of
3× PBS (0.45 M), the binding between antibody and
antigen was suppressed approximately 50% in the assay
of compound 9, and almost completely inhibited for the
assay of compound 8. These results are consistent with
the previous reported observations for atrazinemercap-
turic acid, which was assumed to result in disrupting
Ab-Ag interaction (32).
These two assays also showed similar tolerance to the
pH of the solutions (Figures 3A and 4A). Compared with
PBS buffer (0.15 M, pH 7.5), no influences were observed
at pH 6-8, indicating that a slight difference in the pH
of sample or buffer (in this range) would not affect the
Ma tr ix Effects. Since these assays were intended to
analyze urine samples, it was necessary to study the
influence of pH and ionic strength in this matrix. Both
assays are very sensitive to higher concentrations of salt

ELISA for Esfenvalerate Metabolites
Chem. Res. Toxicol., Vol. 12, No. 11, 1999 1039
F igu r e 4. ELISA competition curves of PBA-glycine (9)
prepared with various (A) pHs, (B) ionic strengths, and (C) %
urine. Reagent concentrations were as follows: coating antigen
(PBA-BSA), 1:10000; antibody (Ab 19), 1:20000 (final concen-
tration in wells); and goat anti-rabbit IgG-HRP, 1:3000.
F igu r e 3. ELISA competition curves of FA-glycine (8) prepared
with various (A) pHs, (B) ionic strengths, and (C) % urine.
Reagent concentrations were as follows: coating antigen (3-
BSA), 1:5000; antibody (Ab 17), 1:10000 (final concentration in
wells); and goat anti-rabbit IgG-HRP, 1:3000.
Ta ble 5. Sp ik ed Recover y
accuracy in the quantitation of 8 and 9. However, both
assays were severely suppressed at the more acidic pH
of 4.0. Therefore, these assays required the urine samples
to be adjusted by dilution or by a desalting method prior
to ELISA detection.
spiked
concentration
compound 9
SPE dilution
<0.2 0.62 ( 0.44 <0.2
1.09 ( 0.22 1.80 ( 0.82 1.15 ( 0.26 1.60 ( 0.90
2.16 ( 0.32 2.85 ( 0.87 2.06 ( 0.40 2.78 ( 1.0
compound 8
SPE dilution
0.84 ( 0.73
(µg/L)
0
1.0
2.0
5.0
10
The effects of the urine matrices were evaluated for
both assays. In tests with four different urine samples,
the assay for 8 could tolerate 4% urine, while the assay
for 9 required 100-fold dilution prior to the ELISA to
reduce the effects of urine matrix interference (Figures
3C and 4C). The results suggest that these assays may
actually be inhibited by endogeneous urinary compounds.
Therefore, dilution prior to ELISA would be necessary
for both assays. When the variation of the different urine
samples is considered, a 150-fold dilution of the urine
before the assay was chosen for both assays. In recovery
experiments for the direct dilution of urine samples, urine
matrix had little effect on accurate quantitation for urines
5.31 ( 0.42 5.88 ( 1.0
10.5 ( 0.10 11.7 ( 0.94
4.89 ( 0.29 5.40 ( 0.67
9.54 ( 0.30 11.2 ( 0.89
48.7 ( 1.0
50
49.0 ( 1.5
52.0 ( 1.5
54.0 ( 2.8
spiked with g5.0 µg/L 8 (18.5 nmol/L) and g5.0 µg/L 9
(18.3 nmol/L) (Table 5). The recovery rates for 8 and 9
at 5.0 µg/L are 108 + 12.3% and 117 + 21.0%, respec-
tively. However, urine interfered significantly with the
quantitation of unspiked samples and urines spiked with
8 or 9 at a concentration of <5.0 µg/L (Table 5). According
to the recommended LOQ determination guideline (42),
the approximate limit of quantitation is 5.0 µg/L for both
8 and 9. This is consistent with the estimated concentra-

1040 Chem. Res. Toxicol., Vol. 12, No. 11, 1999
Shan et al.
F igu r e 6. Relationship between analyte levels spiked into
urine and measured by SPE/ELISA. Urine samples were from
individuals with no known exposure to pyrethroids. Studies
were carried out in a blind fashion. For sFA-glycine (8), y )
1.06x - 1.48, r2 ) 0.99, and n ) 16. For PBA-glycine (9), y )
0.99x + 0.77, r2 ) 0.97, and n ) 15.
F igu r e 5. ELISA inhibition curves for PBA-glycine (9) and FA-
glycine (8). Reagent concentrations were as follows: the assay
of 8 using antiserum 17, 1:10000 (final dilution in wells); and
coating antigen 3-BSA, 1:5000; and the assay of 9 using
antiserum 19, 1:20000 (final dilution in wells); and coating
antigen PBA-BSA, 1:10000. Both standard curves represent
the average of 24 curves.
needed. In conclusion, a highly sensitive, specific, and
reproducible ELISA has been developed and character-
ized for the detection of the fenvalerate metabolites 8 and
9. The linear ranges of the extent of inhibition of 8 and
9 in buffer are approximately 0.03-60 and 0.04-50 µg/
L, respectively, and the I50 values for 8 and 9 are 0.40
(1.47 nmol/L) and 0.42 µg/L (1.56 nmol/L), respectively.
Organic solvents, temperature, incubation time, and
human urine had strong matrix effects on the optimized
immunoassays. The ELISAs were successfully applied to
the quantitative detection of trace amounts of 8 and 9 in
human urine by using either direct dilution or the SPE
method. Since phenoxybenzyl (PB) is a common group
in conventional pyrethroids, the assay of compound 9 is
class-specific for PB-containing pyrethroids, which makes
this assay more useful and significant as a biomarker of
exposure to pyrethroids. Finally, further studies of hu-
man urine from individuals who have been exposed to
esfenvalerate or other pyrethroids are necessary for
toxicological and analytical confirmation.
tion that corresponded to the absorbance of the control
(zero analyte) minus 3 times the standard deviation of
control, when standard analytes were prepared in a
buffer system with 0.67% unspiked urine.
Solid -P h a se Extr a ction (SP E). A C18 column was
used for urine sample extraction in this study. The SPE
method was based on previously reported methods for
isolating atrazinemercapturic acid from human urine
(32). These assays are sensitive to acidic conditions;
therefore, the final washing step was changed by using
deionized water and 20% methanolic water (deionized).
Good recovery and less interference were achieved for
both assays using this washing step (Table 5). However,
SPE under these conditions failed to eliminate all matrix
effects, and dilution of the concentrated sample prior to
the ELISA was required for accurate quantitation. Ac-
cording to the test of four urine samples, a 25-fold dilution
could eliminate the interference of the urine matrix.
When the variability of different urine samples was
considered, a 50-fold dilution was used for both assays
in this study. Similarly, the LOQ for the detection of 8
and 9 can be defined as 1.0 µg/L (3.70 nmol/L) (Table 5).
Assa y Va lid a tion . The assay validation was per-
formed in a blind fashion by direct dilution or SPE
sample preparation of the urine samples, which were
spiked with 8 or 9 ranging in concentration from 0 to 50
µg/L (Figure 5). The linear regression analysis of ELISA
results showed a very good correlation in all four cases
(Figure 6). The data from samples prepared by SPE had
a slightly better correlation (r2 ) 0.99 and 0.97 for 8 and
9, respectively) than those prepared by direct dilution (r2
) 0.89 and 0.90 for 8 and 9, respectively). All recoveries
were >93% of the spiked values. The results demonstrate
that these assays are suitable for the quantitative
detection of esfenvalerate metabolite conjugates at trace
levels in urine samples.
Ack n ow led gm en t. We thank A. Daniel J ones (Penn-
sylvania State University, University Park, PA) for mass
spectral determinations and the Department of Chem-
istry (University of California, Davis) for the use of its
NMR instrument. This project was funded by the Na-
tional Institute of Environmental Health Sciences (NIE-
HS) Superfund Research program (5 P42 ES04699-08),
the NIEHS Center for Environmental Health Sciences,
and the Environmental Protection Agency Center for
Ecological Health Research (CR819658).
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